Although some epidemiologic studies found inverse associations between alcohol drinking and Parkinson's disease (PD), the majority of studies found no such significant associations. Additionally, there is only limited research into the possible interactions of alcohol intake with aldehyde dehydrogenase (ALDH) 2 activity with respect to PD risk. We examined the relationship between alcohol intake and PD among Japanese subjects using data from a case-control study.From 214 cases within 6 years of PD onset and 327 controls without neurodegenerative disease, we collected information on "peak", as opposed to average, alcohol drinking frequency and peak drinking amounts during a subject's lifetime. Alcohol flushing status was evaluated via questions, as a means of detecting inactive ALHD2. The multivariate model included adjustments for sex, age, region of residence, smoking, years of education, body mass index, alcohol flushing status, presence of selected medication histories, and several dietary factors.P for trend = 0.001). There was no significant interaction of alcohol intake with flushing status in relation to PD risk.Alcohol intake during peak drinking periods, regardless of frequency or amount, was not associated with PD. However, when we assessed daily ethanol intake separately for each type of alcohol, only Japanese sake (rice wine) was significantly associated with PD (adjusted odds ratio of ≥66.0 g ethanol per day: 3.39, 95% confidence interval: 1.10-11.0, We did not find significant associations between alcohol intake and PD, except for the daily amount of Japanese sake. Effect modifications by alcohol flushing status were not observed. While smoking has been consistently associated with a decreased risk of Parkinson's disease (PD), a relationship between alcohol intake and PD still remains controversial. Some epidemiological studies reported that alcohol drinking was inversely associated with PD -4. Convein vitro and in vivo [Alcohol drinking status, including frequency and amount, can be influenced by activities of alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases (ALDHs). ALDH2, a key enzyme in the elimination of acetaldehyde, has a polymorphism that is prevalent in East Asians, but rare in Caucasians or Africans . Those w in vivo ,21, it m in vivo .In the present study, we examined the association between alcohol drinking and PD risk using data from a hospital-based case-control study among Japanese. We also investigated whether flushing status, as a proxy variable of ALDH2 activity, modified the association.Methodologies used in this study have been described previously ,23. CaseRecruitment of control subjects was conducted at 3 of the same 11 hospitals: 1 university hospital in Fukuoka Prefecture, and 1 university and 1 national hospital in the Kinki region. Eligible control subjects were inpatients and outpatients without neurodegenerative diseases recruited from departments other than neurology  during the period from April 1, 2006 to March 31, 2008. Controls were not matched to cases either individually or by group. A total of 528 patients were approached by their attending physicians or our research nurses for recruitment as controls; 372 agreed and 156 declined (response rate: 70%). The study protocol was approved by the ethics committees of the 11 collaborating hospitals and written consent was obtained from all subjects.Cases and controls filled out a set of 2 self-administered questionnaires and mailed them to the data management center or handed them to research nurses. Research technicians completed missing or corrected illogical data by telephone or direct interview. One questionnaire was used to collect information on "peak", as opposed to average, alcohol drinking status during a subject's lifetime. Subjects were asked to provide information on their drinking habits during the period in which alcohol consumption was the highest. We assessed drinking frequency, as well as volume of alcohol intake according to beverage types. The volume was subsequently converted to grams of ethanol, and values for each beverage type were added. The ethanol contents for calculation were as follows: 4.5% for beer, 15.5% for Japanese sake (rice wine), 25% for shochu , 12% for wine, and 43% for whisky. Both the daily and the weekly amounts of alcohol intake during the peak drinking period  were estimated for each subject. The questionnaire also elicited information on several factors such as sex, age, smoking, education, and presence of medication history for hypertension, hypercholesterolemia, and diabetes. Although its validity has not been investigated, the questionnaire for this study was developed based on a comprehensive literature review of epidemiologic studies of factors associated with PD.Alcohol flushing status was evaluated via questions as a proxy for detecting inactive ALHD2 , as follDietary habits during the preceding month were assessed using a self-administered, semi-quantitative, comprehensive diet history questionnaire (DHQ). Details of the DHQ's structure, calculation of dietary intake, and validity for commonly studied nutritional factors have been published elsewhere . DietaryBecause we failed to obtain information on peak alcohol drinking from 69 former drinkers (32 cases and 37 controls), they were eliminated from the analysis. Also excluded were 2 cases without available information on peak drinking prior to the onset of PD and 1 case and 4 controls whose information on any of the alcohol-related factors was missing. After further exclusion of 1 case and 4 controls due to missing data on other factors under study, 214 cases and 327 controls were subjected to final analysis.Frequency and amount of alcohol drinking were categorized into 3 levels based on the distribution of controls: 1) never drinkers, 2) current drinkers who consumed alcohol <6 days per week for frequency, 0.1-65.9 grams ethanol per day for daily amounts, and 0.1-219.3 grams per week for weekly amounts, and 3) current drinkers who consumed alcohol ≥6 days per week for frequency, ≥66.0 grams ethanol per day for daily amounts, and ≥219.4 grams per week for weekly amounts. Logistic regression analyses were employed to estimate ORs and 95% CIs of alcohol, using non-drinker as the reference category. The trend of association was assessed by assigning ordinal scores to the levels of independent variables.The primary multivariate model included adjustments for sex, age, region of residence, pack-years of smoking, years of education, body mass index, alcohol flushing status, and presence of medication history  as potential confounders. Region of residence was classified into 2 categories (Fukuoka and Kinki); pack-years (1 pack/day for 1 yr) of smoking into 3 ; years of education into 3 ; flushing status into 2 (never and former/current flushing); and presence of medication history into 2 (yes or no for each disease category). Age and body mass index were included as continuous variables. The fully adjusted model additionally incorporated several dietary factors including caffeine, cholesterol, vitamin E, vitamin B6, iron, and the dietary glycemic index as continuous variables. We also tested for statistical interactions of alcohol intake with flushing status by including product terms as independent variables in a fully adjusted model. All hypothesis testing was conducted assuming a 0.05 significance level and a 2-sided alternative hypothesis. SAS version 9.1  was used throughout the analysis.Thirty-five percent of the subjects were male and the mean age was approximately 70 years Table . CompareP for trend = 0.03). The confounding was primarily due to smoking, not flushing status (data not shown). This positive relationship disappeared, however, when caffeine, cholesterol, vitamin E, vitamin B6, iron, and the dietary glycemic index were further included as confounders . Likewise, no significant associations were observed between frequency or daily ethanol intake and the risk of PD in the fully adjusted model: the adjusted OR of the highest category (≥6 days per week and ≥66.0 g ethanol per day) was 0.96 for the frequency  and 1.46 for the daily amount .In crude analyses, neither frequency nor amount of alcohol intake during the peak drinking period was significantly associated with the risk of PD among all subjects Table . After aP for trend = 0.39), 3.39 for Japanese sake , 1.29 for shochu , 6.11 for wine  and 2.25 for whisky . We could not examine the frequency or weekly amount of alcohol intake for the individual types of alcohol because we did not ask information on drinking frequency separately for each type of alcohol.When we assessed daily ethanol intake separately for each type of alcohol, there were no significant associations with PD, except for Japanese sake Table . The OR P for interaction = 0.22, 0.37, and 0.18 for the highest category of frequency, daily amount, and weekly amount of alcohol intake, respectively). With respect to the separate types of alcohol, no interactions with flashing status were observed.In the stratified analysis according to flushing status, former and current flushers were combined due to small numbers of former flushers Table . We did In this study, alcohol intake during the peak drinking period was not associated with PD when ethanol intake was combined from all types of alcohol. There was no significant interaction of alcohol drinking with flushing status in relation to PD risk. Similarly to the present study, a substantial body of previous case-control studies failed to find significant associations between alcohol and PD. In each study, alcohol as an exposure variable was assessed differently, including as a binary category of "ever vs. never" -8,12,13,Our finding is not in agreement with previous studies that observed significant inverse associations between alcohol intake and PD -4. A metWhen we examined daily ethanol intake separately for each type of alcohol, only Japanese sake was positively associated with PD. To our knowledge, there has been no report that Japanese sake increased the risk of PD. Although our finding could be due to chance, it has been shown that relationships between alcohol intake and PD varied according to different kinds of alcohol ,28,29.In vitro studies showed that aldehydes might react with dopamine. Ethanol enhanced the toxicity of 6-hydroxydopamin (6-OHDA) when ethanol and 6-OHDA were simultaneously applied to cultured cells [ed cells . In an aed cells . By conted cells . These iA strength of the present study is that cases were identified using strict diagnostic criteria, minimizing disease misclassification as much as possible. We also designed our study with extensive data collection, based on comprehensive literature review, allowing us to adjust for several potential confounders, including dietary factors. However, residual confounding cannot be ruled out. In contrast to previous studies that looked at average drinking habits, we examined subjects' peak drinking status as exposure variables. Some PD patients experience several gastrointestinal symptoms as non-motor manifestations. It was shown that constipation, occurring as early as 20 or more years before the onset of motor symptoms, was associated with an increased risk of PD . Given tP <0.0001), older , smokers , more educated , and have a medication history for diabetes . We previously revealed an inverse association between smoking habits or medication history for diabetes and PD in this study population [Our study was limited by our failure to collect information on peak drinking from former drinkers . A comparison of the 541 subjects included in the final analysis with 69 former drinkers revealed that former drinkers were more likely to be male  for frequency, 1.17  for daily amount, and 1.66  for weekly amount. Furthermore, cases in the