Bartonella species are emerging infectious organisms transmitted by arthropods capable of causing long-lasting infection in mammalian hosts. Among over 30 species described from four continents to date, 15 are known to infect humans, with eight of these capable of infecting dogs as well. B. bacilliformis is the only species described infecting humans in Peru; however, several other Bartonella species were detected in small mammals, bats, ticks, and fleas in that country. The objective of this study was to determine the serological and/or molecular prevalence of Bartonella species in asymptomatic dogs in Peru in order to indirectly evaluate the potential for human exposure to zoonotic Bartonella species. A convenient sample of 219 healthy dogs was obtained from five cities and three villages in Peru. EDTA-blood samples were collected from 205 dogs, whereas serum samples were available from 108 dogs. The EDTA-blood samples were screened by PCR followed by nucleotide sequencing for species identification. Antibodies against B. vinsonii berkhoffii and B. rochalimae were detected by IFA (cut-off of 1∶64). Bartonella DNA was detected in 21 of the 205 dogs (10%). Fifteen dogs were infected with B. rochalimae, while six dogs were infected with B. v. berkhoffii genotype III. Seropositivity for B. rochalimae was detected in 67 dogs (62%), and for B. v. berkhoffii in 43 (40%) of the 108 dogs. Reciprocal titers ≥1∶256 for B. rochalimae were detected in 19% of dogs, and for B. v. berkhoffii in 6.5% of dogs. This study identifies for the first time a population of dogs exposed to or infected with zoonotic Bartonella species, suggesting that domestic dogs may be the natural reservoir of these zoonotic organisms. Since dogs are epidemiological sentinels, Peruvian humans may be exposed to infections with B. rochalimae or B. v. berkhoffii. Bartonella are bacteria transmitted by fleas, ticks, sandflies and other insects capable of infecting humans, domestic animals, livestock and wildlife, including marine mammals. In humans, they cause diseases such as trench fever, cat scratch disease, endocarditis, fever of unknown origin and have been recently associated with neurologic and neurocognitive abnormalities. Bartonella bacilliformis was first described in Peru in 1913, and it has never been detected in animals. Despite the fact that 14 other Bartonella species have been detected infecting humans around the world, no other Bartonella species has yet been described from Peruvian humans or domestic animals. We documented a significant number of healthy domestic dogs in Peru infected or exposed to two Bartonella species , which are known to cause disease in humans. These same species were previously detected in human fleas and dog ticks in Peru, suggesting that vector transmission between dogs and humans may be possible. While the role of dogs as a source of Bartonella species for direct transmission to humans is not well understood, preventive measures including vector control in dogs should be implemented to prevent human infection. Bartonella species are gram-negative bacteria associated with an increasing array of disease manifestations in humans and animals. They are small, obligate intracellular organisms that adhere and invade erythrocytes and endothelial cells of mammalian hosts, causing long lasting bacteremia Bartonella are known to infect humans. Among these, nine species have been documented in dogs, based on culture isolation or DNA-based methods: B. clarridgeiae, B. elizabethae, B. henselae, B. koehlerae, B. quintana, B. rochalimae, B. vinsonii subsp. berkhoffii (hereafter B. v. berkhoffii), B. volans (including volans-like) and B. washoensisB. bacilliformis is the most frequent species of Bartonella in Peru. Humans are considered the reservoir host and infection in animals has not been reported Bartonella have been detected from Peruvian humans to date. However, a new bacteria, B. rochalimae, was isolated in 2007 from an American woman who became sick 16 days after returning from a 3-week trip to Peru, where she received numerous insect bites B. rochalimae has been detected by culture and/or molecular techniques from three asymptomatic rural dogs in California B. rochalimae demonstrated that only dogs became highly bacteremic without any disease expression, suggesting that dogs could be the natural reservoir for this species Bartonella infection in humans due to several factors: exposure to similar household and recreational environments of humans, potential parasitism by the same vectors, wide diversity of Bartonella species identified in canines, development of a strong organism-specific antibody response to many vector-borne pathogens; and