Intestinal parasitic infections are amongst the most common infections worldwide. Epidemiological research carried out in different countries has shown that the social and economical situation of the individuals is an important cause in the prevalence of intestinal parasites. Previous studies in Turkey revealed a high prevalence of intestinal parasitic infection. The objectives of the current study were to determine the prevalence of intestinal parasitic infections in Aydin among 7–14 years old school children and to identify associated socio-demographic and environmental factors, behavioral habits and also related complaints.Multistage sampling was used in the selection of the study sample. A questionnaire, cellulose adhesive and a stool specimen examination were done.E. vermicularis, G. intestinalis and E. coli. Intestinal parasite prevalence was higher in rural area, in children with less than primary school educated mother, in children who use hands for washing anal area after defecation, and in children who use toilet paper sometimes or never. The relation between child health and mother education is well known. Children were traditionally taught to wash anal area by hand. Toiler paper usage was not common and might be due to low income or just a behavioral habit also. Most of the complaints of the study population were not significantly related with the intestinal parasitic infection.A total of 456 stool specimens were collected. 145 students (31.8%) were infected with one or more intestinal parasites. 29 (6.4%) of the students were infected more than one parasite, 26 (5.7%) with two parasites and 3 (0.7%) with three parasites. The three most common were Intestinal parasitic infection is an important public health problem in Aydin, Turkey. Rural residence, mother education less than primary school, sometimes or never usage of toilet paper, and washing anal area by hands after defecation were the significant associations. Interventions including health education on personal hygiene to the students and to the parents, especially to mothers are required. The ratio of uneducated women should be declined with specific programs. A multisectoral approach is needed. Intestinal parasitic infections are amongst the most common infections worldwide. It is estimated that some 3.5 billion people are affected, and that 450 million are ill as a result of these infections, the majority being children. These infections are regarded as serious public health problem, as they cause iron deficiency anemia, growth retardation in children and other physical and mental health problems .Epidemiological research carried out in different countries has shown that the social and economical situation of the individuals is an important cause in the prevalence of intestinal parasites. In addition, poor sanitary and environmental conditions are known to be relevant in the propagations of these infectious agents -4.Previous studies at various institutions in Turkey revealed a high prevalence of intestinal parasitic infections among the following populations: 6–16 year olds 10.8%), 12–16 year olds (48.0%), 7–15 year olds (55.1%), and 6–12 year olds (88.0%) %, 12–16 .The objectives of this current study were to determine the prevalence of intestinal parasitic infections in Aydin among 7–14 year old school children and its relation to socio-demographic factors, environmental factors, behavioral habits and complaints related to intestinal infections.The data for the present study was acquired from the primary schools in urban and rural areas of Aydin, a city in the western part of Turkey. The study design was cross-sectional.The sample size was calculated on a prevalence of 30%, d = 0.05 at a confidence level of 95%. A design effect of 2 was used to allow for multistage sampling . The calThe questionnaire contained four sections: 1. Socio-demographic data: age, gender, residence, education and occupation of parents, number of adults and children in the family and birth order of the child; 2. Environmental factors: housing conditions  and water supply; 3. Behavior habits: type of toilet commonly used, hand washing (no washing/washing with only water/washing with soap), washing anal area by hands after defecation (yes/no), usage of toilet paper ; 4. Complaints: abdominal pain, nausea/vomiting, lack of appetite, abdominal distention, intestinal dismotility, salivation during sleeping, headache, irritability in sleeping, perianal itching, teeth grinding, and history of parasitic infections.An informational document about the study, including how to supply a stool specimen and a cellulose tape slide, was given to each participant for their family.Mothers were asked to perform one cellulose tape test on their child who was participating in the study. Laboratory slides were provided with cellulose tape attached to them. The mother collected material for examination in the early morning prior to bathing or defecation. On the same morning a field worker collected these slides to be microscopically examined.The stool specimens (0.5–1.5 gr) were collected in labeled plastic vials without preservatives and transported to the laboratory within four hours after collection. They were examined for the presence of parasites by direct wet mount, Lugol's iodine solution and modified formaline-ethyl acetate sedimentation techniques.The presence of parasites was confirmed when observed by any of the methods above.A computer program was used for data analysis. The descriptive data was given as a mean ± standard deviation (SD). The chi-squared test and Student t-test were used for the analytic assessment. The differences were considered to be statistically significant when the p-value obtained was less than 0.05.A total of 456 71.4%) samples for both stool specimens and cellulose tape slides were collected. The response rate to the questionnaire was lower with 367 (57.4 %). The mean age was 10.34 ± 2.27, 10.17 ± 2.30 for girls and 10.51 ± 2.23 for boys. Important socio-demographic characteristics, housing conditions and hygienic habits of children are given in Table .4% samplEnterobius vermicularis (E. vermicularis) with 63 (13.8%) pure and 20 (4.4%) with multiple infections, in total 83 (18.2%) infected children. The second was Giardia intestinalis  with 28 (6.1%) pure and 21 (4.6%) with multiple infections, in total 49 (10.7%) infected children. The third one was Entamoeba coli (E. coli) with 21(4.6%) pure and 15 (3.3%) multiple infections, in total 36 (7.9%) infected children. G. intestinalis was the most commonly found parasite in multiple infections. The distribution of parasites is given in Table In all, 145 students (31.8%) were infected with one or more intestinal parasites. 29 (6.4%) of the students were infected with more than one parasite, 26 (5.7%) with two parasites and 3 (0.7%) with three parasites. The most common was No statistically significant difference was observed between presence of intestinal parasites and gender, (p = 0.805), and also age (p = 0.916). The prevalence of intestinal parasites were significantly higher (p = 0.042) in the rural area 36.9%) than in the urban area (27.9%). A summary of significant relations observed in overall intestinal parasitic infections for the study population are given in Table 6.9% thanG. intestinalis, where sometimes or non-usage of toilet paper was significant for G. intestinalis (p = 0.008) and for E. vermicularis (p = 0.024) both. Family size was larger in the group infected with G. intestinalis (p = 0.029). The prevalence of intestinal parasitic infection was 31.0% in the group using a municipal water network and 34% in the group lacking a municipal system (p = 0.592). The prevalences of G. intestinalis in these two groups were 9.9% and 16.0% (p = 0.106) respectively. No further significant relationships were found between intestinal parasitic infection and environmental or behavioral factors.Parasite-specific significant relations were the following: The prevalence of E. coli infections was significantly higher (p = 0.010) in the rural area (11.1%) than in the urban area (5.0%). Mother education less than a primary school education (p = 0.012), washing anal area by hands after defecation (p = 0.013) were the significant relations for E. vermicularis infections than G. intestinalis and E. coli.The most frequent complaint related with any parasite infection was intestinal dismotility (40.0%). Nausea/vomiting (37.7%) was second and abdominal distention (37.1%) was third. All of the complaints were seen in higher prevalence for E. vermicularis (16.0%) and G. intestinalis (11.9%) being the two most common infections, as was observed in Aydin [E. vermicularis, 21% for G. intestinalis, 10% for H. nana, 4.3% for E. coli, 0.03% for Ascaris lumbricoides (A. lumbricoides), and 0.03% for Trichuris trichiura (T. trichiura). No Taenia was found [E. vermicularis, G. intestinalis, and E. coli, 18.2%, 10.7% and 7.9%, respectively. Higher prevalence was found in the studies from the eastern part of Turkey, where the socio-economic and environmental conditions were lower. Additionally, it was observed that the types of parasites found in this study where different than those found in the eastern part of Turkey. A survey conducted among 1001 children in four elementary schools in Sanliurfa found parasites in 88% of the stool samples examined ; unfortunately, no data on E. vermicularis was given, because samples with cellulose tape slides were not taken [A. lumbricoides, 4.43% T. trichiura and 15.35% G. intestinalis were found [It was found that approximately one-third (31.8%) of the students, ages 7 to 14, in Aydin were infected by intestinal parasites. In a sample within the same age group in Izmir, a city also in the western part of Turkey, the prevalence for intestinal parasites was 22.4%, with stinalis .9% beingas found . In thisre found . In the Ascaris, Strongyloides and Trichuris) were of lower prevalence in the western part of Turkey, but of higher prevalence in the eastern part. This difference may be due to improper toilet facilities which require individuals to defecate in areas around their homes as well as the use of fecal material for fertilizer in gardens in the eastern parts of Turkey [A. lumbricoides, 11.7% T. trichiura infections in these children while there was 12.2 %A. lumbricoides and 6.6% T. trichiura infections in the control groups [Geohelmint infections  washing the anal area by hand with tap water (2) a piece of cloth is used to clean the anal area after defecation. The cloth is used multiple times until the person decides that it has become too dirty after which it is washed and reused. These improper cleaning practices after defecation could be the probable causes behind autoinfection. The higher prevalence of E. vermicularis could also be explained by the highly infectious nature of the parasites.Enterobiasis occurs worldwide, usually involving school-aged children . In geneG. intestinalis and E. coli were the most common intestinal protozoa among the study population. Both can be transmitted orally by drinking infected water and both are environmental contaminants of the water supply. The water supply is really an important risk