present study were prevalent rather than incident cases. When we conducted a sensitivity analysis confined to cases less than 3 years from onset (n = 88), interpretation of our results was not markedly changed. The adjusted OR in the highest category was 0.60  for frequency, 1.28  for daily amount, and 1.50  for weekly amount. We were also concerned that medication history might affect alcohol drinking habits, so we performed a sensitivity analysis among subjects without medication history for hypertension, hypercholesterolemia, or diabetes (152 cases and 173 controls). The associations between alcohol intake and PD were not considerably altered. The adjusted OR in the highest category was 0.91  for frequency, 1.34  for daily amount, and 1.61  for weekly amount. Thus, the possible influence of medication history on alcohol intake was, if any, likely to be minimal. Finally, all the information in this study relied on self-reports rather than on interviews by trained investigators, and information on alcohol intake and non-dietary factors was collected via a non-validated questionnaire, which could affect the present results.Another possible limitation is that our controls were not fully representative of the population from which our cases arose, as they were selected from only 3 of the 11 collaborating hospitals where cases were recruited. The results of a sensitivity analysis restricted to cases who were recruited from the three hospitals associated with control recruitment (n = 130) were similar to those in the overall analysis: the adjusted OR in the highest category was 0.78 ; Hideyuki Sawada, Eiji Mizuta, and Nagako Murase ; Tsuyoshi Tsutada and Hiroyuki Shimada ; Jun-ichi Kira ; Tameko Kihira and Tomoyoshi Kondo ; Hidekazu Tomimoto ; Takayuki Taniwaki ; Hiroshi Sugiyama and Sonoyo Yoshida ; Harutoshi Fujimura and Tomoko Saito ; Kyoko Saida and Junko Fujitake ; Naoki Fujii ; Masatoshi Naito and Jun Arimizu ; Takashi Nakagawa, Hirofumi Harada, and Takayuki Sueta ; Toshihiro Kikuta and George Umemoto ; Eiichi Uchio and Hironori Migita ; Kenichi Kazuki, Yoichi Ito, and Hiroyoshi Iwaki ; Kunihiko Siraki and Shinsuke Ataka ; Hideo Yaname and Rie Tochino ; Teruichi Harada ; Yasushi Iwashita, Motoyuki Shimizu, Kenji Seki, and Keiji Ando .The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2377/10/111/prepub


Retinitis pigmentosa is the most important hereditary retinal degenerative disease, which has a high degree of clinical and genetic heterogeneity. More than half of all cases of retinitis pigmentosa are autosomal recessive (arRP), but the gene(s) causing arRP in most families has yet to be identified. The purpose of this study is to identify the genetic basis of severe arRP in a consanguineous Chinese family.EYS was used to determine the disease-causing mutation, and to demonstrate that the mutation co-segregates with the disease in the family.Linkage and haplotype analyses were used to define the chromosomal location of the pathogenic gene in the Chinese arRP family. Direct DNA sequence analysis of the entire coding region and exon-intron boundaries of A single nucleotide substitution of G to T at nucleotide 5506 of EYS was identified in the Chinese arRP family. This change caused a substitution of a glutamic acid residue at codon 1,836 by a stop codon TAA (p.E1836X), and resulted in a premature truncated EYS protein with 1,835 amino acids. Three affected siblings in the family were homozygous for the p.E1836X mutation, while the other unaffected family members carried one mutant allele and one normal EYS allele. The nonsense mutation p.E1836X was not detected in 200 unrelated normal controls.EYS gene is a recently identified disease-causing gene for retinitis pigmentosa, and encodes the orthologue of Drosophila spacemaker. To date, there are only eight mutations in EYS that have been identified to cause arRP. Here we report one novel homozygous nonsense mutation of EYS in a consanguineous Chinese arRP family. Our study represents the first independent confirmation that mutations in EYS cause arRP. Additionally, this is the first EYS mutation identified in the Chinese population.The  Mutations in ABCA4, CDHR1, CERKL, CNGA1, CNGB1, CRB1, IDH3B, LRAT, MERTK, NR2E3, NRL, PDE6A, PDE6B, PRCD, PROM1, RBP3, RGR, RHO, RLBP1, RP1, RPE65, SAG, SPATA7, TULP1, USH2A and most recently EYS have been identified to be the cause for arRP [Retinitis pigmentosa  is characterized by the constriction of the visual fields, night blindness, changes of the fundi including 'bone corpuscle' lumps of pigment, and the loss of central vision. The worldwide prevalence of RP is about one in 4,000 . RP is iEYS gene. EYS is predicted to be a 3,165 amino acid multi-domain protein, which contains at least 28 epidermal growth factor (EGF)-like domains in its N-terminus, and C-terminal laminin A G-like domains. It is an evolutionarily conserved protein from Drosophila to humans. Finally, eight EYS mutations were detected in eight arRP families from Spanish and Dutch origins at almost the same time in 2008 [RP25, a genetic locus for arRP, was mapped to a ~ 16 cM region on chromosome 6p12.1-q15 in four Spanish families by Ruiz et al. in 1998 -12. By u in 2008 ,8.EYS. Our data confirmed the conclusion from the two original reports that mutations in EYS can cause arRP, and expanded the mutation spectrum of EYS to the Chinese population.In this study, we characterized a consanguineous Chinese family with a severe form of arRP. After linkage analysis with known arRP causative genes and loci, we excluded all other arRP loci except for the RP25 locus. Further direct DNA sequence analysis revealed a homozygous mutation in the The study subjects from a consanguineous Chinese arRP family were recruited from Hubei Province, P. R. China. Informed consent was obtained from the participants in accordance with the study protocols approved by the Ethics Committee of Huazhong University of Science and Technology. Diagnosis of RP was carried out by clinical and ophthalmological examinations. Peripheral blood was collected from the family members and 200 normal Chinese Han controls. Total human genomic DNA was isolated with the DNA Isolation Kit for Mammalian Blood .ABCA4, CERKL, CNGA1, CNGB1, CRB1, EYS, I8DH3B, LRAT, MERTK, NR2E3, NRL, PDE6A, PDE6B, PRCD, PROM1, RGR, RHO, RLBP1, RPE65, SAG, TULP1, and USH2A. The microsatellite markers flanking these loci were selected from Linkage Mapping Set MD-10 , and genotyped using an ABI 3100 Genetic Analyzer. Genotypes were analyzed by the GeneMapper 2.5 Software program . PCR and genotyping were performed as previously described [EYS gene, additional SNPs  were employed for the linkage analysis to get unambiguous linked haplotype. The EYS was then subjected to mutational analysis. All coding exons and splice sites of EYS were amplified by PCR and sequenced. The intronic primers and corresponding PCR conditions were as described in the paper of Abd El-Aziz et al. [Linkage and haplotype analyses were applied to test the linkage of the family to 22 arRP loci, including escribed . When thThe RP in the consanguineous Chinese family is inherited in an autosomal recessive mode figure . ParentsThe proband exhibited night blindness at the age of 15 years, and progressively lost his visual acuity to 20/400 at approximately the age of 35-40 years. Funduscopic examinations revealed obvious attenuation of the retinal arteries, waxen color of discs, and bone-spicule pigmentation in the peripheral-mid retina figure . ElectroThe other two affected individuals also had the similar symptoms as the proband. Night blindness appeared late in the second decade of life, low visual acuity between 20/400 and hand movement, and a flat ERG confirmed the diagnosis and severity of the RP disease. No additional symptoms or ocular defects were observed in the affected individuals of the family except for patient II5, who was affected with cataract in both right and left eyes.EYS may be the pathogenic gene in this family.After linkage analysis for known arRP causative genes and loci, there was only the RP25 locus that showed a definite linkage with the disease in the family. Homozygosity mapping with haplotype analysis revealed that all three affected siblings  inherited the affected alleles separately from their carrier parents, whereas their unaffected brother II4 inherited two normal chromosomes, and the proband's daughter III1 only one affected allele  found a single nucleotide transversion of G to T at nucleotide 5506 of EYS, and it resulted in a substitution of a glutamic acid residue at codon 1836 of EYS by a stop codon TAA (p.E1836X) -like domains, and 5 LamG domains at the C-terminus. All 5 LamG domains and 7 EGF-like domains in the C-terminus of EYS were truncated in the arRP family, which may significantly disrupt the normal physiological function of EYS in the retina.A novel EYS gene has recently been identified as the disease-causing gene for retinitis pigmentosa, and encodes an orthologue of Drosophila spacemaker. So far, only eight mutations have been reported in arRP families by two groups in 2008. El-Aziz et al identified six mutations, including four homozygous mutations p.D904QfsX17, p.S754AfsX6, p.T657TfsX5, p.W2640X and two compound heterozygous mutations p.E1953X and p.R589RfsX5 [EYS mRNA through a nonsense-mediated decay mechanism.The EYS [EYS mutations. Until recently, mutations of EYS have been detected in eight families [EYS gene [EYS mutation, too [RP 25 could be a major locus for arRP because 10-20% of Spanish typical arRP families were found to have mutations in EYS ,13. Consfamilies ,8. ThreeEYS gene . SimilarEYS contains 44 exons covering 2.0 Mb, and it is considered to be the largest gene identified and expressed in the human eye as well as the fifth largest overall in the human genome. EYS is highly expressed in the retina, but its biological function remains unknown. In Drosophila, the EYS homological protein spam, and its interactor protein prom, plays a critical role in retina morphogenesis. Drosophila mutant lines for spam and prom can result in a failure of inter-rhabdomeral-space separation [PROM1 were associated with arRP, macular degeneration, and cone-rod dystrophy [EYS mutations causing arRP and the data from Drosophila strongly suggest that EYS may have an important function in the photoreceptor morphogenesis. Further investigations of the biochemical function of EYS in retinas will lead to a significant understanding of the pathological mechanism(s) of retina degeneration.paration . Mutatioystrophy -19. EYS EYS mutation found in the Chinese population. This research confirms that mutations of EYS can cause arRP. It has also expanded the spectrum of EYS mutations in arRP.A novel homozygous nonsense mutation in EYS, p.E1836X, was responsible for using arRP in the consanguineous Chinese family. This is the first EYS novel mutations in patients with retinal dystrophy. Abd El-Aziz et al. [EYS mutations in French patients with rod-cone dystrophies. These studies supported that EYS is critical for retinal function and confirmed that EYS is a major causative gene for retinal dystrophies in populations of various ethnicities.Very recently, three groups of investigators described the identification of z et al.  identifiz et al.  reportedz et al.  found EYThe authors declare that they have no competing interests.YH recruited the members of arRP pedigree, performed clinical studies for whole family members, and relationship studies between genotype and phenotype. JZ carried out the extraction of genomic DNA and genotyping assay. CL was responsible for the extraction of genomic DNA and sequencing for mutation detection. GY was involved in linkage analysis and the discussion of the manuscript. ML participated in the original conception of this study, and contributed to the revision of the draft. QKW conceived and designed the experiments and helped the critical revision of the manuscript. ZT designed the experiments, analyzed the data, interpreted the results, and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/11/121/prepub