accessibility for safe handling and sample collection Bartonella species by defining the serological and molecular prevalence of these pathogens in asymptomatic domestic dogs in various geographic regions of Peru. Additionally, this study sought to define the genetic relationship among Bartonella species, subspecies and strains detected from Peruvian dogs and previously described Bartonella species from humans and other hosts from Peru and other countries. We have demonstrated that Peruvian dogs are exposed to zoonotic Bartonella species, and this study may suggest that the human population is at risk of infection with the same species detected by DNA amplification and genetic characterization.Domestic dogs may represent excellent epidemiological sentinels for Bartonella DNA was detected (data not shown) either lack of isolation of Bartonella or major bacterial contamination was observed. Therefore, the present study only tested canine Peruvian blood samples by DNA-based methods.All animals were humanely treated during sample collection. Dogs were manually restrained during blood withdraw, in accordance with the rules of the Medical Ethics and Animal Care Committee of the Universidad Nacional Mayor de San Marcos (UNMSM), Lima, Peru (no protocol number was assigned), which adheres to the Public Health Service (PHS) policies of the Office of Laboratory Animal Welfare, Office of Extramural Research, National Institutes of Health (OLAW-OER-NIH), USA (PHS approved assurance number A5934-01). In addition, this study was also approved by the Medical Ethics and Animal Care Committee of the Universidad Peruana Cayetano Heredia (UPCH), Lima, Peru (study protocol number 60501), in accordance with the guidelines of the PHS policies, OLAW-OER-NIH, USA (PHS number A5146-01). The purpose of the study was explained to each individual and they were informed that participation was voluntary and data collected were confidential. Informed consent was obtained from all owners of enrolled dogs in the oral format, because some participants were illiterate. In a pilot study of blood cultures of five dogs where Bartonella species. It was conducted in five cities and three small communities located in four distinct districts of Peru  at WesternU. DNA of samples from San Juan de Miraflores, Paita, Huaraz, and Caraz were purified at UPCH using a phenol-chloroform method.Bartonella species was performed as previously described B. quintana strain ND1 (DQ648598) B. quintana per reaction tube 100% of the time (10/10 times). Canine samples with DNA amplification at the expected size were further genetically characterized by the amplification and DNA sequencing of a fragment (approximately 610 bp) of the heat-shock protein gene (groEL) as previously described A conventional PCR assay designed to amplify a fragment (approximately 700 bp) of the 16S–23S ribosomal RNA (rRNA) intergenic transcribed spacer (ITS) of Amplicons were purified from PCR products or from specific bands on the gel  and sequenced with a fluorescence-based automated sequencing system . Chromatogram evaluation, primer deletion and sequence alignment were performed . Bacteria species and strain were defined by comparing similarities with other sequences deposited in the GenBank database using BLAST Bartonella vinsonii subsp. berkhoffii genotype I . Contingency table analyses were performed to evaluate association with age or gender and any difference in prevalence by geographical region by using the Fisher's exact test for 2×2 comparisons and the Fisher-Freeman-Halton test for row×column comparisons. Contingency table analysis was also performed to evaluate the correlation between serology and PCR results by Cohen's Kappa agreement test. A p-value<0.05 was considered statistically significant. Data analysis was performed using JMP Pro 10 .Molecular prevalence of B. rochalimae and 6 of 205 (2.9%) were infected with B. v. berkhoffii .Based on DNA amplification and sequence of the ITS region, 15 out of 205 dogs (7.3%) were infected with rkhoffii . Co-infeB. rochalimae and 43 of the 108 dogs (40%) had antibodies against B. v. berkhoffii by IFA  were seroreactive for B. v. berkhoffii. Dogs seropositive for B. v. berkhoffii were also seropositive for B. rochalimae; however, the frequency of dogs seroreactive for B. rochalimae was higher than dogs seroreactive for B. v. berkhoffii from titer 1∶128 until 1∶2048  had antibodies against i by IFA  using thl 1∶2048 . No statB. rochalimae and B. v. berkhoffii , whereas for B. v. berkhoffii the highest titer detected was 1∶1024 (three dogs). Ninety four dogs had concomitant PCR and IFA results. Among 15 dogs bacteremic for B. rochalimae by PCR, only