factor for the Giardiasis, and several large outbreaks of giardiasis have resulted from the contamination of municipal water supplies with human waste [Giardia spp. has been increasingly recognized over the past 10 years as a cause of water-borne diseases in humans [Giardia cysts have been isolated from water supplies in different parts of the world [G. intestinalis and E. coli are most common in the western part of Turkey [G. intestinalis was found 11.6% in the western part of Turkey [G. intestinalis was lower in the group using municipal water (9.9%) than the other group (16.0%) in the current study. It is thought that an in-depth assessment should be done on the ways that drinking water becomes contaminated.an waste . The ingan waste . Contamin humans . Giardiahe world ,21. Epidhe world . G. intef Turkey . In a stf Turkey . From thTaenia spp. Since the consumption of pork and pork products are forbidden for Muslims this may account for the absence of T. solium cases in the population. It is also a common practice to eat uncooked meat in the eastern part of Turkey, but not in the western part. T. saginata infections are observed in the eastern studies, with prevalence of 13.8% and 12.9% [In this current study, there were no cases of nd 12.9% ,24. HoweDifferences due to gender were not observed in this current study. These results were similar to a study conducted in the central part of Turkey . In thisIn this current study, most of the complaints by the study population were not significantly related with the intestinal parasitic infection. For example, perianal itching was noted in 15.8 % of the study population. There was no significant difference in the prevalence of this symptom in pinworm infected and non-infected children. Furthermore, no association was found between the prevalence of pinworm infection and a history of teeth grinding, colic, enuresis, and irritability. The complaints may not have been assessed effectively through the use of only a questionnaire without an interview, or they might not be useful for every individual's diagnosis.The prevalence of intestinal parasites was higher in groups where the mother in the household had less than a primary school education, where the hand is habitually used for the cleaning of the anal area and where toilet paper is seldom or never used. The relation between a child's health and the mother's education is well known. Health indicators of children whose mother's education level is lower are always worse . In the The major limitation of the current study was a low response rate. The assessment would have been more valuable if a higher response rate could have been obtained. But, it was thought that the results were still important because there is little knowledge on the data of the region. Additionally, the current study was the first population-based study for the region. Although an important number of risk factors were discussed, a few risk factors (e.g.: shoe wearing) were not evaluated in the current study. This might have been another limitation of the study. The important risk factors for the region were evaluated in the study.As a conclusion, intestinal parasitic infection is an important public health problem in Aydin, Turkey. Rural residence, households where the mother has less than a primary school education, periodic or non-usage of toilet paper, and the washing of the anal area by hand after defecation were the significant associations. Interventions including health education on personal hygiene to the students and to the parents, especially to mothers are required. The ratio of uneducated women should decline with specific programs. A multisectoral approach is needed.The author(s) declare that they have no competing interests.PO planned the research, performed the sampling and statistical analyzes and wrote draft and final version of the manuscript. SE contributed in planning the research, performed parasitic examinations and contributed discussing the results and writing manuscript. BG performed parasitic examinations and contributed discussing the results. OO organized the work in the schools and participated in its design. EB participated in initial study design, coordinated the study and revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Injuries are becoming a major health problem in developing countries. Few population based studies have been carried out in African countries. We examined the pattern of nonfatal injuries and associated risk factors in an urban and rural setting of Tanzania.A population-based household survey was conducted in 2002. Participants were selected by cluster sampling. A total of 8,188 urban and 7,035 rural residents of all ages participated in the survey. All injuries reported among all household members in the year preceding the interview and resulting in one or more days of restricted activity were included in the analyis.A total of 206 (2.5%) and 303 (4.3%) persons reported to have been injured in the urban and rural area respectively. Although the overall incidence was higher in the rural area, the incidence of major injuries (≥ 30 disability days) was similar in both areas. Males were at a higher risk of having an injury than females. Rural residents were more likely to experience injuries due to falls  and cuts  but had a lower risk of transport injuries. The most common causes of injury in the urban area were transport injuries and falls. In the rural area, cuts and stabs, of which two thirds were related to agriculture, formed the most common cause. Age was an important risk factor for certain types of injuries. Poverty levels were not significantly associated with experiencing a nonfatal injury.The patterns of injury differ in urban and rural areas partly as a reflection of livelihoods and infrastructure. Rural residents are at a higher overall injury risk than urban residents. This may be important in the development of injury prevention strategies. Injuries have been recognised as a major public health problem in both developed and less developed countries . It is gStudies on the magnitude of injuries and the groups at risk, have been conducted world-wide, and especially in developed countries. Hospital based studies, which are commonly reported from developing countries, presumably provide a representative picture of the prevalence and incidence of serious injury, but only a partial picture of the circumstances in which injuries occur. Given the limited access to hospital care in poor countries, however, data based on health facility data are not likely to be representative. In contrast, population-based studies are costly and rarely carried out particularly on topics such as injury, which are not high on the public health agenda in developing countries at present.Several studies have been conducted in high-income countries to examine factors associated with injury morbidity -7. In deThe survey was conducted in Dar es Salaam city (an urban area) and Hai District . These areas are part of a health and demographic surveillance system carried out by the Adult Morbidity and Mortality Project (AMMP) in six districts in Tanzania from 1992 to 2004. One aim of the project was to measure rates and causes of morbidity and mortality. At the time this investigation was carried out, the areas were being prospectively monitored through repeated censuses to ascertain the resident population at risk. Deaths were recorded through an active reporting system and probable cause of death determined by a validated verbal autopsy ,16.Dar es Salaam city lies on the east coast of Tanzania. The AMMP demographic surveillance population was situated in two of the city's three municipalities. These areas contained 8 'branches' (an urban administrative unit) and covered 63,330 persons in 15,269 households living in urban and peri-urban neighbourhoods. Hai District lies on the South-Western slopes of Mount Kilimanjaro in Northern Tanzania. The AMMP demographic surveillance area in Hai covered 51 out of 61 villages in the district and around 62% of the total district population . Agriculture, livestock keeping and commercial mining are the main economic activities there. Details of the study population have been described elsewhere .In the urban surveillance area, an initial cluster sample of 500 households was randomly selected. Because initial data collection yielded fewer injuries than expected in the first two enumeration areas, the sample size was increased to 2,000 households with the difference made up of households selected at random from the remaining six surveillance branches. Thus, the final sample under-represented the first two branches. Information was sought on all individuals residing in the selected households. A two-stage cluster sampling method was adopted in selecting the rural sample. In the first stage, using existing AMMP data on mortality and poverty, six out of 51 villages were selected to represent different levels of socio-economic status and injury mortality. A random sample of 2,000 households was obtained from the selected villages in the second stage. All individuals in the selected households were included in the survey.Ethical clearance for this study was given by the Tanzania Commission for Science and Technology and the Regional Committee for Medical Research Ethics in Norway. Informed verbal consent was sought from each family. For children below age 15, parents or guardians were interviewed; for adolescents aged 15 to 18, consent was obtained from both the parent and the child.The survey tool was translated into Swahili, back translated into English, and pre-tested before use in the field. Two questionnaires were used in the study. Questionnaire 1 was a screening form used to identify whether a household member had an injury in the past one year that resulted in losing one or more days of 'normal' activity (e.g. not being able to work or go to school). The head of household or any other responsible person was interviewed to obtain information about the household members. Variables included were age, sex, relationship to head of household, level of education, religion, marital status and occupation.Questionnaire 2 was used to record the circumstances in which the injury occurred. Some of the variables included were: month and year when the injury occurred; cause of the injury; place of occurrence; activity at time of injury; length of disability; and health facility use. Efforts were made to interview the injured person if an adult, otherwise we interviewed an informed member of the injured person's household. The number of days with restricted activity (disability days) was considered as a measure of severity of injury. Poverty was assessed at the household level using data from the 2000–2001 National Household Budget Survey and variables from AMMP data. The measure of poverty used was a predicted value of monthly consumption expenditure per adult equivalent for each household included in the study ,18.Data analysis was done using STATA (version 7). Bivariate analyses were performed by cross tabulations and the chi-squared test was used to test for homogeneity. In the case of multiple injuries, the most recent injury episode