Needle length plays an important role for the success of ophthalmic block. The standard practice is to use 25 mm needles length; however, unnecessarily long needles may increase the risk of complications especially in the presence of staphyloma or previous scleral buckle.This work was designed to compare the efficacy of using 15 and 25 mm needle in performing extraconal block for patients undergoing vitreoretinal surgery.Prospective randomized double blinded study.A total of 120 patients were enrolled in this study and were divided in two groups. In group (1) extraconal block was performed using 25 mm needle, while in group (2) 15 mm needle was used. After primary injection, assessment of the block was done by an anesthesiologist who was unaware of the needle used. If satisfactory akinesia was not achieved a supplementation was provided. At the end of the procedures, patients and surgeons were asked to assess their pain and satisfaction with the anesthetic technique.t-test, and chi-square test, respectively.The sample size calculation using N-Quary version 4. Numerical and categorical data were analyzed using an independent sample, a two-tailed The volume of primary injectable was significantly higher in group 2. The two groups were comparable as regards total volume of local anesthetic, supplementation rate, akinesia, pain score, and surgeon satisfaction.Using 15 mm needle length to perform extraconal blockade for posterior segment procedures is equally effective to 25 mm needle. A majority of patients presenting for retinal procedures are either elderly or having several co-morbidities in which general anesthesia carries a higher risk. A peribulbar technique was successfully used for this type of patients. PosterioAlthough peribulbar block is considered as an effective technique, the blind administration of long needle into the orbital cavity has been associated with sight-threatening complications including inadvertent globe penetration, optic nerve injury, brainstem anesthesia, retrobulbar hemorrhage, and retinal vascular occlusion. On the other hand, life-threatening complication including seizures and cardiopulmonary arrest has been reported. It had bNeedle length is important for the safe conduct of peribulbar blocks. Theoretically, shorter needles may reduce damage to vital orbital structures and decrease complications. Previously, some authors recommended that maximum penetration of needle from orbital rim should not be greater than 31 mm. The aim After approval of the hospital Research and Human ethics committees as well as informed patient consent, 120 patients undergoing pars plana vitrectomy under regional anesthesia were enrolled in this randomized double-blinded prospective study. The patients were randomly divided into two groups using a sealed envelope technique (60 each). In group 1, peribulbar block was performed using standard sharp disposable 25 mm, 25 G needle while in group 2, 15 mm, 25 G needle was used. Patients allergic to local anesthetic solutions, with local sepsis, serious impairment of coagulation, and orbital abnormalities, or who were unable to cooperate in maintaining a relatively motionless supine position or who refused the anesthetic technique were excluded from the study.The patients were requested to be fasting for 6 h preoperatively and were premedicated with hydroxyzine, paracetamol and, codeine (Revacod) 1 h before surgery according to the standard policy of the hospital. On arrival at OR holding area, standard monitoring of vital signs were commenced and an intravenous cannula was placed. Initial globe movements in all directions of gaze  were assessed.Our technique of peribulbar block involves insertion of the needle through the lower eyelid as far lateral as possible in the inferotemporal quadrant. After negative aspiration, a volume of 8-10 ml of local anesthetic solution  was injected. Digital pressure was applied by the thumb and index fingers around the needle hub during injection. Ocular movements were assessed at 5 and 10 min after the block. A simple akinesia score was used for assessment of the block.[Anesthesia-related scores of satisfaction was determined postoperatively by surgeons using horizontal visual scale from 0  to 10 . For bettt-test. On the other hand, categorical data were expressed as number and percentage. Ordinal and nominal data were compared using the chi-square test. A P value of 0.05 was used as the level of significance.The results were analyzed using the Statistical Package for Social Science for Windows version12 . The sample size calculation using N-Quary software version 4 indicated that 120 patients were required to detect a 0.5 difference in the mean of the simple akinesia score between the two groups, when alpha error =0.05, and type II error 0.1. Numerical data were expressed as a mean and standard deviation (SD) and were analyzed using an independent sample, two-tailed A total of 120 patients were enrolled in this study. Demographic and clinical data are shown in P value=0.035). However, no difference detected as regard the total volume of local anesthetic used . The number of patients who required supplementation and total number of supplements were comparable between the two groups . No significant difference was observed between the groups as regard akinesia score after 5 and 10 min following injection .The volume of local anesthesia injected, supplementation rate, and akinesia score are shown in P value=0.21 and 1.00, respectively). The groups were comparable as regards intraoperative supplementation rate, time after initial block, and volume of the supplement .Intraoperative data are shown in No major life-threatening or block-related complications were noted.et al,[The results of the current study showed that the use of a 15 mm needle for posterior segment procedures gives a comparable result to a 25 mm needle in terms of volume injected of local anesthetic, akinesia, supplementation rate, intraoperative pain, and surgeon/patient satisfaction. Gills et al, divide tet al, Hamiltonet al, Ben- Davet al, Scott anet al, demonstret al,[The findings of this work are consistent with Rizzo et al, who demoet al, demonstret al, The sameet al,[1315The observation in this study that relatively larger volumes of local anesthetic (approximately 11 ml) are used after supplementation compared to lower volume (5-6.5 ml) used by Rizzo et al, could beet al, On the oet al, used a met al,131516 shPeribulbar anesthesia with 15 mm needle, using the combination of 2% lidocaine, 0.5% bupivacaine, and 5 unit/ml hyaluronidase, provides excellent surgical conditions, akinesia, and analgesia in patients undergoing posterior segment surgery and it is equally effective to 25 mm needle.


Caenorhabditis elegans, we have investigated how two major life-history traits, (i) the development of environmentally resistant dauer larvae and (ii) reproduction, respond to environmental stress (high population density and low food availability), and how these traits vary between lines and the genetic basis of this variation.Organisms live in environments that vary. For life-history traits that vary across environments, fitness will be maximised when the phenotype is appropriately matched to the environmental conditions. For the free-living nematode C. elegans vary in their phenotypic plasticity of dauer larva development, i.e. there is variation in the likelihood of developing into a dauer larva for the same environmental change. There was also variation in how lifetime fecundity and the rate of reproduction changed under conditions of environmental stress. These traits were related, such that lines that are highly plastic for dauer larva development also maintain a high population growth rate when stressed. We identified quantitative trait loci (QTL) on two chromosomes that control the dauer larva development and population size phenotypes. The QTLs affecting the dauer larva development and population size phenotypes on chromosome II are closely linked, but are genetically separable. This chromosome II QTL controlling dauer larva development does not encompass any loci previously identified to control dauer larva development. This chromosome II region contains many predicted 7-transmembrane receptors. Such proteins are often involved in information transduction, which is clearly relevant to the control of dauer larva development.We found that lines of C. elegans alters both its larval development and adult reproductive strategy in response to environmental stress. Together the phenotypic and genotypic data suggest that these two major life-history traits are co-ordinated responses to environmental stress and that they are, at least in part, controlled by the same genomic regions. Drosophila melanogaster body size metrics across temperature ranges ). These possible explanations were investigated further.We wished to determine how s Figure . There wper capita food availability. To investigate how the fecundity of C. elegans was affected by this we firstly determined the lifetime fecundity of 12 RILs under ad libitum food conditions. This showed that these RILs had significantly different lifetime fecundities ; fecundity did not differ between experimental blocks . We calculated the rate of reproduction, q, a measure of the rate of increase in the proportion of lifetime fecundity, of the RILs and found that this also differed significantly between RILs  and between experimental blocks .One of the principal features of high density environments is a reduction in the 2 = 0.548, p = 0.002), but not on days 5 and 9 . This may suggest that at day 7, growing populations of C. elegans are not constrained by density-dependent effects, but at day 9 that they are. The absence of a significant correlation on day 5 may be due to inter-RIL variation in the rate of reproduction.These data were used to calculate the expected population size of the lines in the absence of any limits on population growth on days 5, 7 and 9. In all cases, the population size was greater than that which was observed. These predicted population sizes correlated with the observed population size on day 7  and was significantly negatively affected by food concentration . But, the lines did not differ in how their lifetime fecundities were affected by different food concentrations . The rate of reproduction, q, did not differ between lines , but it was significantly affected by the food concentration , such that at lower food concentrations q was lower. The lines also differed significantly in how q was affected by food concentration . Together, these results therefore show that the rate of reproduction was affected by food concentration independently of effects on lifetime fecundity.We next considered how lifetime fecundity and the rate of reproduction, and hence the expected population sizes of the lines, was affected by low concentrations of food, a likely feature of high-density populations. Analogously, as above, the lifetime fecundity differed between the lines (LINE: FC. elegans is reduced when there is limited food available. We sought to investigate whether this occurs due to (i) direct effects of caloric restriction per se or (ii) indirect effects on reproduction due to the perception of food availability, i.e. altered resource allocation in response to perceived food limitation. To do this we compared reproduction under a range of food concentrations in mutants defective in pharyngeal pumping with reproduction in wild-type worms. This was done with strains with mild pharyngeal defects (DA573 eat-14(ad573) and