three had serum available for testing, with two dogs seropositive for this species at titers 1∶256 and 1∶512. Conversely, among 6 dogs bacteremic for B. v. berkhoffii by PCR, only two had serum available for testing, with one dog being seropositive at titer 1∶128  at the time of sample collection, with antibodies against B. rochalimae detected in 71% of 69 adult dogs and 46% of 39 young dogs . Conversely, bacteremia for B. rochalimae was associated with young age (characterized as <1 year old), with B. rochalimae DNA detected in 16% of 50 young dogs and 1.8% of 114 adult dogs . No association was detected between B. v. berkhoffii and age, and no association was detected between sex and serology or PCR results for both Bartonella species.Seropositivity for B. rochalimae isolate obtained from the sick woman (ATCC isolate BAA-1498 Pulex sp.) collected in 1998 from a person in the city of Cusco, Peru B. rochalimae detected from fleas (Pulex irritans) from red foxes (Vulpes vulpes) in Andalusia, Spain Urocyon cinereoargenteus) in northern California (531/534 bp) Multiple DNA sequence alignment and phylogenetic analysis of ITS region from Peruvian dogs  demonstrB. v. berkhoffii was obtained from six Peruvian dogs. Phylogenetic analysis indicated a high homology with DNA sequences from genotype III  was 99.6% homologous (562/564 bp) to a previously reported DNA sequence from another flea (Pulex sp.) collected in 1998 from another human in Cusco, Peru B. v. berkhoffii sequence from Peruvian dogs was 99.7% homologous (619/621 bp) to B. v. berkhoffii genotype III isolate obtained from a human with endocarditis from the United Kingdom A 621 bp sequence of ITS region of type III . SimilarB. rochalimae (JX846497) being 100% homologous (565/565 bp) to the original B. rochalimae isolate obtained from the sick woman Bartonella sp. sequence detected from rat fleas from Egypt Bartonella sp. isolated from an American red squirrel B. rochalimae were not available in GenBank database at the time of writing. A 614 bp sequence of groEL gene was obtained from two out of the six dogs infected with B. v. berkhoffii (JX846496) being 97.2% homologous (549/565 bp) to B. vinsonii subsp. vinsonii isolate obtained from a vole from Canada , and 96.3% homologous (544/565 bp) to B. arupensis isolate obtained from an American cattle rancher with bacteremia  in sick referral or healthy (<1%) dogs in the USA B. v. berkhoffii, modulation of the host immunity by the pathogen, or cross-reactivity with other bacteria. B. v. berkhoffii has co-evolved with domestic dogs and wild canids, and we have previously reported that only 50% of dogs infected with B. v. berkhoffii were seroreactive by IFA testing B. v. berkhoffii were detected in this study is a consequence of the inclusion criteria used, and does not rule out the possibility that sick dogs or humans in Peru may be infected with this pathogen.Additionally, approximately 40% of the dogs in this study were exposed to B. v. berkhoffii documented in the six Peruvian dogs in this study was closely related to genotype III originally detected from an endocarditis case in a human in the United Kingdom and in a dog in the USA Bartonella species, as detected in this study, has been previously described in humans and animals, and it is suggested to be associated with an anergic immune response of the host based on the “stealth” properties of Bartonella species, or substantial antigenic variation among various Bartonella strains, resulting in false-negative IFA assay results B. v. berkhoffii and B. rochalimae. Similar results were recently detected from gray foxes in Texas, where 54.5% of 132 foxes were seroreactive to antigens of B. v. berkhoffii and B. clarridgeiae, which is a suitable antigen marker for B. rochalimae detection B. v. berkhoffii and B. rochalimae cannot be excluded; however, in an experimental infection with these Bartonella species in two dogs, no cross reactivity occurred between host anti-B. v. berkhoffii or anti-B. rochalimae antibodies and antigens from other Bartonella species by IFA (titers ≤1∶16) B. v. berkhoffii was later experimentally inoculated with B. rochalimae, cross reactivity to other Bartonella species was documented Bartonella could substantially increase the serological cross reactivity between species Bartonella species has been documented in several animal species, including dogs and humans The genotype of B. rochalimae or B. v. berkhoffii have never been described in humans or animals from Peru, even though other known and unknown species of Bartonella have been described from small mammals, bats, body lice, fleas and ticks from Peru B. rochalimae and 7.7% of dogs infected with B. v. berkhoffii