was considered in all the analysis. Multiple logistic regression was used to examine the influence of socio-demographic and socio-economic factors on the risk of being injured, controlling for potential confounding variables. Odds ratios are reported with 95% confidence intervals. Tests for trend in associations over groups defined by other factors were performed where appropriate.Preliminary analysis indicated that a long recall period underestimated annual injury rates, with the effect being greater for injuries resulting in <30 disability days while the rates for injuries resulting in 30 or more disability days were quite stable. We therData were gathered on a total of 15,223 individuals residing in 3,653 households. The urban sample included 8,188 individuals while the rural sample included 7,035 persons. The response rates were 89% and 92% for the urban and rural areas respectively. The rural sample had a larger proportion of individuals aged 44 years and above (24%) compared to the urban area (11%). This was comparable to national averages as reported by the 2002 national census in Hai (17%) and Dar es Salaam (10%)[Of the total sample, 509 persons reported to have sustained an injury in the past one year preceding the survey, representing an injury incidence of 32.7 per 1,000 persons per year (95% CI= 29.9 – 35.7). The incidence for all, minor and major injuries was 24.5, 16.4 and 8.1 per 1,000 persons per year for Dar es Salaam, and 42.5, 32.8 and 9.7 for Hai district. The mean age of the injured was 27.6 years (standard deviation 20) and 62% were males. Almost all injuries were unintentional (96%). On average, 14 days of normal activity were lost per person because of an injury.In Dar es Salaam, the most common cause of injury reported in both males and females was transport injuries, followed by falls and cuts Table . In Hai,As expected, the cause of injury varied by age Figure . In the Major injuries accounted for 33% and 23% of all injuries in Dar es Salaam and Hai respectively  of all injuries in both areas. Children aged less than five years were 8 times as likely to sustain injuries due to burns as adults aged 15 to 44 years . Although the magnitude of association appears to be large, the estimate was based on small numbers .In this study, we found major differences between urban and rural residents with respect to cause and severity of injury and the circumstances in which they occurred. This has great implications in setting priorities when planning for intervention strategies.Transport injuries formed the most common injury category in the urban area. The low risk of transport injury in the rural areas is probably a reflection of the relatively lower level of motorization in this mainly agricultural area. However, they will often have more serious consequences than other types of injury. A number of studies have reported similar findings. In a study from Pakistan, farmers were found to be at a lower risk of traffic injury than labourers and vendors . A studyCuts and stabs by instruments such as axes and machetes constituted the most frequent injury category in the rural area, which is due to the fact that rural residents engage in agricultural activities using unprotected equipment. Cuts and stabs also contributed significantly among children aged 5 to 14 years with farm work being the common activity in the rural setting while play was the main contributing factor in the urban area. As emphasized in other studies, there is a need for safe space for play among children. In addition, the issue of a working child in developing countries like Tanzania needs to be addressed.Falls were also a significant contributor among young children and older adults. A better understanding of the circumstances in which falls occur would assist in planning for fall-prevention programmes in Tanzania. Injuries due to accidental poisoning were infrequently reported in both areas and hence grouped under 'others'. It is possible that people were not comfortable reporting such events. In addition, near drowning did not feature among the causes of nonfatal injuries although the urban area has a port.Except for transport injuries which were the commonest cause of fatal and nonfatal injuries in the same surveillance areas, the distribution of nonfatal injuries differed essentially from those of fatal injuries reported for 1992–1998 in the same surveillance areas . In addiA significant risk factor for injury turned out to be the place of residence. The likelihood of self reported injury was 66% higher for rural residents compared to urban residents. Similar findings have been reported from other studies ,23. HoweAge is an important risk factor for many injuries but its influence varies between specific injury groups. Our findings show that adults aged 15–44 years are at a high risk of transport injuries. This has great economic impact since these are people in their most productive years and the injuries impose a considerable burden on their families and the society as a whole. Children below 15 years were at greater risk of injuries due to falls. This may be due to high risk environments such as lack of proper play facilities. This is in keeping with studies from other developing countries whereby falls among children have been reported to be a common cause of injury seen in hospitals . As expeSocio-economic status has been documented to be an important determinant of injury, although the effect depends on the socio-economic indicator considered, the cause and severity of injury . We founThe findings of this study are subject to a number of limitations. The information is based on self-reported data elicited through interviews, which is subject to recall bias ,29. A 12Intentional injuries such as assaults and domestic violence are probably underreported since they would not be adequately captured in such a survey. This may lead to injury rates being underestimated. In this study, a clinical injury severity assessment was not possible. Disability days were used instead as a measure of severity of injury. One should be careful in generalizing the findings to other urban and rural settings of the country. Dar es Salaam is in many ways different from other urban areas of Tanzania and Hai is a relatively wealthy rural area. Furthermore, it might be difficult to generalize the findings to the surveillance areas due to the selection process that was employed. However, from our knowledge of the areas, we see no obvious reasons indicating that our samples should be very different from the urban and rural surveillance areas. Despite its limitations, this study has generated information that could be useful for targeted prevention at the local level.This study, the first of its kind in Tanzania, describes the patterns of nonfatal injuries and associated socio-demographic and socio-economic factors. It has attempted to identify specific groups of individuals as having a greater risk of experiencing certain types of injuries. This information is important for raising the level of awareness among policy makers and the public in general since the problem of injury receives little attention in most of the developing world including Tanzania. It is also useful in setting priorities for cause-specific prevention strategies. More detailed qualitative studies are required, however, on sensitive events such as assault. A nationally representative sample is also essential to measure the health burden due to nonfatal injuries.The author(s) declare that they have no competing interests.CM designed and conducted the study, performed statistical analysis, wrote the initial draft and revisions of the manuscript after consultation with other authors. IH, AN and GK participated in the design of study and revision of the manuscript. PS and YH participated in study design and co-ordination, and in revision of manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Cough is the most common symptom presenting to doctors. The quality of cough (productive or wet vs dry) is used clinically as well as in epidemiology and clinical research. There is however no data on the validity of cough quality descriptors. The study aims were to compare (1) cough quality (wet/dry and brassy/non-brassy) to bronchoscopic findings of secretions and tracheomalacia respectively and, (2) parent's vs clinician's evaluation of the cough quality (wet/dry).Cough quality of children (without a known underlying respiratory disease) undergoing elective bronchoscopy was independently evaluated by clinicians and parents. A 'blinded' clinician scored the secretions seen at bronchoscopy on pre-determined criteria and graded (1 to 6). Kappa (K) statistics was used for agreement, and inter-rater and intra-rater agreement examined on digitally recorded cough. A receiver operating characteristic (ROC) curve was used to determine if cough quality related to amount of airway secretions present at bronchoscopy.Median age of the 106 children  enrolled was 2.6 years (IQR 5.7). Parent's assessment of cough quality (wet/dry) agreed with clinicians' . When compared to bronchoscopy (bronchoscopic secretion grade 4), clinicians' cough assessment had the highest sensitivity (0.75) and specificity (0.79) and were marginally better than parent(s). The area under the ROC curve was 0.85 (95%CI 0.77–0.92). Intra-observer (K = 1.0) and inter-clinician agreement for wet/dry cough  was very good. Weighted K for inter-rater agreement for bronchoscopic secretion grades was 0.95 (95%CI 0.87–1). Sensitivity and specificity for brassy cough  were 0.57 and 0.81 respectively. K for both intra and inter-observer clinician agreement for brassy cough was 0.79 (95%CI 0.73–0.86).Dry and wet cough in children, as determined by clinicians and parents has good clinical validity. Clinicians should however be cognisant that children with dry cough may have minimal to mild airway secretions. Brassy cough determined by respiratory physicians is highly specific for tracheomalacia. Cough is the most common symptom presenting to medical practitioners in Australia, the UK and USA -3. Coughin-vivo visual assessment of larger airways usually to the 3rd (lobar bronchi) or 4th generation  in young children.The sound of a cough is due to vibration of larger airways and laryngeal structures during turbulent flow in expiration ,17. It iThe study aims were to compare (1) cough quality (wet vs dry and brassy vs non-brassy) with bronchoscopic findings of secretions and tracheomalacia respectively and, (2) parent(s) vs clinician's evaluation of the cough quality (wet and dry). We hypothesised that clinical assessment of cough is good compared to bronchoscopic findings and that a wet cough is related to presence of airway secretions.Children electively admitted for bronchoscopy without a known underlying respiratory diagnosis were seen by a member of the research team 0.5–3 hours prior to bronchoscopy. The clinician's assessment of cough quality (wet or dry) was recorded on a standardised sheet (based on the cough present on the day of the bronchoscopy), before the parent(s) independently evaluated the current  cough quality (wet or dry) of their child. For clinician's assessment of