DA602 eat-15(ad602)), with severe pharyngeal defects (DA522 eat-13(ad522) and DA606 eat-10(ad606)) [i.e. the comparison allowed the separation of the acquisition of food from the perception of food. We hypothesised that if caloric restriction  controlled the food concentration-dependent change in the rate of reproduction, q, the rank order of the food-dependent reduction in q would be eat-10 and eat-13; eat-14 and eat-15; N2. In contrast, if food perception  controlled the food concentration-dependent reduction in the rate of reproduction, q, then no relationship between strength of the pharyngeal defects and food-dependent reduction in fecundity would be observed.The fecundity of wild-type (ad606))  and N2 aeat-10, eat-13) had the lowest rate of reproduction, q, and this was reduced most under lower food concentrations, compared with mutants with mild pharyngeal defects (eat-14 and eat-15) and compared with N2  analysis .,25.C. elComparative interval mapping (CIM) of dauer larvae formation in response to different food concentrations in 153 RILs identified six QTLs: two each for the plasticity of dauer larva formation, and for dauer larva formation at high and low food concentrations , but not with the plasticity in response to food concentration . Secondly, there is a region on chromosome II near marker 11 (see below) that significantly affects both the population size on day 5 and dauer larva formation at low food concentrations. However, these traits do not correlate .Combined, the analyses of dauer development and population size have therefore identified two regions of the We undertook further mapping to investigate the extent of the co-incidence of the chromosome II QTLs affecting both population size and dauer larva formation.i.e. RILs with a DR1350 genotype at the chromosome II QTL) formed a significantly lower proportion of dauer larvae compared with those lines either with an N2 genotype at both markers and recombinants (i.e. RILs with a DR1350 genotype at marker 11 and an N2 genotype at marker 13)   Figure .5,87 = 8.42, p < 0.001) , a significantly lower proportion of dauer larvae developed in NILs-9, -68 and -110 and DR1350 than in NIL-10 and N2 (F) Figure , i.e. thI Figure . This coI Figure  thereforThis chromosome II QTL region in N2 is predicted to contain 72 genes and 10 pseudogenes . Within this region there are a number of genes known to be involved in dauer larva development, d dyf-12 ,23. The 4,96 = 17.86, p < 0.001)  Figure . The genC. elegans and has been observed for a number of other life-history traits . ,25. C. ets  loss of function phenotype.Our genetic mapping analyses identified 10 QTL that affect either dauer larva development or population size Figure . There aThe QTLs for population size appeared, on average, to have greater phenotypic effects compared with those affecting dauer larva development Table . HoweverC. elegans RILs. These analyses have been undertaken on natural variation in these traits, thereby reflecting some aspect of how these traits have evolved and, or are maintained. These results further suggest the hypothesis that the same, or closely linked genes, may co-ordinate the response of these different life-history traits to environmental stress and that there are different evolved strategies by which these traits can be deployed. Further, detailed genetic analyses are required to fully resolve this.This work has shown that the larval dauer/non-dauer developmental decision and adult reproduction are affected by environmental conditions and that this varies among C. elegans alters both its propensity to form dauer larvae and its adult rate of reproduction and lifetime fecundity, in response to environmental stress.1. C. elegans isolates vary in their dauer larva formation and adult reproductive phenotypes and how these vary in response to stress. Changes in dauer larva development and adult reproductive strategy are related, shown by the correlation of these traits among RILs.2. 3. QTL analyses have identified regions on chromosome II and X that control dauer larva development and population size. The chromosome II QTL was further resolved to genetically separate the control of these two phenotypes.c. 210 Kbp region containing no genes known to be involved in dauer development.4. Genetic analysis of the chromosome II QTL affecting dauer larva formation, resolved this to a C. elegans has a co-ordinated larval and adult response to environmental stress controlled, at least in part, by physically close genomic regions.5. C. elegans isolates N2 and DR1350 were used for the construction of recombinant inbred lines (RILs) and nearly isogenic lines (NILs). All C. elegans lines were maintained on standard NGM plates with an Escherichia coli OP50 food source [Caenorhabditis Genetics Center. RILs were generated as previously described [d source . Strainsescribed . BrieflyC. elegans dauer pheromone and that daumone induces the lowest level of dauer larva formation, compared with the other components of dauer pheromone [Dauer pheromone extract was prepared from N2 liquid culture media as previously described . A singlheromone . All dauheromone , with thheromone .E. coli OP50 were grown overnight to saturation, centrifuged and the media removed, and resuspended, in water, at 2 and 1% w/v, respectively. RILs were assayed in 10 separate assays, with N2 and DR1350 controls included in each assay. Within each assay, there were three or four replicate plates for each RIL, and between five and ten replicate plates for N2 and DR1350 each at both food concentrations. Plates on which there were fewer than 20 worms and plates on which larvae could not be classified as dauer or non-dauer larvae with confidence, were excluded from all further analyses. Further, when data were only available for a single plate at one combination of conditions, these data were also excluded. The dauer formation phenotype of the NILs was measured at low food concentrations as described above.For the RILs, the dauer larva formation phenotype was measured at both high and low food concentrations against a standard concentration of dauer pheromone (15 μL/mL agar). To prepare the high and low food concentrations, liquid cultures of To measure the population growth of 48 randomly selected RILs, L1 stages were obtained by allowing eggs liberated from hypochlorite treated gravid hermaphrodites , to deveq , were determined for 12 RILs chosen to be representative of the range of dauer development phenotypes observed among the 48 RILs whose population growth had been determined. Late-stage L4s were transferred individually to NGM plates that had been previously seeded with an excess of OP50 food source, and the worms were then transferred to fresh plates every 8 hours, until egg laying had ceased. The plates from which the hermaphrodites had been removed were then maintained for 24 hours to allow the eggs to hatch and larvae to develop, which were then counted. This was done in two experimental blocks, with the parental lines, N2 and DR1350, included in each block, with 15 replicate plates for each for line at 19°C.The lifetime fecundity and the rate of reproduction, To determine the effect of different concentrations of food on lifetime fecundity and reproductive timing, these analyses were repeated except that the NGM plates were seeded with a food source consisting of 100 μL of 100, 50, 25, 12.5 or 6.25% w/v OP50 diluted in LB medium. Prior to the addition of these food sources, all plates were supplemented with streptomycin to a final concentration of 50 mg/mL, to prevent the growth of OP50. At low food concentrations, worms leave the site of the food and can be lost from an experiment; to prevent this, heated, sterile, 1 cm diameter brass rings were individually heated and melted into the agar prior to the application of the food and the worms, which were placed, and contained, within the rings . This waeat mutants (DA606 eat-10(ad606), DA522 eat-13(ad522), DA573 eat-14(ad573), DA602 eat-15(ad602)) [The lifetime fecundity and reproductive timing of five (ad602))  and N2 wDauer development and lifetime fecundity were analysed using generalized linear models (GLMs), with LINE and, as appropriate, FOOD and, or PHEROMONE concentration, ASSAY and TIME fitted as factors. When included in analyses, ASSAY was fitted such that LINE was nested within ASSAY. All analyses were performed in Minitab and all proportion data were arcsine transformed prior to analysis. All data presented in the text, figures and tables has been back transformed.q, the daily cumulative fecundity, expressed as a proportion of lifetime fecundity, was determined for each worm. This gives a relationship where the proportion of eggs laid increases from 0 on day 0 and converges to an asymptote of 1 on the final day of egg laying. The reproductive timing, expressed as the cumulative proportion of eggs laid, can then be described by y = 1 - eqx) that differentiated N2 and DR1350 ; SNPs th2 analysis for each marker with Bonferroni-corrected significance levels.Marker order was based on physical position in the N2 genome . Geneticp < 0.05, for each trait were determined by performing 1000 permutations on the data. Effects size and R2 values for significant QTLs were determined from these CIM analyses. To better control for the analysis of multiple traits, each of the three experiments, were also analysed by multiple trait CIM (MT-CIM) [p < 0.05, were determined by performing 1000 permutations on the data. GxE interactions were investigated in two ways. Firstly, the plasticity of dauer larvae formation, calculated as described above, was analysed by CIM as a separate trait. Secondly, GxE interactions were analysed as part of the multiple trait CIM analyses. Epistasis between identified QTLs was assessed by multiple interval mapping (MIM).QTL mapping was performed using QTL cartographer . In tota(MT-CIM) ,37. AnalMEV conceived and directed the study. SCH devised and executed the dauer larva phenotyping, genotyping, all molecular genetic and genetic analyses and data analyses; AS undertook the population growth and fecundity assays. The manuscript was written by SCH and MEV. All authors read and approved the final manuscript.Genomic regions and markers screened in DR1350. (A) Genomic regions sequenced in DR1350, showing the physical location, the size of the amplified region and the PCR oligonucleotide primers (5'-3') and, where appropriate, the candidate SNP contained within the amplified region [d region . (B) N2/d region , the chrClick here for fileCandidate genes underlying the chromosome II QTLs. Genes and pseudogenes predicted to occur in N2 within the defined regions of the chromosome II QTLs for (A) dauer larva development and (B) for population size on day 5. For each gene, the Wormbase ID number, gene name, gene description and position and, where available, any RNAi phenotypes, the expression pattern and gene ontology (GO) terms are given [re given . 7TM, 7-Click here for fileModel used in the calculation of predicted population size. The equation used in the calculation of the predicted population size and the definition of the parameters.Click here for file