from Caraz and Huaraz. In that same region, 127 human cases of bartonellosis were documented in 2008 based on serology methods, with the isolation of B. bacilliformis from 11 human specimens B. rochalimae or B. v. berkhoffii, their exposure status to these pathogens is unknown. In addition, culture methods used for isolation of Bartonella in Peru may bias the detection of B. bacilliformis since culture media and temperature used for isolation of B. bacilliformis (28°C) are different from B. rochalimae and B. v. berkhoffii isolation (35°C). Growth differences between isolates of the Bartonella genus have been shown in vitro, where culture medium and temperature differences as low as only 2°C can select the growth of a given species B. bacilliformis has been a well-established human pathogen in Peru for over a century, our data suggest that other Bartonella species may also cause illness in humans in Peru.Infections with B. rochalimae and B. v. berkhoffii obtained from Peruvian dogs matched 100% and 99.6%, respectively, with Bartonella sequences obtained from human fleas (Pulex irritans) previously collected from schoolchildren and adults in Peru Bartonella species for dogs and humans in that country. The human flea is an aggressive and promiscuous ectoparasite found on a wide range of hosts, including domestic dogs B. rochalimae was also recently detected in a brown dog tick  from a dog in the province of Callao B. rochalimae was detected in our study. Both Callao and San Juan de Miraflores are suburbs of the country's capital (Lima), where human and canine population densities and social and economic conditions may favor the multiplication and dissemination of vectors, increasing the risk of human infection. The role of ticks as vector for B. rochalimae or B. v. berkhoffii in dogs or humans is still unclear. However, clinical studies have associated canine infection with B. v. berkhoffii and the presence of R. sanguineus ticks R. sanguineus is the most frequent urban tick in tropical regions, and its ability to feed on humans have been documented, including its role in a recent outbreak of Rocky Mountain Spotted Fever in humans in the United States The fact that the DNA sequences of B. henselae or B. clarridgeiae to humans Bartonella species to humans using similar ways. Bartonella DNA was detected in dog saliva Bartonella infection Cat bites and scratches can transmit B. rochalimae and B. v. berkhoffii, suggesting that asymptomatic domestic dogs in Peru are exposed to these zoonotic pathogens and may be their natural reservoir. Consequently, our results indirectly suggest that the local human population may be exposed to infections with B. rochalimae or B. v. berkhoffii and support public health actions for vector control in dogs and humans.In conclusion, our results expand the current knowledge about Text S1Alternative Language Abstract. Translation of the abstract of “Infection of domestic dogs in Peru by zoonotic Bartonella species: a cross-sectional prevalence study of 219 asymptomatic dogs” into Spanish by author Cesar M. Gavidia: “La infección de los perros domésticos en Perú por especies zoonóticas de Bartonella: Un estudio de prevalencia en 219 perros asintomáticos”.(DOC)Click here for additional data file.


This study was designed to assess the prevalence of Attention-Deficit/Hyperactivity Disorder (ADHD) and Oppositional Defiant Disorder (ODD) in a representative sample of second grade students from a country in a region where no previous rates are available (Turkey). The second aim is to evaluate the differences in ADHD and ODD prevalence rates among four different waves with one-year gap in reassessments.Sixteen schools were randomly selected and stratified according to socioeconomic classes. The DSM-IV Disruptive Behavior Disorders Rating Scale (T-DSM-IV-S) was delivered to parents and teachers for screening in around 1500 children. Screen positive cases and matched controls were extensively assessed using the K-SADS-PL and a scale to assess impairment criterion. The sample was reassessed in the second, third and fourth waves with the same methodology.The prevalence rates of ADHD in the four waves were respectively 13.38%, 12.53%, 12.22% and 12.91%. The ODD prevalence was found to be 3.77% in the first wave, 0.96% in the second, 5.41% in the third and 5.35% in the fourth wave. Mean ODD prevalence was found to be 3.87%.The prevalence rates of ADHD in the four waves were remarkably higher than the worldwide pooled childhood prevalence. ADHD diagnosis was quite stable in reassessments after one, two and three years. A mean ODD prevalence consistent with the worldwide-pooled prevalence was found; but diagnostic