wet/dry cough, when no spontaneous cough was heard or if child was too young to elicit a cough, cough quality (wet or dry) was deemed 'non-assessable'. Clinicians also rated cough as 'brassy' or 'non-brassy' based on coughs heard anytime before bronchoscopy. For assessment of reliability of cough quality (wet/dry and brassy/non-brassy), 21 cooperative children had their coughs digitally recorded  using music compact disc quality format  on the morning of their bronchoscopy. These stored cough sounds were later replayed  from a computer and re-scored in a blinded manner  for wet/dry and brassy/non-brassy qualities. Written consent was obtained from a parent and the study approved by the hospital's ethics committee on human research.Flexible bronchoscopy was performed under general anaesthesia as previously described -22. BrieA respiratory consultant (ABC or IBM) blinded to the child's history and cough quality scored the bronchoscopy sheet quantifying the amount of secretions at the time of the bronchoscopy in real time. When no scorer was available, the session was videotaped and played back. A secretion quantification card figure  was visiBS Grade 1 = Nil secretionsBS Grade 2 = Near dry = Bubbles only in < half total number of bronchi involvedBS Grade 3 = Minimal = Bubbles found in > half total number of bronchi involved or Secretion type-I in < half total number of bronchi involvedBS Grade 4 = Mild = Secretion type-I, > half total number of bronchi involved or Secretion type-II, < half total number of bronchi involvedBS Grade 5 = Mod = Secretion type-II, > half total number of bronchi involved or Secretion type-III, < half total number of bronchi involvednd assessor blinded to the child's condition.BS Grade 6 = Large = Secretion type-III, > half total number of bronchi involved Inter-rater reliability of BS grading was assessed by replaying the videotapes of the recorded bronchoscopy of 20 children, with the 2BAL was obtained from the macroscopically most abnormal lobe; when changes were generalised, BAL was obtained from the right middle lobe. Cell count was performed on the cell suspension, cytocentrifuge slides were prepared and stained (modified Wright's stain) for cell differential profile. All cellular examinations were performed by cytologists blinded to the children's medical history.Data were not normally distributed and thus non parametric analyses were used; medians and inter-quartile range (IQR) were used for all descriptive data and Kruskal Wallis for comparisons between groups. Cohen's kappa (K) with 95%CI was utilised for inter and intra-observer reliability and graded from 'poor' (K<0.2) to 'very good' (K = 0.81–1.0). For calMedian age of the 106 children  enrolled was 2.6 years (IQR 5.7). Indications for bronchoscopy were chronic cough , wheeze , stridor , investigation of persistent radiological changes , recurrent pneumonia , suspicion of aspiration lung disease , BAL and suspected foreign body . In four children, BS grades were not obtained (session was inadvertently not recorded and 'blinded' clinician not present at bronchoscopy). Scores of BS were done in real time in all but 9 children.In 30 children, cough was non-assessable. Agreement between clinicians and parents assessment of cough quality (wet/dry) was good . For cough quality of 'wet/dry', cough assessed by clinicians had the highest specificity, sensitivity, NPV, PPV and positive likelihood ratio for both BS cut-offs tables  and 2. PThere was little difference in sensitivity and specificity between children grouped by indication for bronchoscopy (cough or other indications). Values were marginally better in older children . There was only one disagreement in wet and dry cough between clinicians and in this child the cough was mildly wet (BS grade of 3). Kappa scores for intra-observer and inter-observer clinician agreement for brassy cough was good, K in both was 0.79, 95%CI 0.73, 0.86. Inter-rater agreement for BS grades was 'very good' .Cellularity for total cell count, percentages of neutrophils and macrophages were significantly different between children grouped by BS grade cut-offs of 3 and 4 as well as wet/dry cough . When cough is wet, secretions were always present; when cough was dry secretions if present, were usually minimal or mild. Clinicians were marginally better than parents at assessing wet/dry cough and agreement between the 2 groups was good. When clinicians detected presence of a brassy cough, tracheomalacia was usually present. Inter-rater clinician agreement for cough qualities of dry/wet and brassy/non brassy was good.Accuracy and reliability of symptoms are important in clinical and research settings. Cane and colleagues ,26. founWhen compared to bronchoscopic findings, this study showed that a wet cough is always associated with BS grades of 3 or more. Dry cough is less valid; the presence of dry cough does not necessary indicate absence of secretions. However BS grades are less in dry cough as shown in the ROC curve. The generation of cough sounds and some factors that influence cough sounds have been examined in the laboratory ,30. UsinOne possible limiting factor of our study is the choice of cut offs for BS grades in determining presence or absence of significant secretions. We chose to use a cut off of 3 as a minor amount of bubbles in the airways can be present from trickling of lignocaine into the airways or spillage from the upper airways. BS cut-off at grade 4 resulted in improved specificity but decreased sensitivity. Children grouped by both BS cut-offs (3 and 4) had significantly different airway cellular profile. The clinical significance of minimal BS grades and appropriate cut-offs can only be determined in a prospective follow-up study which is not an aim of this study. This study did determine that our BS scoring method was easy to use  and had very good inter-rater agreement. The clinical outcomes of wet and dry cough were not the aims of this study and thus cannot be determined here. To relate clinical outcomes to cough descriptors would ideally require a randomised controlled trial with dry and wet cough as entry criteria. A follow-up cohort study with strict clinical diagnostic categories would be useful and we have shown in a preliminary study that dry cough was significantly more likely to naturally resolve than wet cough .In addition to the limitation of quantifying airway secretions using a bronchoscopic method, this study is also limited by a number of factors. Firstly, clinical repeatability or agreement of cough sounds was assessed by doctors in a tertiary setting. Whether or not these findings can be extrapolated to the secondary and primary setting can only be speculated. Hay and colleagues showed that inter-observer agreement for clinical signs of fever, tachypnoea and chest signs were poor to fair (kappa of 0.12–0.39) in the primary care setting but these signs are known to have good agreement in secondary care settings . HoweverDetermining the validity of cough quality in children is important not only because of the commonality of the clinical problem of cough but also its use in guidelines and research studies ,27,28. AWe conclude that the description of a cough as wet or dry cough as determined by clinicians and parents has good clinical validity as it has good agreement with, and relates to, quantification of airway secretions. However as minimal amount of secretions may be present in children with dry cough, clinicians should be cognisant that a dry cough may eventually become wet if airway secretions increase. Thus it should not be assumed that airway secretions are absent in children with chronic dry cough and cough quality in these children should be reviewed. We also conclude that the brassy cough determined by respiratory physicians is highly specific for presence of tracheomalacia.BAL Bronchoalveolar lavageBS Bronchoscopic secretionK KappaNPV Negative predictive valuePPV Positive predictive valueROC receiver operating characteristicAC conceived the idea, designed the study, performed the data analysis and drafted the manuscript. JG participated in data acquisition and coordination of project. ME participated in electronic acquisition of data and software for sound recordings. JF and NC designed the microbiology and cytological components respectively and both helped draft the manuscript. IBM helped in formulation of overall study design, data acquisition and drafting of the manuscript. All authors read and approved the manuscript.Figure 3: ROC curve ROC curve with 95%CI relating cough quality (wet/dry) to bronchoscopic secretion (BS) grades from 1–6 in children grouped according into age (a) ≤ 2 years and (b) > 2 years.Click here for fileTable 4: Assessment of cough quality vs bronchoscopic findings in children grouped by indication for bronchoscopy 4a: Assessment of cough quality vs bronchoscopic findings in children whose indication for bronchoscopy was cough 4b: Assessment of cough quality vs bronchoscopic findings in children whose indication for bronchoscopy was others (ie not cough) Table 5: Assessment of cough quality vs bronchoscopic findings in children grouped by age 5a: Assessment of cough quality vs bronchoscopic findings in children aged ≤ 2 years 5b: Assessment of cough quality vs bronchoscopic findings in children aged > 2 yearsClick here for file


Non-invasive monitoring of respiratory muscle function is an area of increasing research interest, resulting in the appearance of new monitoring devices, one of these being piezoelectric contact sensors. The present study was designed to test whether the use of piezoelectric contact (non-invasive) sensors could be useful in respiratory monitoring, in particular in measuring the timing of diaphragmatic contraction.Experiments were performed in an animal model: three pentobarbital anesthetized mongrel dogs. The motion of the thoracic cage was acquired by means of a piezoelectric contact sensor placed on the costal wall. This signal is compared with direct measurements of the diaphragmatic muscle length, made by sonomicrometry. Furthermore, to assess the diaphragmatic function other respiratory signals were acquired: respiratory airflow and transdiaphragmatic pressure. Diaphragm contraction time was estimated with these four signals. Using diaphragm length signal as reference, contraction times estimated with the other three signals were compared with the contraction time estimated with diaphragm length signal.The contraction time estimated with the TM signal tends to give a reading 0.06 seconds lower than the measure made with the DL signal , with a standard deviation of 0.05 seconds . Correlation coefficients indicated a close link between time contraction estimated with TM signal and contraction time estimated with DL signal . In general, correlation coefficients and mean and standard deviation of the difference were better in the inspiratory load respiratory test than in spontaneous ventilation tests.The technique presented in this work provides a non-invasive method to assess the timing of diaphragmatic contraction in canines, using a piezoelectric contact sensor placed on the costal wall. Non-invasive monitoring of respiratory function is