PedGenie software, introduced in 2006, includes genetic association testing of cases and controls that may be independent or related (nuclear families or extended pedigrees) or mixtures thereof using Monte Carlo significance testing. Our aim is to demonstrate that PedGenie, a unique and flexible analysis tool freely available in Genie 2.4 software, is significantly enhanced by incorporating meta statistics for detecting genetic association with disease using data across multiple study groups.Meta statistics  were calculated using formal Cochran-Mantel-Haenszel techniques. Simulated data from unrelated individuals and individuals in families were used to illustrate meta tests and their empirically-derived p-values and confidence intervals are accurate, precise, and for independent designs match those provided by standard statistical software.PedGenie yields accurate Monte Carlo p-values for meta analysis of data across multiple studies, based on validation testing using pedigree, nuclear family, and case-control data simulated under both the null and alternative hypotheses of a genotype-phenotype association.PedGenie allows valid combined analysis of data from mixtures of pedigree-based and case-control resources. Added meta capabilities provide new avenues for association analysis, including pedigree resources from large consortia and multi-center studies. In the study of common diseases and genes with modest effects, large consortium and multi-center efforts hold the promise of increased power to detect associations, but present analysis challenges. The study populations for the multiple centers may differ geographically and ethnically, and considerable differences in case-control ascertainment and pedigree structures between studies are likely. PedGenie, available as an analysis option in Genie 2.4 software, requires Java 1.6 for execution and extends the functionality previously available in PedGenie 1.2 .In addition to offering all the capabilities of version 1.2, PedGenie (version 2.4) includes allele and genotype association analysis across studies based on formal Cochran-Mantel-Haenszel (CMH) techniques to calculate the following meta statistics: chi-squared test of independence, chi-squared test of trend, and odds ratios and confidence intervals. An illustration of a meta-association analysis as implemented in PedGenie can be found in Curtin et al [PedGenie is a flexible, easily implemented, and freely available analysis tool that is enhanced significantly in Genie 2.4 software  by the ith and 97.5th percentile of odds ratios from the null genotype configurations.In epidemiologic studies, data are often collected that can be summarized in three-way contingency tables, the presence or absence of a disease phenotype cross-classified with allele or genotype, and controlling for a third categorical variable or 'group' . In a mui. The Mi meta statistic is from the null distribution since it was derived from data simulated under the null hypothesis. To match the information content of the real data, we limit calculation of the statistic of interest in the simulated data to only those individuals with genotype data in the observed sample. In this way, the missing data structure is captured. Steps 2 and 3 are repeated multiple (N) times, and the series of null meta statistics stored. Hence an empirical null distribution is created for the meta statistic of interest, conditional on the particular pedigree and phenotype structures contained across all studies in the total resource.An outline of steps for meta statistics in PedGenie follows. First, allele frequencies for the markers of interest are estimated from the data. This is performed separately for each study. As before, these can be estimated by four different methods: genotyped founders only, all genotyped individuals, all founders with statistically inferred genotypes, and user-defined . Second0, is computed based on the true genotype and phenotype data using the same meta statistic of interest, again ignoring any pedigree relationships that exist. This observed meta statistic is then compared to the empirical null distribution to determine significance as follows:Finally, the observed statistic, Mp-value is less than or equal to the required level of type I error (α).The specified null hypothesis is rejected if the To evaluate the accuracy of the meta association statistics available in PedGenie  and corresponding empirical p-values and 95% confidence intervals (95% CI), two simulated meta-datasets were constructed, each comprised of three studies, as described below. Briefly, two test set designs were used. The first 'independent' design was comprised of three separate studies of varying sample size, each being the standard association design of independent cases and 1:1 frequency matched controls. Results from this design should be in exact agreement with results from standard statistical packages for test statistics, and in asymptotic agreement for their associated p-values and confidence intervals. The second 'mixed' design was comprised of mixtures of study designs (independent and family-based), both within and between studies. This 'mixed' design is used to illustrate PedGenie's versatility.Within each of the two meta-analytic datasets (independent or mixed design), a variety of sample sizes and minor allele frequencies (MAFs) for one bi-allelic single nucleotide polymorphism (SNP) were used across the component studies as detailed in the following section. In each component study, a dataset was simulated under two scenarios: one under the null hypothesis of no association, and another under the alternative hypothesis of association of an allele of interest and phenotype. Under the null scenario, a sporadic rate of 0.10 for affected phenotype status was randomly assigned, regardless of genotype. Under the alternate scenario, penetrances for each genotype  were determined based on a multiplicative genetic model with a genotypic relative risk (GRR) of 1.5 and a sporadic rate of 0.10, according to the methodology described in Camp .® version 9.1). For each PedGenie analysis, either 1,000 configurations (for the null scenario) or 10,000 configurations  were used to determine the empirical null distribution and p-values and confidence intervals for meta (across studies) and within-study test statistics. Generally, the number of gene-dropping configurations was guided by the expected size and desired precision of a p-value. Clearly, the accuracy of the empirical p-value increases with the size of the empirical null distribution simulated; for example, an N of 2,000 gives a 95% confidence interval around α = 0.05 with a width of 0.02 under the null hypothesis.Since PedGenie is an empirical approach, we repeated this procedure 1,000 times for each test design (independent or mixed) and scenario  by randomly re-seeding the initial assignment of alleles in each analysis to demonstrates the reproducibility of the Monte Carlo method, as equivalent results are obtained if a different random number seed is used. The means and standard deviations of the 1,000 empirical p-values and confidence intervals for each meta study simulation were examined for stability and accuracy. For the 'independent' test design, the meta statistics and p-values were compared to statistics and p-values from a standard distribution using an established statistical software package  were simulated with the following sample sizes and MAFs: 1,000 independent cases and 1,000 random controls, MAF = 0.05 (study 1.1); 500 independent cases and 500 random controls, MAF = 0.10 (study 1.2); and 250 independent cases and 250 random controls, MAF = 0.20 (study 1.3). Data were constructed for this design under both the null and alternative scenarios as described above.Three separate studies with a mixture of both family-based and independent resources were simulated with the following sample sizes and MAFs: 1,000 independent cases and 1,000 random controls, MAF = 0.05 ; nuclear families consisting of 200 affected sib-pairs plus parents, 200 discordant sib-pairs plus parents, and 200 independent controls, MAF = 0.10 (study 2.2); and four-generation pedigrees consisting of 53 family members per pedigree ; and including 500 independent controls, MAF = 0.20 (study 2.3).Study characteristics resulting from the independent and mixed design simulations under the null and alternative scenarios are shown in Tables For the nuclear family study study 2.2), alleles were first randomly assigned to parents in families with two siblings in a "population" of over 15,000 families  based on the specified MAF. Second, alleles were assigned to siblings according to Mendelian inheritance. Third, phenotype was assigned to all individuals randomly, but according to penetrances under either the null or alternate scenario. Finally, 400 nuclear families (200 affected and 200 discordant sib-pairs and their parents) were selected randomly from the "source" population. In addition, 200 independent controls were randomly selected from case-control study 1.2 from the appropriate scenario  to create study 2.2, a mixed family and independent resource. Genotypes on all parents, affected and unaffected, were also included in the simulation to demonstrate the flexibility of PedGenie to handle any pedigree structure and use all information available in each pedigree. This resulted in additional family-based cases and controls as noted in Tables .2, allelSimilarly, in extended family study study 2.3), the initial step consisted of randomly assigning alleles to all founders in a "population" of 150 four-generation pedigrees (14 founders and 53 members per pedigree) based on the MAF specified previously. Second, a Mendelian gene-drop was performed in which alleles were assigned to offspring randomly based on parental alleles. Third, phenotype was assigned to individuals randomly, but according to penetrances under the either the null or alternate scenario. Genotype data for generations 1 and 2 were removed, such that genotypes were only considered available for the bottom two generations. Finally, pedigrees containing at least four related, genotyped cases were randomly selected until ~500 affected family members had been ascertained. This resulted in 90 pedigrees in the null scenario, and 83 pedigrees in the alternate scenario containing 501 and 503 family-based cases, respectively. In addition, 500 unaffected independent controls were randomly selected from a source "population" corresponding to the null or alternate scenario samples in case-control Study 1.3 to create a mixed pedigree and independent resource. All family members with genotypes, both affected and unaffected, were included in the simulation to demonstrate the flexibility of PedGenie to handle large pedigree structures and use all information available in each pedigree. This flexibility resulted in additional pedigree-based controls as detailed in Tables .3, the iThe Monte Carlo p-value and confidence interval assessment results, for 1,000 randomly seeded runs using the multi-study, independent-design meta-analytic datasets under both null and alternate scenarios, are shown in Tables For the independent design, all statistics were shown to be equivalent to those from a standard package Tables  and 4. TThe PedGenie software implements a Monte Carlo approach that corrects the null distribution, and not the observed statistic, for family relationships and hence the statistics for the mixed design are also the standard statistics. As shown in Tables ® Precision 670 computer with 4 gigabytes of memory, using a Red Hat® linux operating system. Runtime  with 10,000 null configurations took approximately 15 minutes per independent-design meta analysis and ~35 minutes per mixed-design meta analysis.To illustrate performance, runtime is reported. Runtime for PedGenie (null scenario) with 1,000 null configurations took approximately 1 minute, 30 seconds per single independent-design meta analysis and under 4 minutes per single mixed-design meta analysis on a DellFor common traits involving small effect genes, consortia and multi-center efforts are becoming commonplace. For example, CMH statistics for ordered genotypes have been used to assess association in multiple case-control studies in which cases are independent, either as probands or as randomly selected affecteds . ComplexPedGenie can incorporate all relationships in family-based resources, therefore all family members with phenotype