stability was much lower compared to ADHD. Attention-Deficit/Hyperactivity Disorder (ADHD) and Oppositional Defiant Disorder are among the most common psychiatric disorders of childhood either in community or clinical samples -3. MoreoAs in any medical conditions, the development of health strategies directed to early diagnosis and treatment of ADHD depends on robust epidemiological data . It is iDespite its clinical relevance, surprisingly very little data is available about ODD prevalence. One possible reason for this is the fact that several studies implemented the evaluation of ODD in combination with CD under a category named “conduct problems”. This situation has its roots in a usual tendency to view ODD under the umbrella of CD. With rare exceptions, the epidemiology, comorbidity pattern and course of ODD were investigated in combination with CD . HoweverStudies on ODD prevalence produced a wide range of rates from 2 to 15% -14. NockOne of the most important gaps in the epidemiology of ADHD and ODD is the fact that age related changes of these disorders were not thoroughly investigated in longitudinal community studies . It is kThis study was conceptualized to provide the following specific data lacking in the literature: Firstly, to find out the prevalence rates of ADHD and ODD in a non-referred representative sample from a country in a region where no previous rates are available (Turkey). Turkey has also a specific importance with an interesting geographical position as a bridge between Europe and Asia. Secondly, to evaluate the differences in ADHD and ODD prevalence rates between four different waves with one-year gap in reassessments. So, the data obtained from the study has the potential of increasing our knowledge on the developmental epidemiology of ADHD and ODD.This study is a 4-year longitudinal ADHD and ODD prevalence investigation. The first year assessment started with a screening phase in April 2008 when the ADHD and Disruptive Behavior Scales were sent to parents and teachers. A study sample was derived at the end of the screening phase and clinical evaluations (screening positive cases and matched controls) were performed in May 2008, configuring the first wave of the study. The sample was reassessed in May 2009, May 2010 and May 2011 in the second, third and fourth waves of the study.th grade), a total number of students of about 155.266 and the total number of 2nd graders was 12.667.This study has been performed in the central district of İzmir, which is the third biggest city of Turkey. The central district of İzmir had a total population of 782.309 inhabitants, 67 government primary schools  was used as a screening instrument in this study. The items in the scale are identical to the list of symptoms described in the DSM-IV criteria for ADHD , ODD (8 items) and CD (15 items). The T-DSM-IV-S was developed by Turgay  and tranT-DSM-IV-S was sent to the parents and teachers of the 1500 randomly selected cases. Cases with completed forms were admitted to the study. The cases with incomplete parent or teacher scales were excluded. Parent and teacher forms of 1455 cases were taken back and considered valid, making the response rate 97%.For the screening of ADHD; students with at least 5 symptoms of inattention and or hyperactivity/impulsivity on both teacher and parent scales were considered to have positive screening for ADHD. This method was called as “and rule” and it is known to be a conservative approach. This cutoff point was reported to have a high sensitivity (82%) and the highest negative predictive power (96%) for the screening of ADHD in a previous study . All posSimilarly, ODD screening relied on the “and rule” method. Students with at least 3 symptoms of ODD on both teacher and parent scales were considered to have positive screening for ODD. The aim was again to decrease the false negative ratio. In addition, some investigators suggested that the threshold of 3 symptoms might be enough for ODD diagnosis ,27. LikeAmong the ADHD screening positive cases (n = 86) and ODD screening positive cases (n = 43), 59 cases were only screening positive for ADHD, 16 cases were only positive for ODD and 27 cases were positive for both ADHD and ODD. The control groups for ADHD and ODD screening positive cases were constituted by screening negative children individually matched for sex and parental education level. For ADHD, all cases both in the study group and with one exception in the control group were enrolled into the study, totalizing 171 cases . One screen negative case was excluded since his parents did not want to participate in the study. In the ODD group, all the study and control group cases agreed