an area of increasing research interest, resulting in the appearance of new monitoring devices . At presThe purpose of this study is to evaluate a non-invasive method to study the timing of the diaphragmatic function, using an animal model (dogs). Accordingly, the present study was designed to test whether the use of contact (non-invasive) piezoelectric sensors, placed on the dogs' costal wall, could be useful in monitoring the diaphragm contraction period in different respiratory conditions, comparing it with other physiological signals such as transdiaphragmatic pressure, diaphragm length measured by sonomicrometry, and respìratory airflow. Diaphragm contraction time is expected to be very close to inspiratory time, which is one of the most utilized parameters in the studies of breathing pattern under experimental or clinical conditions.Three mongrel dogs (15–20 kg) were surgically instrumented under general anesthesia given via a femoral vein catheter . Respiratory flow was recorded with a Fleisch pneumotachograph. Diaphragm shortening was measured via two piezoelectric crystals , as described in . The diaAll analogical signals were amplified (HP 8802A), filtered and digitised with a 12 bit A/D system at a sampling rate of 4 kHz. Inspiratory airflow (FL), diaphragm length (DL), thoracic cage motion (TM), transdiaphragmatic pressure (DP) and diaphragmatic electromyography (EMGdi) signals were decimated at a new sampling rate  and digitally filtered . The sampling frequencies and filter bands were selected to be adapted to the frequency content of the signals. Figure ti, tf), the integral of TM signal and the first derivative of the DL, FL and DP signals were computed. Initial contraction time is detected when these signals reach 10 % of the maximum. In a similar way, final contraction time is detected when the signals reach 10 % of their minimum. We also computed contraction period (TC = tf - ti). The EMGdi signal was included initially in the study but later was rejected because the postinspiratory activity present in this signal hindered the detection of the end of the diaphragm shortening contraction time , standard deviation (STD). DL signal was used as a goldstandard  for muscle shortening, since it is a direct measure of the reduction of the diaphragm muscle length, contrary to flow and trandiaphragmatic pressure which are more remote markers of muscle contraction.Relationships between contraction periods obtained by means DL and TM signals, DL and FL, and DL and DP were analyzed by diverse statistical methods. Different correlation coefficient were calculated: the Pearson correlation coefficient (r), the intra-class correlation coefficient (or reliability coefficient: R) , the sloThe obtained results of the comparison of contraction period estimated with the DL signal (a direct measure of diaphragm shortening) with the contraction periods estimated with the TM, FL and DP signals, are shown in Tables In the Fig. In Table Table In the SVN and SVW respiratory tests (except for the SVN test of the first dog), results showed a moderate relationship, but, in general, correlation coefficients estimated in TM signal were better than estimations in FL and DP signal. Relationship between the contraction period estimated with TM signal and contraction period estimated with DL signal showed Pearson correlation coefficients between 0.60 and 0.91, reliability coefficients between 0.23 and 0.77, slope of the linear regression lines between 0.55 and 1, and Spearman's rank-order correlation coefficient between 0.58 and 0.84.In Fig. Finally, Figure In the present work we have compared the rib cage motion recorded by surface sensors with the changes in diaphragm activity registered by sonomicrometry, transdiaphragmatic pressure and airflow recorded in dogs during spontaneous and inspiratory load breathing. Diaphragmatic time contraction measured with a surface sensor has a good correlation with the rest of signals, especially during the inspiratory load test.In a recent study we observed that the beginning and the end of diaphragmatic contraction were indicated with inflexion points in the thoracic cage motion signal, acquired with a piezoelectric contact sensor placed on the costal wall of the thorax ,7. An alSpinal anaesthesia was used in the present study as a means to isolate diaphragmatic activity by eliminating the activity of the intercostals muscles. In this way, TM, FL and DP signals are directly related with the contraction of diaphragm muscle in the SVW and ILW respiratory tests . In the SVN test the morphologies of TM, FL and DP signals are influenced by the activity of intercostal muscles. However, time differences between contraction periods measured in SVN and SVW respiratory tests were very similar (except for the SVN test of the first dog). This could be explained taking into account that during spontaneous breathing the activity of intercostals and respiratory accessory muscles is minimal ,7.A close relationship between the different methods of diaphragm contraction time estimation has been found during the inspiratory load test. However, during spontaneous ventilation, the correlation was lower than that obtained in the resistive load test. This difference could be explained by the fact that during spontaneous ventilation, the motion of the respiratory system is very low and in consequence, the signal recorded by the contact sensor  has not sufficient intensity to detect with the same precision the beginning and ending of diaphragmatic activity. Besides, during inspiratory resistive loading, there is a marked distortion of the rib cage  and this produces a more marked thoracic-cage motion signal.Another important finding of this work was that time contraction differences between signals were less than 0.1 s for spontaneous ventilation and inspiratory load (except in the irregular case of the SVN respiratory test of the first dog), which indicates that the application of contact sensors constitutes an indirect way to detect the diaphragmatic activity. This could be seen in the graphs of Fig. The contraction of the costal diaphragm acts to displace the rib cage through its insertions at the costal margins and by changing the pressure on the inner surface of the rib cage in the area of apposition. The crural diaphragm is not inserted on the rib cage, but it is considered to have an action through the central tendon. Therefore, in spontaneous respiration the crural part has an important inflationary action on the lower rib cage , and altFinally, to acquire the diaphragm shortening signal, it has been necessary to surgically isolate their diaphragms. The effect of diaphragm isolation could be to favour the operation of the piezoelectric contact sensor to measure diaphragmatic contraction timing. This should be kept in mind when extrapolating these results to human.The technique presented in this work represents a non-invasive method to assess the timing of diaphragm contraction in dogs. We believe that in the future this technique could provide a new potentially useful method for non-invasive respiratory timing monitoring in humans.None declaredJAF, JM, RJ and AG conceived the study, and participated in its design and coordination. JAF and AG designed and conducted the experiments. AT participated in the design of the study and performed the signal processing and statistical analysis. BG and JG provided advice on analysis of the data and manuscript writing. JAF, AT and RJ wrote the first draft of this manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Gecko (Gene Expression: Computation and Knowledge Organization) is a complete, high-capacity centralized gene expression analysis system, developed in response to the needs of a distributed user community.Based on a client-server architecture, with a centralized repository of typically many tens of thousands of Affymetrix scans, Gecko includes automatic processing pipelines for uploading data from remote sites, a data base, a computational engine implementing ~ 50 different analysis tools, and a client application. Among available analysis tools are clustering methods, principal component analysis, supervised classification including feature selection and cross-validation, multi-factorial ANOVA, statistical contrast calculations, and various post-processing tools for extracting data at given error rates or significance levels. On account of its open architecture, Gecko also allows for the integration of new algorithms. The Gecko framework is very general: non-Affymetrix and non-gene expression data can be analyzed as well. A unique feature of the Gecko architecture is the concept of the Analysis Tree , in which all successive results in ongoing analyses are saved. This approach has proven invaluable in allowing a large (~ 100 users) and distributed community to share results, and to repeatedly return over a span of years to older and potentially very complex analyses of gene expression data.. In totality or in parts, the Gecko framework should prove useful to users and system developers with a broad range of analysis needs.The Gecko system is being made publicly available as free software  In recent years, in response to the needs of our scientific community we have developed a comprehensive, company-wide gene expression data analysis platform based on a centralized client-server architecture Figure . This plAnalysis Tree  which provides a complete historical and hierarchical display of all analyses conducted to date by the users. In particular, in support of this concept, Gecko permanently stores the results of all analyses performed. A second organizing concept is that of the agglomeration syntax, an "Erector Set" of operations for flexibly creating, combining and subsetting data matrices. The third organizing concept is the pervasive use of experimental designs, which are associated with each data matrix, and which enable the application of a wide range of statistical and pattern recognition tools.Three organizing concepts have guided the construction of Gecko. The first is the use of the It is the aim of this paper to give an idea of the user's view of Gecko and how one conducts analyses using the system, as well as to provide a software-level overview of the Gecko system architecture. Indeed, we believe that Gecko presents a number of innovative features well-worth presenting, and in connection with this publication, we are making available a public release of the Gecko software.In what follows, we first go "behind the scenes", and present the system architecture in some detail, including overall data organization, database structure, computational engines, statistical tools and models, and finally utility programs. We then present a focused discussion of a specific analysis example, so as to give the reader a more immediate impression of the Gecko system.Gecko is based on a client-server architecture, with a global structure shown in Figure The exchanges between the client and the server occur through HTTP requests, which transit through a web server running on the server platform. A central aspect of the Gecko client is that it contains an embedded Internet Explorer browser, a feature which