and genotype data that are available can be included. For example, if only one affected sibling was chosen from each pedigree as some investigators are forced to do to appropriately utilize standard statistical packages, the sample size in study 2.2 would have been reduced under the alternate scenario from 694 cases, 200 independent controls, and 706 family-based controls to 400, 200, and zero, respectively. This may be particularly useful for linkage consortia interested in pursuing association testing, as PedGenie can provide valid meta analyses across multiple studies each containing family-based cases and family-based controls, in addition to any supplemental independent controls available. The ability to more fully utilize all data on hand both increases the utility of prior linkage resources and provides increased power to detect associations, particularly in stratified and subset analyses that likely lead to small sample sizes in individual studies. We anticipate that the new meta capabilities in PedGenie will provide new avenues for linkage consortia to explore in terms of association testing.A potential limitation of PedGenie meta analysis is the fixed-effects nature of the CMH approach, and interpretation of the meta odds ratio is dependent on an assumption of homogeneity in genetic effects across studies. Future work will include providing an option to test for heterogeneity across studies such as described in ,22,23, aIn this study, we have described allele and genotype tests for single SNPs. PedGenie also incorporates haplotype and composite genotype tests; these are based on the most likely haplotype configurations from expectation-maximization  rather than a full likelihood approach based on all possible haplotypes (which is not a feasible approach for pedigrees of arbitrary size). We are currently working towards more sophisticated haplotype methods. Future efforts will also include meta extensions for other quantitative and transmission statistics, such as the TDT test, and multiple logistic regression analysis (including a random-effects extension) with covariates.We have demonstrated PedGenie is a valid statistical software tool that offers a comprehensive way of performing valid meta association testing across studies of mixed design. PedGenie (freely available in Genie 2.4 software) can accommodate independent and family-based resources, allowing for more power to detect associations by allowing for valid combined analyses and maximal use of available data.[PedGenie is an analysis option in Genie 2.4 software, University of Utah Genetic Epidemiology Division, freely available at . Java 1.KC carried out the validation testing and statistical analysis, participated in software development, and drafted the manuscript. JW performed the Java programming and maintains the software home page. KAB participated in the design of the study and helped to draft the manuscript. NJC conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.genie. A compressed archive file of Genie 2.4.2, including Genie.jar, genepi.jar, and all PedGenie example files.Click here for filegeniesource. A compressed archive file with Genie 2.4.2 source code only.Click here for file


This case describes the increased anticoagulation effect associated with the use of high-dose ketoconazole. A 59-year-old man treated with warfarin for aortic valve replacement was prescribed high-dose ketoconazole and hydrocortisone for the treatment of prostate cancer. Despite lowering the warfarin dosage by 35% during the start of high dose ketoconazole, an additional dose reduction was required subsequently when the INR rose from 2.62 to 3.82 within nine days. After a total dose reduction of 43%, the INR returned to therapeutic range within two weeks. The Naranjo probability scale revealed a probable adverse reaction of increased anticoagulant effect associated with high dose ketoconazole. Due to the inhibition of warfarin metabolism by ketoconazole, patients taking high dose ketoconazole concomitantly with warfarin may need their warfarin dosage reduced by more than is currently recommended, as well as receive more frequent INR monitoring to avoid over anticoagulation. In advaynthesis . As a reAs an antifungal, ketoconazole is dosed at 200 mg daily, however, for HRPC therapy; doses of 200–400 mg three times a day are used to produce castrate levels of testosterone within 24 hours . Since kA 59-year-old man had been treated with warfarin with an INR goal of 3.0 for the last five years after receiving a mechanical aortic valve replacement. His past medical history includes diabetes mellitus type 2, hyperlipidemia, hypertension, depression, chronic obstructive pulmonary disease, gastroesophageal reflux disease, osteoarthritis, and prostate cancer. His medications included albuterol and ipratropium inhaler, benztropine, calcium/vitamin D, guaifenesin/dexamethorphan, docusate, furosemide, glyburide, goserelin, hydrocodone/acetaminophen, lisinopril, lorazepam, metoprolol tartrate, nitroglycerin sublingual, omeprazole, potassium chloride, prochlorperazine, quetiapine, simvastatin, terazosin, and trazodone. His INR had been therapeutic in the past two months on a usual weekly dose of 52.5 mg. His only other elevated INR prior to this period was when antibiotics were started for an acute infection, and his INR increased from 3.03 to 4.27.In March 2006, the patient was prescribed ketoconazole 400 mg three times a day and hydrocortisone 10 mg daily by his urologist for treatment of prostate cancer. As part of the pharmacist-managed anticoagulation program, the pharmacist decreased the warfarin weekly dose by 35% to avoid overanticoagulation. This dose adjustment was based on the recommendation from standard clinical references to reduce the warfarin dose by one-third . The patIn this case, despite lowering the warfarin dosage by 35% during the start of HDK, an additional dose reduction was required when the INR increased to 3.82 within nine days. A total dose reduction of 43% was required to bring the anticoagulant effect to therapeutic goal and to avoid bleeding complications. No new medications besides ketoconazole and hydrocortisone were started during this period of time. The medications listed previously were chronic medications that the patient had been taking for years, except for calcium/vitamin D, guaifenesin/dexamethorphan, and glyburide which were started within two months prior to the initiation of ketoconazole and hydrocortisone. Among the concurrent medications with warfarin, only omeprazole, glyburide, and ketoconazole show an enhanced anticoagulation effect . The druTo our knowledge, this is the first report of the potentiation of oral anticoagulants by HDK. The mechanism of the drug interaction is most likely due to the inhibition of warfarin metabolism. Ketoconazole is a potent inhibitor of cytochrome P450 1A2 and 3A4, with moderate inhibitory effect on isoenzymes 2A6, 2C9, 2C19, and 2D6 , 13. As While clinicians may readily acknowledge the interaction between ketoconazole and warfarin, supporting data are sparse. In the widely cited evidence-based overview of warfarin drug interactions, Holbrook and colleagues indicate that ketoconazole has no effect on warfarin at the probable level, based on a pharmacokinetic study assessing two patients receiving concurrent warfarin and ketoconazole , 14. OnlThe use of HDK therapy for hormone refractory prostate cancer has increased. Clinicians must be cognizant and use caution when HDK is used with warfarin as it can greatly potentiate the anticoagulation effect, leading to an increased risk of bleeding. This drug-drug interaction may be minimized by more frequent INR monitoring and considering reducing the warfarin dosage by more than the currently recommended 35% in order to achieve the therapeutic goal and to avoid overanticoagulation.


Alterations in circulating large neutral amino acids (LNAAs), leading to a decrease in the plasma ratio between branched-chain and aromatic amino acids (BCAA/AAA ratio), may be involved in sepsis-associated encephalopathy. We hypothesised that a decrease in the BCAA/AAA ratio occurs along with a net cerebral influx of the neurotoxic AAA phenylalanine in a human experimental model of systemic inflammation.Escherichia coli lipopolysaccharide (LPS) in 12 healthy young men.The BCAA/AAA ratio, the cerebral delivery, and net exchange of LNAAs and ammonia were measured before and 1 hour after a 4-hour intravenous infusion of LPS induced systemic inflammation, reduced the BCAA/AAA ratio, increased the cerebral delivery and unidirectional influx of phenylalanine, and abolished the net cerebral influx of the BCAAs leucine and isoleucine. Furthermore, a net cerebral efflux of glutamine, which was independent of the cerebral net exchange of ammonia, was present after LPS infusion.Systemic inflammation may affect brain function by reducing the BCAA/AAA ratio, thereby changing the cerebral net exchange of LNAAs. Sepsis-associated encephalopathy (SAE) is often one of the first manifestations of sepsis  and is aSepsis is characterized by increased peripheral protein breakdown, notably in skeletal muscle ,6, and hAt present, neither the physiological implications of alterations in the BCAA/AAA ratio nor the effects of systemic inflammation on the cerebral net exchange of LNAAs has been investigated in humans. Applying a human experimental model of systemic inflammation, we hypothesised that the BCAA/AAA ratio decreases with a concurrent net cerebral influx of the neurotoxic AAA phenylalanine, and that this attenuates the net cerebral influx of BCAAs.Twelve healthy male volunteers aged 20 to 33  years participated in the study after providing oral and written informed consent. All had an unremarkable medical history, with no signs of infection within 4 weeks ahead of the trial day, and none took regular medication. Before inclusion, volunteers underwent a thorough physical examination; a 12-lead electrocardiogram (ECG) was obtained, and standard biochemical tests were performed; all tests were normal. The study was approved by the Scientific Ethical Committee of Copenhagen and Frederiksberg Municipalities, Denmark (file number (KF) 01 290011) and was performed in accordance with the Helsinki Declaration.Volunteers reported to the laboratory at 7.00 a.m. after an overnight fast and were placed in bed. They were subsequently catheterized with antecubital catheters bilaterally , respectively), a peripheral arterial line and a jugular bulb catheter, of which the two latter were inserted by using local anesthesia with lidocaine. The jugular bulb catheter was inserted into the right internal jugular vein with the tip pointing cranially and by using ultrasound guidance. Correct placement in the jugular bulb was ascertained by feeling a resistance to further advancement of the catheter, as well as the volunteer hearing a purl during a bolus injection of saline; x-ray confirmation was not used. One of the authors (KM) inserted all catheters, including the jugular bulb catheters. After catheter insertion, the volunteer rested in the supine position with slight head elevation for 30 minutes before measurements. Heart rate (via a three-lead ECG), blood pressure, and capillary oxygen saturation were continuously monitored. Volunteers were discharged from the unit after 12 hours after removal of catheters and a light meal. No complications occurred.Escherichia coli LPS ; Batch G2 B274, US Pharmacopeial Convention, Rockville, MD, USA). In this model, plasma tumor necrosis factor (TNF)-α peaks at approximately 1 hour after cessation of infusion [After an overnight fast, subjects were catheterized, and CBF measurements and paired arterio-jugular venous blood samples were obtained at baseline and after a 4-hour continuous intravenous infusion of purified