to participate in the study, so totally 86 cases were recruited for the study . Since 27 cases were positive for both ADHD and ODD, the total number of cases that were enrolled into the study was 203  Since the sample of cases that matched for sex and parental education with the screen positive cases was larger; a random sample was selected among them to serve as controls.Positive and negative screening cases for ADHD and ODD diagnoses completed clinical assessments that included the Schedule for Affective Disorders and Schizophrenia for School Age Children Present and Lifetime version (K-SADS-PL) . The K-SIn the evaluation of impairment criteria, firstly parents were interviewed about 4 areas: the child’s relationships with his/her sibling(s), friends, ability to do his/her homework and general adjustment at home. In the interviews with teachers, again 4 domains were evaluated: Whether the patient was considered problematic or not, his/her relations with friends at school, his/her general success in subjects and lastly the self-esteem level of the children. The case was considered to be impaired if he/she was rated as very problematic in at least one area or a little problematic in two or more areas. Similar approaches were previously used in other epidemiologic studies ,32.All the original interviews were made blind to the screening status of the cases. “A best estimate procedure” was used to determine final diagnoses . “Best end, 3rd and 4th waves of the study, subjects from both the study and control groups of the first year were called back for reevaluation in the same school settings. All the procedures of the original study except than WISC-R were performed to the study participants by the same researchers in these waves.In the 2In the ADHD group, 3 cases from the study group and 2 cases from the control group could not be reached in the 2nd wave (response rate of the 2nd year of the study was determined to be 97%). In the 3rd wave, same 3 cases from the study group could not be reached again but the two cases of the control group who were not reached in the 2nd wave were reached this time, making the total number of cases evaluated in the 3rd wave 168 (response rate = 98%). In the 4th wave, 5 cases from the study group and 4 cases from the control group could not be reached that decreased the response rate to 94.7%.th wave. Thus the response rates in the 2nd, 3rd and 4th waves of the study were 96.5%, 100% and 94.2% respectively.In the ODD group, 3 cases from the study group could not be reevaluated in the 2nd wave because their parents refused to take part in this wave; making the total number to be 83. However, all the cases including the 3 excluded cases of the 2nd wave agreed to take part in the 3rd wave and the entire original sample were assessed. Three cases from the study group and 2 cases from the control group could not be reached in the 4Prevalence rates of ADHD and ODD were calculated according to a standard formula as suggested by Rohde et al. : Pe = cdThe cases were included in analyses only if both the parent and teacher scales were returned and if they were correctly filled out. Thus out of 1500 children, 1455 (between 8 to 12 years of age) were included in the screening phase and the response rate was found to be 97% .Using a best estimate procedure, 89 subjects were diagnosed as having ADHD  in the wave 1. In the second wave of the study, 68 subjects from screen (+) group and 7 subjects from screen (−) group were diagnosed as ADHD ; in the third wave, 65 subjects from screen (+) group and 7 subjects from screen (−) group were diagnosed to be ADHD  and in the fourth wave, 53 subjects from screen (+) group and 8 subjects from screen (−) group were diagnosed to be ADHD . Prevalence rates in each wave were calculated as the number of cases detected in the diagnostic phase + number of screening negatives that were not assessed in diagnostic phase × /sample size. The prevalence rates were calculated in such a complex way, because simply dividing the number of cases by the sample size would not count the cases that have the diagnosis when the screening was negative. In other words, since no screening test has 100% accuracy, there are always false negatives. The formula presented adjusts prevalence for the performance of the screening instrument. So, ADHD prevalence was calculated to be 13.38% (95% CI = 11.75-15.43) in the first wave, 12.53% (95% CI = 11.02-14.53) in the second wave, 12.22% (95% CI = 10.77-14.18) in the third wave and 12.91% (95% CI = 11.48-16.44) in the fourth wave.For ODD, 31 cases (29 from positive screening group and 2 from negative screening group) were diagnosed to be ODD in the first