greatly simplifies the task of building user interfaces. Thus, forms for submitting parameters to the server are typically built in HTML dynamically generated by server-side Perl CGI or Java servlet programs, and displayed in the embedded browser.The Gecko server itself runs on a UNIX platform, and consists of the four main components indicated in Figure The set of tables in the Gecko database can be partitioned into three main groups, which we call the "Scan", "Chip" and "Analysis" groups in accordance to their functional roles.The Scan group of tables stores attributes of the individual scans of microarray data entered into the system. These attributes include a unique scan identifier (the scan name), as well as many parameters  applied and treatment duration, hybridization protocols, etc), which record the nature of the biological sample used and how it was processed, and place the scan in a tree with experimental and biological context.While the Scan group of tables captures many items in common with the so-called MIAME (Minimum Information about a Microarray Experiment) annotation standards, it shouThe Scan group of tables also records numerical data in the form of summary statistics for each scan, including several measures of chip brightness and measures of noise and saturation. However, the bulk numerical data for each scan is stored as a file in the flat file repository, with only a file pointer stored in the database.The Chip group of tables stores the attributes of the Affymetrix chip designs currently known to the system. These include the names of the chip designs used, and for each chip design, all the qualifiers pertaining to it. The tables also store sequence annotation information on a qualifier-by-qualifier basis, including a short description line, as well as a URL that provides a link to more general annotation information for each qualifier. The annotation information is generated externally to the Gecko system, with periodic updates via flat files which can be automatically uploaded.The Analysis group of tables is central to all the analysis functions available in Gecko. Any analysis object generated in the system has a set of attributes which are saved under five categories of information: 1) its parent/child relations, 2) an internal pointer to the machine-generated file which contains the bulk numerical data, 3) the parameters for the operation which created the analysis object, 4) general parameters , and 5) experimental design parameters. Knowledge of the experimental design ,,Get Statk-nearest-neighbor classifier(GENNC)Data transformations are frequently required in the course of analyses. Among those available in Gecko are point transformations, where each element of the data matrix is independently transformed , as well as more global transformations, including variance stabilization, standarNew analysis methods, if already available as executables or applications running from the UNIX command line , can be internally added to the Gecko system by straightforward programming steps. These steps include i) providing for a user interface, generated by server Perl CGI or Java servlet programs, and displayed as HTML in the client Browser window; and ii) constructing a server-based driver program, that will execute the UNIX command, using the parameters communicated by the user interface. We note that while an application programming interface (API) has not been formalized, a Gecko API is already well-approximated, by the existence of a modular set of methods for accessing the database, and for reading and writing to numerical flat files.For external analysis using other applications, direct streaming of all internal Gecko types is currently implemented for Spotfire and MicrA central concern in the design of Gecko was to enable the user to perform and especially to later recall complex analysis work flows . In general, graphs of analyses conducted in Gecko, with nodes corresponding to datasets and edges to operations on these datasets, result in directed acyclic graphs (DAGs). A DAG is unlike a tree, in that each of its nodes can have multiple parents, whereas in a tree each node has a unique parent; for simplicity however, we refer to the DAG generated by Gecko as the Analysis "Tree". Furthermore, in the Gecko client the DAG is actually displayed as a tree: the DAG topology is correctly maintained by replicating, for nodes with multiple parents, the corresponding subgraphs under each of the parent nodes.Once generated, the data file corresponding to a node in the Gecko Analysis Tree is permanently stored (unless the node is explicitly deleted by the user at some later time). This approach enables users to return at any time to potentially very large and complex panels of analysis results, without requiring them to regenerate all final and intermediate results on-the-fly, as might be required in an alternative real-time "dataflow" approach . We have found that the dataflow approach can entail a prohibitive computational cost and waiting time, whenever a large number of analyses are being simultaneously considered, as in the examples of Figure The permanent storage of all analysis results might seem an extravagant use of computer resources, but experience shows that it results in reasonable use of server memory over time. For an expression analysis community of roughly 100 scientific users, over a span of 5 years memory use has been limited to about 150 GB , reached with slow linear growth over time. Furthermore, should it be absolutely required, implementing a file archival and retrieval system for the oldest analyses would be a straightforward task.The Gecko noise model is based on the so-called PFOLD joint noise model and ratio estimation algorithm. This moindependent of microarray technology, with a generalized storage of intensity and noise information for each chip qualifier or microarray spot. Programming modifications needed for a new technology thus primarily occur in the design of the new raw-file parser . Note that for the two-color technologies mentioned above, data for each channel is entered as a separate intensity scan. Channel-to-channel ratios between matched scans are then computed downstream by the users, using the Cat Ratio Agglomeration tool mentioned above.While currently all scans uploaded into Gecko are generated by Affymetrix technology, in the past the system has also been used with other types of expression data, for instance generated by two-color hybridizations on spotted arrays. This has been possible at low programming cost, because the internal representation of scan data in Gecko is An alternative and very flexible method for data entry into Gecko, which entirely bypasses scan entry, is to directly upload a tabular file through the Gecko client. In particular, this method enables one to upload gene expression data from the many public sources where it is provided only in spreadsheet format. Furthermore, as already stated, it also enables one to use the Gecko analysis tools in contexts unrelated to gene expression.As Gecko was designed as a centralized resource, but also for service of geographically remote sites Figure , it was First, users register scans through an interface provided in the Gecko client, using an appropriate submission window. This registration step stores the scan attributes in the Gecko database , but does not transfer the scan numerical data  itself. In the second, independent step, the users send the scan numerical data, in the form of Affymetrix CEL files, to a spThe Gecko scan processing pipeline, run as a periodic "cron" job on the UNIX platform, automatically converts the Affymetrix CEL file data to Affymetrix MAS5 estimateThe processing pipeline has proven to be very robust, and can be readily modified to accept other sources of gene expression data, as already mentioned above. Thus, it has also been used to process cDNA microarray data in the pAs an example of an analysis workflow conducted in Gecko, we describe a study of a cancer cell line treated with a panel of compounds which are inhibitors of cell proliferation. Cultures of the A498 cell line (a cell line derived from kidney carcinoma and part of the NCI60 panel) were trFigure The left-hand window pane (Tree window) provides a tree representation of the data objects existing in Gecko; in the figure, it currently displays the Analysis Tree, which provides a full and permanent record of analysis operations and resulting datasets executed so far. This window can also display the Scan Tree, a hierarchical display of all scans in the system, by selection of the corresponding Scan Tree tab (upper left-hand corner).k-nearest neighbor classifier  design compound_panel.AGG which result from the combinations (compound × time_hr) and (moa × time_hr), respectively. The number of replicates for every combination of levels is indicated in each cell of the tables. The factorial design (compound × time_hr) is of particular interest for finding genes with expression differentially regulated by the treatments with the different compounds, with or without concommittant time variation. In the A498 analysis workflow, this design was used to generate a two-way analysis of variance (ANOVA) compound-panel_compound_time_hr.ANOVA2 can be revisited for selection of statistically significant data using a generic utility called Get Stats. In particular, Get Stats internally computes receiver operating characteristics   compoundcompound_panel.AGG, was then filtered to the rows corresponding to the 517 signifi-cant qualifiers contained in compound-panel_compound_time_hr-517.ANOVA2, in preparation for down-stream clustering and supervised classification operations. This step, implemented by the subsetting tool Reduce on Qlist, results in the filtered data matrix compound_panel-517.AGG.Following the ANOVA operations, the original data matrix, Note that for all of the datasets discussed above, prior to each operation, a tentative output name was automatically created , and then presented to the user in a preview page. The tentative name can then be modified, if desired, before final submission.compound_panel-517.AGG , three clustering methods were first applied, resulting in i) a self-organized maps (PCA)  k-nearest-neighbor classifier5k-nearescompound effects in the two-way ANOVA (compound-panel_compound_time_hr.ANOVA2) is displayed in the Browser window. In Figure compound_panel-517RmedNR_feature_sel_FILTER_60_moa.CVEC is displayed as a three-dimensional scatter plot.Gecko provides for flexible visualization of analysis results, with results either directly displayed in the client Browser window, or streamed to external visualization tools such as Spotfire. In FiguConstructed around the three organizing concepts of the Analysis Tree, the agglomeration syntax, and the pervasive use of experimental designs, Gecko has proven to be a robust analysis platform for a large and distributed scientific community. Gecko has allowed for flexible incorporation of new analysis methods over time, and has insured intelligible access to older, complex analyses, successfully answering the question of "where is my data?".It should be emphasized that the analysis