infusion , at whicinfusion .2), carbon dioxide tension (PCO2), pH, plasma glucose, and hemoglobin were determined on a blood-gas analyser . For the subsequent calculation of cerebral metabolic rates (CMRs), arterial and jugular venous PO2 and plasma glucose were converted to whole-blood oxygen and glucose content, respectively. The oxygen content (CxO2) in arterial and jugular venous whole blood was calculated asArterial and jugular oxygen tension  containing the internal standard for the analysis, norleucine. Samples were then placed on ice for 15 minutes, after which they were centrifuged at 4°C, 3,000 rpm for 30 minutes. With this approach, the amino acid concentration of plasma and red blood cells is fully equilibrated, so that the plasma concentration of a given large neutral amino acid can be considered identical to the whole-blood concentration . Accordio-phthalaldehyde-mercaptoethanol [LNAAs  were separated by a single-column gradient lithium cation-exchange high-performance liquid chromatography with fluorescence detection , by using post-column derivatization with oethanol . The coe4+) and uncharged (NH3) species.To explicate whether a putative cerebral glutamine efflux depended on a cerebral influx of ammonia, the cerebral delivery and net exchange of ammonia were evaluated. The term "ammonia" is used here to depict the total of the charged  is quantitated. The range of the assay is 6 to 587 μmol/L, and internal validation revealed a coefficient of variation of 5% for values greater than 60 μmol/L and of 11% for values less than this. The whole-blood ammonia concentration was calculated by Conn's formula [Immediately after each CBF measurement, paired blood samples from the radial artery and the jugular vein were simultaneously drawn into EDTA tubes and placed on ice. Within 30 minutes, they were centrifuged at 4°C, 3,600 rpm for 15 minutes; plasma was stored at -80°C. The plasma concentration of ammonia was determined by use of microdiffusion, quantitation by reaction with bromophenol blue, and spectrophotometry at 600 nm  at the Department of Clinical Biochemistry, Rigshospitalet. In this assay, NHnditions , is dire formula :and converted from μg per 100 mL into μmol/L.White blood cell and platelet counts were analyzed with standard laboratory methods. The plasma concentration of TNF-α was measured by using ELISA . Plasma was obtained by centrifuging whole blood in EDTA-containing tubes at 3,600 rpm at 4°C for 15 minutes and was kept at -80°C until analysis. Samples were analyzed in duplicate, and mean concentrations were calculated. Interassay variability (CV) was assessed by using two internal controls (human plasma); one in the lower end of the standard curve , and one in the upper end of the standard curve , as the variability differs throughout the standard curve. Interassay CVs were 32.9% for "low" and 12% for "high" TNF-α. The TNF-α detection limit is 0.12 ng/L, according to the manufacturer.The BCAA/AAA ratio was calculated as the ratio between the arterial plasma concentrations of the BCAAs valine, leucine and isoleucine and the AAAs phenylalanine and tyrosine:2) and glucose (CMRglc) were calculated according to the Fick principle [The cerebral delivery of a given LNAA was calculated as the product of the arterial concentration and CBF. Cerebral net exchange  values of LNAAs and the CMR of oxygen  of a given substance x; a-jvDx depicts the arterio-jugular venous concentration difference of x in whole blood. By convention, a positive value of Jx signifies a net influx (uptake) of x, whereas a negative value indicates a net efflux (release) [in which Jrelease) .in, Phe) can be calculatedGiven that the BBB transport of a substance can be described accurately by means of a single-membrane model in which the cerebrovascular endothelium behaves as a single membrane, which is the case for LNAAs , the uni1 is the permeability-surface area product of phenylalanine from the capillary into the brain interstitial space. Because the kinetic constants for the transfer of phenylalanine from blood to brain have been estimated in humans in vivo [1 can be estimated by means of the Michaelis-Menten equation:where PS in vivo , and hav in vivo , PS1 canmax, was assumed to be 46.7 nmol/g/min, the apparent Michaelis-Menten constant, Km, was assumed to be 0.328 mmol/L, and KD, the nonsaturable diffusion constant, was assumed to be 0.01 ml/g/min [In the present context, the maximum transport velocity, Vml/g/min .t tests, before and after LPS infusion, to detect an effect of the intervention, and between arterial and jugular venous concentrations at a given time point to determine whether a calculated cerebral net exchange value was different from 0, that is, whether a cerebral influx or efflux was present. Data are presented as mean (95% CI) or as geometric mean (95% CI) in case data had to be log-transformed to achieve normality. Significance was established at P < 0.05.Parametric methods were applied throughout by using SAS statistical software, version 9.1. Thus, all analyses were performed as paired-samples 2  mmHg; LPS, 38.8 (36.0-41.6) mmHg; P < 0.01), and an increase in pH ; LPS, 7.42 (7.39-7.44); P < 0.05) was evident after LPS infusion. CMRO2 increased slightly  μmol/g/min; LPS, 2.3 (2.0 - 2.6) μmol/g/min; P < 0.05), whereas CMRglc was unaffected  μmol/g/min; NS). Some volunteers dozed intermittently but remained easily rousable and alert when awakened and were fully awake during measurements; no overt signs of encephalopathy occurred.LPS infusion was associated with a pronounced inflammatory response; immunologic variables are summarized in Table LPS infusion increased plasma phenylalanine and decreased the concentration of all other LNAAs except isoleucine Table , with a The present study is the first to investigate the physiological impact of a decline in the BCAA/AAA ratio after a standardized systemic inflammatory stimulus in humans. In this study, systemic inflammation with an increase in temperature, total white blood cell count, neutrophil count, and plasma TNF-α was associated with a decline in the BCAA/AAA ratio, mainly because of an increase in the arterial concentration of the neurotoxic AAA phenylalanine. A concordant increase in the cerebral delivery and unidirectional cerebral influx of phenylalanine was present during systemic inflammation; this was accompanied by an abolished net cerebral influx of the BCAAs leucine and isoleucine, as well as a cerebral efflux of glutamine. Assuming that the measured cerebral net exchange values are representative for the entire period after LPS infusion, the present findings furthermore indicate that the brain does not contribute to the depletion of glutamine and BCAAs from the circulation during systemic inflammation.per se through a compensatory increase in the unidirectional cerebral efflux of phenylalanine, a parameter that was not assessed in the present study. This could involve the energy-dependent LNAA transporters on the abluminal membrane of the BBB, which exports phenylalanine from the brain [A decrease in the BCAA/AAA ratio was previously demonstrated in patients with sepsis, and this appears to be related to the occurrence of encephalopathy -9. Althohe brain ,29.Because AAAs and BCAAs compete for transport across the BBB into the brain by means of the same saturable LNAA carrier , the incIn the present study, LPS infusion was found to instigate a decrease in the arterial glutamine levels, and a cerebral glutamine efflux accompanied this. The former has repeatedly been demonstrated, both in clinical and experimental studies of sepsis ,6,35,36;3) was recently disputed [4+, the most abundant form of ammonia in the circulation, is indeed transported across the BBB by means of a specific carrier [4+ and NH3, was affected in the present model. Consistent with our findings, both circulating and brain levels of ammonia have been reported to be unaffected by LPS infusion in rats [The cerebral glutamine efflux after LPS infusion was not found to be associated with any changes in the cerebral net exchange of ammonia, which is normally detoxified to glutamine. The classic conception that ammonia merely diffuses across the BBB in its uncharged form (NHdisputed ,39; alth carrier ,38. Neit in rats . Hyperam in rats -42 and m in rats . FurtherRather than ammonia detoxification, the cerebral efflux of glutamine after LPS infusion probably reflects increased cerebral proteolysis or a compensatory astrocytic glutamine release, for example, to reduce osmotic stress in the context of cytotoxic edema. As with phenylalanine, an alternative explanation could be the presence of an inflammation-induced increase in the activity in the energy-dependent abluminal LNAA transporters , phenomein vivo studies of certain aspects of the pathophysiology of early sepsis.Certain limitations exist for the conclusions that can be made from our findings. Based on the methods and findings in the present study, we cannot definitively conclude that the cerebral net exchange of a given amino acid is unaffected in sepsis. The cerebral net exchange values are relatively small at baseline Table , and theWe did not perform cognitive tests in the volunteers. Thus, possible interrelations between cerebral function and the described alterations in arterial amino acids could not be assessed. It is quite possible that the general malaise experienced by the subjects might have affected cognitive performance, had it been tested thoroughly. However, because all subjects remained alert and responsive during the course of LPS infusion, major cognitive disturbances were unlikely to be present.The present study lends further support to the view that LNAAs, particularly phenylalanine, play a pertinent role in the cerebral pathophysiology of sepsis; the arterial levels, cerebral delivery, and unidirectional cerebral influx of phenylalanine increased, whereas the cerebral influx of leucine and isoleucine were abolished, and a cerebral glutamine efflux was induced by LPS infusion in humans. Future studies should address these interrelations and characterize them further, for example through bedside studies on cerebral net exchange, neurotransmitter profiles, BBB function, and the effects of amino acid supplementation on brain function in patients with sepsis.• LPS infusion induces a systemic inflammatory response and reduces the arterial levels of most LNAAs in humans.• The systemic inflammatory response is associated with a decrease in the BCAA/AAA ratio, because a reduction in the arterial levels of the BCAAs valine and isoleucine occurs with a concomitant increase in the arterial levels of the neurotoxic AAA phenylalanine.• The BCAA/AAA ratio decrease is associated with an increase in the cerebral delivery and unidirectional cerebral influx of phenylalanine, an abolished influx of the BCAAs leucine and isoleucine, and an ammonia-independent cerebral efflux of glutamine.AAA: Aromatic amino acid; BCAA: branched-chain amino acid; CBF: global cerebral blood flow; CMR: cerebral metabolic rate; ECG: electrocardiogram; Hgb: hemoglobin; J: flux; LNAAs: large neutral amino acids; LPS: lipopolysaccharide; NH: ammonia; SAE: sepsis-associated encephalopathy.The authors declare that they have no competing interests.RMGB conducted the study, acquired, analyzed, and interpreted the data, performed statistical analyses, and drafted the manuscript. ST, DMB, and CL conducted the study and acquired and interpreted the data. FSL conducted the amino acid analyses. BKP conceived of and designed the research and handled funding and supervision. KM conceived of and designed the research, conducted the study, acquired, analyzed, and interpreted the data, drafted the manuscript, and handled funding and supervision. All authors made critical revisions and read and approved the final manuscript.