wave. Since DSM-IV does not allow ODD diagnosis in the presence of conduct disorder; 8 cases with both ODD and CD diagnosis (“inclusive ODD diagnosis”) were subtracted from the total number; leaving 23 cases  with DSM-IV diagnosis of ODD. In the second wave, 22 of the first 23 ODD (+) cases and 61 of 63 ODD (−) cases could be assessed and among these, 9 ODD (+) cases were still found to be ODD. Five new cases from ODD (−) group in the first wave were diagnosed to be ODD in the second wave making the total number equal 14. None of the screen negative cases were diagnosed as ODD in the second wave of the study. In the third wave, all the subjects of first wave were reached and among them 13 subjects from the positive screening group were diagnosed as ODD. In the third wave of the study, 2 subjects from the negative screening group was detected to be ODD making a total number of 15 subjects to be ODD (+) (10 of these 15 subjects had been assessed as ODD (+) in the first wave and other 5 were new diagnosis). In the fourth wave of the study, 9 subjects from screen (+) group and 2 subjects from screen (−) group were diagnosed as ODD  in the first wave).ODD prevalence was found to be 3.78% (95% CI = 3.48-6.33) in the first year, 0.96% (95% CI = 0.95-3.64) in the second year, 5.42% (95% CI = 5.04-8.27) in the third year and 5.35% (95% CI = 4.79-10.91) in the fourth year of the study. When the findings from 4 waves were evaluated concomitantly; 13.21% of the cases were found to be diagnosed as ODD in at least one wave of the study; while 0.21% of the cases were diagnosed to be ODD in all three waves. The mean ODD prevalence of three waves was found to be 3.87%.It was found that 74 subjects among the ADHD (+) cases of first wave (89 cases) were still found to be ADHD (+) in the second wave (3 cases were not reached). One subject who was ADHD (−) in the first wave received the ADHD diagnosis, making the total number of ADHD diagnosed cases 75 in the second wave. Thus 86% of the ADHD (+) cases of first wave were still ADHD (+) in the second wave. In the third wave, 70 subjects among the ADHD (+) cases of first wave were still found to be ADHD (+) (3 cases were not reached). Two subjects who were ADHD (−) in the first wave received ADHD diagnosis making the total number of ADHD diagnosed cases 72 in the third wave. Thus 81.4% of the ADHD (+) cases of first wave were still ADHD (+) in the third wave. In the fourth wave, 57 subjects among the ADHD (+) cases of first wave and 4 cases of ADHD (−) in the first wave were diagnosed as ADHD.Kappa values to estimate test-retest reliability for ADHD diagnosis were found to be: a) 0.84 between the first and second waves; b) 0.78 between the first and third waves; c) 0.62 between first and fourth waves; d) 0.85 between the second and third waves; e) 0.70 between second and fourth waves and f) 0.69 between third and fourth waves  0.37 between the first and second waves; b) 0.40 between the first and third waves and c) 0.22 between the first and fourth waves . Moreover, no significant difference was found between the ADHD (+) and (−) cases and ODD (+) and (−) cases in socio-demographic variables (parent education and neighborhood) in any wave (data available upon request). Finally, no significant difference was found between the ADHD (+) and ADHD (−) cases and ODD (+) and ODD (−) cases in estimated IQs. (Mean estimated IQ for ADHD (+) cases: 78.60 (SD: 26.70), for ADHD (−) cases: 86.06 (SD: 24.68); t = −1.864, df = 164, p = 0.064. Mean estimated IQ for ODD (+) cases: 86.14 (SD: 29.03), for ODD (−) cases: 89.52 (SD: 28.59); t = −0.477, df = 83, p = 0.635).This study is a 4-year longitudinal investigation on the ADHD and ODD prevalence rates conducted in Turkey which is an interesting geographical area standing on the intersection of Europe, Asia and Middle East. It is important to note that these 3 areas have very few investigations on the prevalence of child mental disorders including ADHD and ODD. Our findings on ADHD for the 4 waves  were remarkably higher than the worldwide pooled childhood prevalence of ADHD (6.48%) . On the The five main findings from this study were: 1) A substantially higher prevalence of ADHD in school-age children in Turkey compared to the one reported in a recent meta-analyses including studies from several different countries . This diIn Turkey, ADHD prevalence was not previously studied in extensive investigations, but two studies with diagnostic procedures including only parent and teacher scales reported the prevalence of ADHD in school age children between 6–12 and 6–15 years as 8.1% and 8.4 respectively ,36. It wOne of the most important aspects of ADHD epidemiology is