framework afforded by Gecko is general and not limited to gene expression data. Data can be uploaded from many other sources, and the analysis methods relevant to the new data types can also be incorporated as needed. Thus, methodologies for the analysis of protein-protein interaction data, or for All components of the Gecko software, including source code, are being made available as a package under SourceForge.net. The GecInstallation of the complete platform will require manual intervention as well as execution of the automated builds provided in the package. Manual intervention is required for installation of the required external software libraries  as well as for setting up the run-time Gecko infrastructure . The automated builds provide for compilation of the C++ and Java source code, for creation of required flat-file directories, and for the creation of the database tables.Existing installations of the Gecko platform are on Sun Enterprise UNIX servers running SunOS 2.8. Transposition to other operating systems, such as Linux, will thus require some additional "tuning" of components during installation. It should also be noted that the Gecko numerical analysis programs can be used in a standalone fashion (i.e. by execution from the command line), without requiring a complete installation of the platform.JT designed and implemented the early browser-based version of Gecko; he then focused on algorithm and tool development and implementation, alongside giving overall scientific direction for the design of the production version. AU designed and implemented the database. AM designed and built the Java servlets which process user requests, as well as implement many analysis tools and utilities. JC designed and built the client application. DX designed and implemented several statistical analysis algorithms. RN and MH have been involved in running, packaging and in creating automated builds of the Gecko platform. CB had a central role in promoting the Gecko platform and in scientific design input. SB initiated and oversaw development of the production version of Gecko, and was closely involved with the design of the earlier browser-based versions.


Streptococcus mutans colonization and the development of dental caries in non-human primates, and to prevent bacterial colonization in human clinical trials.Common oral diseases and dental caries can be prevented effectively by passive immunization. In humans, passive immunotherapy may require the use of humanized or human antibodies to prevent adverse immune responses against murine epitopes. Therefore we generated human single chain and diabody antibody derivatives based on the binding characteristics of the murine monoclonal antibody Guy's 13. The murine form of this antibody has been used successfully to prevent S. mutans.The antibody derivatives were generated using a chain-shuffling approach based on human antibody variable gene phage-display libraries. Like the parent antibody, these derivatives bound specifically to SAI/II, the surface adhesin of the oral pathogen S. mutans in vitro, making it a useful candidate passive immunotherapeutic agent for oral diseases.Humanization of murine antibodies can be easily achieved using phage display libraries. The human antibody fragments bind the antigen as well as the causative agent of dental caries. In addition the human diabody derivative is capable of aggregating  Streptococcus mutans has been identified as the major etiological agent of the disease. Unlike many other diseases, dental caries is as prevalent in the West as it is in developing countries, and therefore attracts significant interest from medical and dental authorities as well as pharmaceutical companies. The first step in the initiation of infection is the attachment of the bacterium to a specific receptor, and this is an ideal point for intervention. Two groups of proteins from mutans streptococci represent primary candidates for a human caries vaccine: i) glucosyltransferase enzymes, which synthesize adhesive glycans and allow microbial accumulation, and ii) cell surface fibrillar proteins that mediate adherence to the salivary pellicle [S. mutans to the tooth surface. Antibodies recognizing this protein prevent colonization of the buccal cavity by the bacterium and could be developed as a vaccine against dental caries. The most suitable vaccination strategy would be passive immunization, in which monoclonal antibodies or fragments thereof are applied to the tooth surface e.g. using toothpaste, mouthwash or chewing gum. This would make active immunization with the S. mutans adhesin unnecessary.Dental caries is one of the most common infectious diseases of humans. The main causative agent is a group of streptococcal species collectively described as the mutans streptococci . Streptopellicle . The bacpellicle , a surfaS. mutans and Streptococcus sobrinus has been used successfully to prevent S. mutans colonization and the development of dental caries in non-human primates [The murine monoclonal antibody Guy's 13  which spprimates . The antprimates ,7. Howevprimates . The proprimates , CDR graprimates  and guidprimates . FurtherThere has been a growing interest in the use of single-chain fragment variable (scFv) antibodies, in which the variable regions of the heavy and light chains are combined in the same polypeptide chain . The advantages of such derivatives are that they can be expressed as single transgenes in various hosts, they fold spontaneously to adopt the correct tertiary structure, and their small size facilitates tissue penetration. The scFv has the heavy and light chain variable regions joined by a flexible peptide linker allowing the two domains to interact, forming a univalent antibody. Alternatively, diabodies have the same structure but the two domains are joined by a shorter, less-flexible linker, forcing dimerization and the formation of divalent antibodies .We have generated human derivatives of the murine Guy's 13 antibody using a chain-shuffling approach based on human antibody variable gene phage-display libraries. We have taken the variable gene regions of the original Guy's 13 monoclonal antibody and created human scFv and diabody derivatives by chain shuffling in human phage-display libraries. Firstly, the heavy chain variable gene of the Guy's 13 construct was introduced into a naïve human light chain phage display library to select human light chains that, in combination with the murine heavy chain, showed binding specificity for the SAI/II antigen. Once such chimeric antibodies had been selected, the murine heavy chain gene was replaced with the human counterpart, by introducing the selected human light chain genes into a human heavy chain phage display library.S. mutans. The stepwise procedure for generating human antibody chains allows the advantages of scFv and diabody antibody fragments to be exploited without suffering the negative effects of non-human antibodies in a clinical setting. The human antibody fragments were expressed in bacteria as scFv and diabody derivatives and used to aggregate S. mutans in vitro. The diabodies were able to aggregate the bacteria and therefore have the potential to be developed as therapeutic agents to treat and/or prevent dental caries.The resulting clones were expressed in bacteria and tested for specificity in ELISAs using both SAI/II antigen and whole 5. Single chain Fv antibody fragments with appropriate binding activities were selected on purified, immobilized SAI/II antigen. Three rounds of selection were carried out and unique candidate antibodies were identified by ELISA  and a combinatorial library with a complexity of 1 × 106 was established. Three rounds of selection were carried out in solution using SAI/II antigen coupled to paramagnetic beads. Eleven human scFvs were identified by ELISA (data not shown). Subsequent sequence analysis identified three human scFvs: clones huscFv B10, huscFv D12 and huscFv H6. Table L domain in chimeric scFv A6 (Vκ1 HK137) was selected in combination with two different human variable heavy chains, giving human scFvs B10 and H6, respectively. The VH domain of human scFv B10 is homologous to VH1 family DP10, and the VH domain of human scFv H6 is homologous to VH3 family DP35. The human VL domain in chimeric scFv B4 (Vκ1 L12) was selected in combination with one human variable heavy chain giving the human scFv D12. The VH domain of human scFv D12 is homologous to VH5 family DP73. Figure S. mutans. Inhibition ELISA showed that the binding of all three human scFvs to SAI/II was inhibited by Guy's 13, suggesting that epitope recognition was maintained.The selected human VS. mutans cells  and has been expressed in plants as a secretory IgA (sIgA) [S. mutans when coated onto the teeth and gums following eradication of the bacteria. The sIgA is probably the most appropriate format for the topical application of antibodies that inhibit the colonization of the tooth surface by S. mutans because this is the predominant form of antibody naturally found in the saliva. However, each sIgA comprises ten polypeptide chains of four different types making it difficult to produce on a large scale in conventional production systems. The more convenient diabody antibody derivatives, which can be expressed in large quantities in microbial culture systems, may be more suitable for the type of production scales that would be required for the routine control of dental caries using this strategy.Antibodies recognizing and neutralizing the oral pathogen A (sIgA) . In ongoAs a first step in this direction, we have taken the variable gene regions of the original Guy's 13 monoclonal antibody and created human scFv and diabody derivatives by chain shuffling in human phage-display libraries. The heavy chain variable gene of the Guy's 13 construct was introduced into a naïve human light chain phage display library to select human light chains that, in combination with the murine heavy chain, showed binding specificity for the SAI/II antigen. Once such chimeric antibodies had been selected, the murine heavy chain gene was replaced with the human counterpart, by introducing the selected human light chain genes into a human heavy chain phage display library.S. mutans. The stepwise procedure for generating human antibody chains allows the advantages of scFv and diabody antibody fragments to be exploited without suffering the negative effects of non-human antibodies in a clinical setting. Small scFv and diabody antibody fragments are easy to express in large quantities, they penetrate tissues easily and they lack the constant domains that promote often-unwanted and usually superfluous effector functions. However, where such antibodies are murine in origin, they can provoke an immune reaction in the human host, leading to rapid clearance and poor efficacy during long-term treatment. Since dental caries tends to be chronic rather than acute, murine antibodies would be of little benefit to patients in the long term.The resulting clones were expressed in bacteria and tested for specificity in ELISAs using both SAI/II antigen and whole S. mutans.Two drawbacks of scFvs compared to the ideal sIgA format are monovalency and