Rabies, encephalitis caused by lyssaviruses, was considered universally fatal until a young, unvaccinated patient with bat rabies survived after a new therapeutic approach EurosurveillanceWe report on a patient with rabies due to Duvenhage virus from a bat in Kenya. A very brief description of this case without clinical details and without results of virological and pathological investigations was reported in A bat flew against the face of a 34-year-old Dutch woman, a medical doctor, on a campsite in Tsavo West National Park, Kenya. She swept the animal away and noticed two small superficial bleeding wounds on the right side of the nose. The wounds were washed with water and soap, and cleaned with alcohol swabs. The park wardens and the personnel at the nearby health facility assured her that locally rabies was only known to be carried by dogs and cats. She was not referred for postexposure prophylaxis.Fourteen days after her return to The Netherlands, 23 days after the incident with the bat, she complained of malaise, dizziness, muscle aches, and headache. She had no fever. Two days later, she experienced difficulty with speech and swallowing. There was hypaesthesia of both cheeks and she felt unsteady. She vomited once. She was admitted in isolation the next day, day 4 of complaints. Her past medical history was unremarkable and she did not take medication.Listeria monocytogenes infection, malaria, and trypanosomiasis were considered and ruled out by appropriate tests  and microscopy of blood slides and CSF, PCR for enterovirus, inclusive of enterovirus 71). Neurological considerations included infection by neurotropic viruses, acute disseminated encephalomyelitis, and, initially, anxiety disorder. On day 2 of admission, her temperature was 38.5°C, the dysarthria had worsened, and she complained of difficulty with swallowing. She was agitated and she had a high frequency postural tremor of her hands.On examination she was anxious, but alert and well oriented. Her temperature and other vital parameters were normal. There was no hypersalivation, hydrophobia, or aerophobia, and no difficulty swallowing. The wounds on the nose were resolved; there was no itch or pain. There was a slight dysarthria and she spoke in a staccato way. Apart from the hypaesthesia of both cheeks, further neurological examination was unremarkable. The deep tendon reflexes were normal. On advice of the National Coordination Center for Infectious Diseases (NCCID), she received human anti-rabies immunoglobulin (HRIG), 20 IU/kg im, divided over both quadriceps muscles, and human diploid rabies vaccine (Mérieux) im . Other infections, such as Rift Valley fever, West Nile fever, lymphocytic choriomeningitis, yellow fever, enterovirus 71 and 3 (n<5), no red blood cells, a protein level of 0.23 g/l (n<0.5) and a glucose level of 3.0 mmol/l (blood glucose 4.9 mmol/l). During the following days her dysarthria increased. She complained of weakness of arms and legs and double vision. She developed hypersalivation and became hyperreactive to noises. Her temperature was variable with bouts of fever, up to 39.2°C. On day 6 of admission, the condition acutely worsened; she had an epileptic seizure and aspirated, and oxygenation dropped due to respiratory insufficiency. She was intubated and admitted to the Intensive Care Unit (ICU). The CSF now showed 64 white blood cells  and 25 red blood cells/3 mm3, a protein level of 0.72 g/l, and an elevated IgM index of 0.35. An MRI showed an area of increased signal intensity on T2-weighted images in the posterior part of the medulla oblongata and pons /3 mmand pons . The nexLyssavirus-specific RT-PCR was negative in those specimens. The nuchal biopsy, obtained on day 2 of admission, showed a weak band of correct size on agarose gel in nested RT-PCR in only one of the quadruplicate samples and therefore required confirmation by cloning and sequencing Rabies seemed the probable cause of the encephalitis, but the pharyngo-cervical-brachial variant of Guillain-Barré syndrome (GBS) was also considered. No rabies-specific antibodies had been detected in serum collected before vaccination or in serum or CSF from day 2 of admission, and Clinical samplesSerum, d2 antibody and PCR neg, d7 antibody neg;CSF, d2 antibody and PCR neg, d 6 and d 28 Lyssa PCR neg;Nuchal skin biopsy, d2 PCR pos, see text, IF rabies neg, d10 PCR neg (repeated because of delay with first biopsy);Saliva, d7 Lyssa PCR pos (reported d 13), d10 neg;Cornea smear, d7 IF neg.(Note: results of tests performed.)On day 11 of admission the presence of Duvenhage virus in the nuchal skin biopsy specimen from the 2nd day of admission was confirmed. This may seem like a long delay, but only one of the two agarose gels tested by PCR showed a faint band, and the repeated test in duplicate was negative in both tests. The amplicon had to be cloned as direct sequencing was not successful. Details of virological investigations are presented in Staphylococcus aureus was treated with flucloxacillin iv, 1 g, four times per day for 7 days. On admission to the ICU, the temperature was elevated up to 40.1°C, but after administration of acetaminophen suppositories  the temperature was normal 24 hours later and remained normal thereafter without further measures. Diabetes insipidus, first apparent on day 12 of admission, day 7 of ICU, was regulated with iv fluids to which desmopressin was added on days 13, 16, and 17. Pentobarbital was stopped after 5 days when no bursts were seen on the EEG and midazolam was stopped after 7 days of administration. Twenty-four hours later, 3 days after stopping pentobarbital and while there was no pentobarbital detectable in the serum, the EEG showed a very low voltage background pattern.Blood pressure, heart rhythm, kidney function, and diuresis were regulated by fluids and low dose noradrenaline; a supervening lung infection with MRI abnormalities of the medulla oblongata had increased and now extended to mesencephalon and both dentate nuclei in the cerebellum . Also, tAt postmortem examination no gross abnormalities were found, apart from the pneumonia. The brain was swollen and weighed 1,360 g. Histopathology of the cerebral cortex  showed eThe Platelia Rabies II Kit  was used to detect and titrate IgG anti-rabies virus glycoprotein in serum or plasma. The test was also used to detect specific antibody in CSF.Lyssavirus antigen was detected by the immunofluorescence examination using Bio-Rad Rabies Anti-nucleocapsid Conjugate . Nuchal skin biopsies and corneal smears were examined by the immunofluorescence assay to detect the presence of rabies virus antigen.Cell culture of the various clinical samples was carried out by inoculation of murine neuroblastoma cells and BHK-21 cells propagated in 24-well microtiter plates. CSF was tested without processing, and saliva was made up to a volume of 2 mL with virus transport medium. The other specimens were ground with virus transport medium up to a volume of 5 mL. After centrifugation a volume of 200 µL was added to each well. Immunofluorescence tests with anti-rabies nucleocapside conjugate (Bio-Rad) showed immunopositivity as presented in A rabies-specific diagnostic RT-PCR was performed as previously described A 367–base pair fragment in the nucleoprotein region was sequenced and phylogenetic analysis was performed using Mega 3.1  It should be noted that a Duvenhage strain had not been present in our laboratory.Newborn NIH outbred mice  were inoculated intracerebrally with the various clinical samples . The preparation of the specimens was the same as for the cell culture. The specimens were homogenized and a volume of 15 µL was introduced into the central part of the upper parietal area of the calvaria. The inoculated mice were observed daily for a maximum of 28 days; the inoculated mice did not show symptoms of infection. Rabies virus antigen could not be detected by immunofluorescence of the mouse brains; furthermore, the brains were also negative by PCR.Antibodies against lymphocytic choriomeningitis virus, Rift Valley virus, West Nile virus, and yellow fever virus were determined by immunofluorescence assays or enzyme-linked immunosorbent assays.Tissue was fixed in 10% buffered formalin and embedded in paraffin. Paraffin-embedded tissue was sectioned at 6 µm, mounted on organosilane-coated slides , and used for hematoxylin and eosin (H&E) staining. Apart from H&E staining, representative sections of all brain specimens were processed for conventional histochemical staining  and immunohistochemistry.Several blocks of different brain regions  were used for immunohistochemical analysis with neuronal nuclear protein .For the detection of the virus, polyclonal rabbit anti-rabies-nucleoprotein antibody  was used. The immunostaining with this anti-rabies antibody was also performed on material from adrenal gland, salivary gland, pancreas, thyroid, heart, and gastrointestinal tract.2O2 diluted in methanol. Antigen retrieval was performed by incubation for 10 min at 121°C in citrate buffer  or for the anti-RNP with proteinase K pretreatment . After incubation with the primary antibodies overnight at 4°C, ready-for-use Powervision peroxidase system  and 3,3′-diaminobenzidine  or 3-amino-9-ethyl carbazole  were used to visualize the antibodies. Sections were counterstained with haematoxylin. Sections incubated without the primary antibody were essentially blank.Immunohistochemistry was carried out as previously described LyssavirusRabies occurs on all continents apart from Antarctica. The causative viruses belong to the family Rhabdoviridae, genus Diagnosis of rabies depends on demonstration of the virus or antibodies to the virus in various specimens . Several methods are available: RT-PCR, immunofluorescence test, virus neutralization test, and virus isolation in cell culture or animals. In the early stages of disease, results may be negative and tests may have to be performed several times. Once neurological symptoms occur, death is  universally inevitable. Only one patient, who had not received pre- or postexposure prophylaxis, has survived We applied the same methods of coma induction with benzodiazepine and phenobarbital with continuous monitoring of bursts on the EEG and administered ketamine, ribavirin, and amantadine as reported by Willoughby et al. Rabies remains a virtually always fatal disease. In a patient with symptomatic rabies with neutralizing antibodies in the CSF early in the disease, an attempt at intensive treatment inclusive of respiratory and cardiovascular support may be successful. Otherwise, as there are no new data from in vitro and animal studies and no advice from experts on combination treatments, the best possible medical and nursing care with relief of symptoms and comfort care of the patient and support of the family is probably what can be offered at present This is the third human case of rabies due to Duvenhage virus infection. This virus was for the first time isolated in 1970 from a human patient in South Africa This tragic incident is another reminder that even after a minor animal-related injury the wound should be washed thoroughly and postexposure prophylaxis should be given. Where limited access to postexposure prophylaxis may be expected, pre-exposure prophylaxis by vaccination is advised for travellers, especially when travelling with children for longer periods. Human diploid cell vaccine protects against classic rabies virus strains and Duvenhage virus, but not Mokola virus and Lagos bat virus; Duvenhage virus is neutralized by RIG, but Mokola and Lagos bat viruses are not