instability. ScFvs are monovalent because the heavy and light chains are joined by a flexible peptide linker, which allows the two domains to fold and interact with each other. We have addressed this problem by converting the scFv antibodies into diabodies, which is achieved by shortening the linking peptide and forcing the heavy and light chain variable domains to seek interaction partners as part of a dimer. As a consequence of this interaction, the diabody is bivalent like the parent immunoglobulin, and therefore has increased binding avidity. We showed that the bivalent binding of the diabody antibody constructs leads to agglutination of The problem of decreased stability may be more difficult to address, because the efficacy of scFv and diabody molecules used to treat dental caries will depend largely on their persistence and effective concentration. Secretory IgAs include a secretory component, which protects the antibody from proteolytic degradation in the saliva. One possible solution is to include this secretory component in any scFv or diabody format through the use of further gene fusion strategies. However, it is envisaged that antibodies for the prevention of dental caries will be administered in the form of toothpaste or mouthwash, or perhaps chewing gum, which will allow the treatment to be refreshed at regular intervals.S. mutans with great specificity, and can successfully aggregate S. mutans cells in the dimeric form in a dose dependent manor. These recombinant therapeutic proteins therefore represent the first step towards an inexpensive and convenient general treatment for dental caries.We have shown that the humanization of a murine monoclonal antibody can be easily achieved using a chain-shuffling approach based on scFv antibody phage display libraries. The human antibody derivatives of the murine Guy's 13 antibody can be expressed and isolated from bacteria, recognize SAI/II and S. mutans 20523 serological group c was purchased from DSMZ  and grown in an S2 containment laboratory in trypticase soy yeast extract medium  at 37°C for 2 d prior to use.spaP gene [SfiI/NotI fragment and inserted into the bacterial expression vectors pCantab5E (Pharmacia) and pSin1 [E. coli TG1. The pCantab5E vector contained an additional sequence encoding the E-tag, facilitating the detection of expressed proteins using the monoclonal antibody 5E (Pharmacia). The pSin1 vector similarly contained sequences encoding a MYC-tag, facilitating detection with the murine monoclonal antibody 9E10 (ATCC CRL 1729), and a His6 tag, allowing purification of expressed proteins by immobilized metal-chelate affinity chromatography (IMAC) and detection using a murine Penta-HIS antibody (Qiagen). SAI/II expressed using pSin1 was used for the selection of antibodies from phage-display libraries. SAI/II expressed in pCantab5E was used for enzyme-linked immunosorbent assays (ELISAs).Nucleotides 214–3048 of the nd pSin1  which haFor the selection of phage-display antibodies, 250 μl of phosphate-buffered saline (PBS)-washed Dynabeads  was resuspended in 500 μl 0.1 M phosphate buffer (pH 7.4) and mixed gently for 2 min. The beads were collected with a magnet, the supernatant discarded and the beads resuspended in 250 μl of the same buffer, followed by the addition of 500 μl SAI/II antigen (1 mg/ml). After incubation for 16 h at 37°C with slow tilt rotation, the beads were collected with a magnet and the supernatant was discarded. The coated beads were washed four times, twice with 0.13 M NaCl, 1% milk powder in 0.01 M phosphate buffer (pH 7.4) for 5 min at 4°C, once with 0.2 M Tris-HCl (pH 8.5) for 4 h at 37°C and again in the same buffer for 5 min at 4°C.SfiI and NotI. The products were inserted into the phagemid vector pSin1, which had been treated with the same enzymes, and the recombinant vector was introduced into E. coli strain TG1.The variable region genes of the heavy and light chain of the murine monoclonal antibody Guy's 13 were amplified using oligonucleotide primers LMB3 (5' CAG GAA ACA GCT ATG AC 3') and fdSeq 1 (5' GAA TTT TCT GTA TG/AG GG 3') followed by digestion with SfiI/SalI fragment in the bacterial expression vector pHenIX containing a light-chain antibody phage-display library derived from naïve human peripheral blood lymphocytes . This vector is based on the phagemid vector pHen1 [E. coli strain TG1.Figure or pHen1  designedThree rounds of selection were carried out using immobilized SAI/II antigen as described by Marks et al. . ElutionApaLI and NotI fragments in pHenIX containing a human variable heavy chain library  and introduced into E. coli TG1. This was achieved by PCR amplification of the human light chain genes using primers Vκ4 ApaLI (5'-TGAGCACACAGTGCACTCGACATCGTGATGACCCAGTCTCC-3'), Vκ1 ApaLI (5'-TGAGCACACAGTGCACTCGACATCCAGATGACCCAGTCTCC-3') and Jκ1 NotI (5'-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATC/TTCCAC/GCTTGGTCCC-3').The selected variable antibody domain genes of the shuffled human light chains were cloned as E. coli TG1 and grown overnight at 30°C on TYE plates containing 100 μg ml-1 ampicillin, 1% glucose. Selection, phage rescue and induction of soluble scFv expression were carried out as described by Marks et al. [Three rounds of selection were carried out using SAI/II antigen immobilized on Dynabeads. Briefly, 150 μg of SAI/II-coated beads was blocked for 1 h with 2 ml 2% milk powder. The beads were collected with a magnet, washed in PBS and incubated with the antibody phage display library for 1 h on a turntable. The beads were washed 15 times with PBS containing 0.05% Tween 20 and 15 times with PBS to remove unbound phage. Bound phage were eluted with 100 μl 100 mM triethanolamine for 10 min on a turntable followed by neutralization in 200 μl 1 M Tris-HCl (pH 8.0). Eluted phage were used to infect exponentially growing s et al. . Antigen-1 ampicillin, 1% glucose) was inoculated with an aliquot of the phage antibody library glycerol stock. The rescue and induction of the phage was carried out essentially as described in Marks et al. [10 units of helper phage VCSM13 (Pharmacia) to the growing phage antibody library. The culture medium was changed to 2× TY containing 100 μg ml-1 ampicillin and 25 μg ml-1 kanamycin and incubated on an orbital shaker overnight at 30°C and 250 rpm. Phage were purified twice by polyethylene glycol (PEG) precipitation  and resuspended in a final volume of 2 ml PBS. The phage were stored at 4°C until further use.One litre of 2× TY . The variable antibody genes from two clones of each restriction pattern were analyzed by PCR cycle sequencing using infrared-labeled primers according to the manufacturer's instructions (Licor). Sequencing reactions were carried out on a Licor automated DNA sequencer (4000 L) and the sequences were analyzed using Sequencher 3.1 (Gene Codes Corporation). The sequences of the VH and VL genes were compared with the germline sequences in the V-BASE database .The number of unique clones was determined by PCR amplification of the recombinant antibody inserts using primers LMB3 (5' CAG GAA ACA GCT ATG AC 3') and fdSeq 1 (5' GAA TTT TCT GTA TG/AG GG 3') followed by digestion with the restriction enzyme The construction of diabodies  was carr4, 1 mM EDTA and incubated for 15 minutes on ice before a final centrifugation step as above. Both supernatants were pooled, dialyzed against PBS and stored at 4°C.Recombinant proteins were recovered from the bacterial periplasm following induction with 0.5 mM final concentration of isopropyl-β-D-galactopyranoside (IPTG) for 3–4 h at 30°C . After c-1 bacto-tryptone, 24 g l-1 bacto-yeast-extract, 4 ml l-1 glycerol) containing 100 μg ml-1 ampicillin and 0.5 mM ZnCl2. The culture was diluted 30-fold in 200 ml of the same medium. When the OD600 nm of the culture reached 2.0, it was supplemented with 0.5 M sorbitol, 4% NaCl, 40 mM glycine betaine and incubated at 26°C for an additional 30–60 min. Expression was induced with 1 mM final concentration IPTG and growth for 6 h at 26°C. Cells were harvested by centrifugation at 30,000 × g for 10 min. The recombinant antibody fragments were isolated from the periplasmic space as described above. The periplasmic and osmotic shock fractions were pooled and dialyzed against PBS. Phenylmethylsulfonylfluoride (PMSF) was added to a final concentration of 1 mM.Recombinant proteins were also expressed in the periplasm under osmotic stress in the presence of compatible solutes as described by Barth et al. . Briefly6 tag as described by Griffiths et al. [280 nm = 1 corresponds to a scFv or diabody concentration of 0.7 mg ml-1.The human scFv and diabody antibody fragments were purified by IMAC using the Hiss et al. . Briefly-1. Aprotinin (6500 Da), cytochrome C , carbonic anhydrase , bovine serum albumin (BSA)  and Dextran Blue  were used as molecular weight standards (Fluka).Gel filtration was used for further purification. A Sephadex 200 column (Pharmacia) was equilibrated with PBS. ScFv or diabody antibody fragments were loaded and run at 1 ml minS. mutans, SAI/II antigen or BSA were coated on ELISA plates (Nunc) at a concentration of 1–10 μg per well in PBS overnight at 4°C. The plates were washed three times with PBS and blocked with 2% milk powder in PBS for 2 h at room temperature. The scFvs were tested either at a concentration of 1 μg per well or 100 μl per well of overnight-induced culture. Recombinant antibodies containing the MYC tag were detected with the murine 9E10 monoclonal antibody (ATCC CRL1729). Antibodies containing a His6 tag were detected using the murine anti-Penta-HIS antibody (Qiagen). The murine antibodies were detected with a goat-anti-mouse (Fc-specific) peroxidase-labeled antibody. The assays were developed with 3,3',5,5'-tetramethylbenzidine (TMB) (Sigma). Reactions were stopped by the addition of H2SO4 after 20 min and readings taken at OD450 nm. Between every incubation step, the plates were washed three times with PBS containing 0.05% Tween 20 and three times with PBS.Inhibition ELISA was carried out by binding of chimeric scFv to SAI/II and replacement with an excess of mAb Guy13. Bound scFv were detected as described above using the murine 9E10 monoclonal antibody detecting the MYC-tag.S. mutans was divided into 20-μl aliquots and incubated with serial dilutions of bacterially expressed recombinant antibodies dia mGuy13 and diaD12, respectively for 2 d at 4°C or 1 h at 37°C on Lab-Tek II chamber slides . As negative control an unrelated human diabody was used. Excess medium was discarded and the cells were air-dried. The bacteria were counterstained with Gram solution (Diagnostica Merck). The slides were mounted with Immunofluor medium  and photographed with a Zeiss Axioskob microscope.Cultured in vitro assays. MH did the upscaled expression of the antibodies. HS assisted in the analysis of data. JKCM assisted in the analysis and interpretation of the data especially in respect to the data obtained with the murine mAb Guy13. SB assisted in the interpretation of the data. RF assisted in the conception and revision of the project. RF* generated the human phage display libraries, developed the concept and supervised the projectMBK generated the diabody formats and performed all