the course of the disorder across the life span. The ADHD prevalence tends to decrease with age and 60% of the cases diagnosed as ADHD during childhood continue to be diagnosed with ADHD in adulthood . But mosOne interesting finding was the fluctuation of ODD prevalence rates among the three waves. It has been known that variations like presence or absence of diagnosis are frequent in longitudinal studies . LavigneWhile the mean ODD prevalence found in this study is very consistent with the literature; distribution of the cases according to sex is quite different than previous literature findings. Female/male ratio of ODD was around 1/10 in all three waves of our study. This is a quite large difference, although statistical significance was not obtained. ODD prevalence was reported to be higher in boys than in girls in most of the previous studies, especially in preadolescent period. Among the previous literature, there is only one study with similar findings to our results in terms of male/female ODD ratio; but nevermore only in a subgroup of the study  . In thatParent education and neighborhood were the only investigated socioeconomic variables in the study and no statistically significant difference was found between controls and both ADHD and ODD groups. In an epidemiological survey from Germany, ADHD was reported to be more frequent among subjects with low socioeconomic status whereas there were no statistically significant differences with regard to geographical characteristics  . It has Our findings should be understood in the context of some limitations. First, this is a regional study of ADHD and ODD prevalence and the results may not generalize to other areas of Turkey. Second, we only included public schools. Therefore, cases found in the top socio-economic group may not be sufficiently represented. Thirdly, socioeconomic status was not extensively evaluated in this study. Finally, we did not assess all possible comorbidities.Our findings describe the ADHD prevalence rates and stability for the first time in a region where no previous study was conducted implementing a careful and extensive diagnostic procedure. Surprisingly, even after using strict criteria with impairment assessments, we found a high ADHD prevalence rate for children at 8 to 10 years of age. In addition, ADHD diagnosis was quite stable in reassessments one, two and three years after. Our results argues against the hypothesis that ADHD is a cultural construct that is uniquely associated with the United States or any particular culture. More studies from regions with no previous epidemiological information on ADHD will improve our knowledge on the real impact of cultural diversity in the disorder.In addition, to the best of our knowledge, this study was the first longitudinal prevalence study which evaluated ODD in particular by assessing it separately from CD. This study was designed with the aim to investigate ODD prevalence in a non-referred school sample, with a well defined and sufficient sample size, by three stage evaluation and by considering impairment criteria and employing best estimate procedure for diagnosis. An ODD prevalence which was very consistent with the worldwide pooled prevalence was found in the study but diagnostic stability was found to be much lower compared to ADHD diagnosis. The results obtained from this longitudinal study should be confirmed with future studies using state of the art methodologies in different regions of Turkey.The findings of the study described the ADHD prevalence rates and stability for the first time in Turkey which is a region where no previous study was conducted implementing a careful and extensive diagnostic procedure. In addition, to the best of our knowledge, this study was the first longitudinal prevalence study which evaluated ODD in particular by assessing it separately from CD. An ODD prevalence which was very consistent with the worldwide pooled prevalence was found in the study but diagnostic stability was found to be much lower compared to ADHD diagnosis.No financial or material support was taken for the study. Dr. Ercan is on advisory boards for Eli Lilly and Janssen. Dr. Rohde was on the speakers’ bureau and/or acted as consultant for Eli-Lilly, Janssen-Cilag Turkey, Novartis and Shire in the last three years . He also received travel awards (air tickets + hotel) for taking part of two child psychiatric meetings from Novartis and Janssen-Cilag. The ADHD and Juvenile Bipolar Disorder Outpatient Programs chaired by him received unrestricted educational and research support from the following pharmaceutical companies in the last three years: Abbott, Bristol-Myers Squibb, Eli-Lilly, Janssen-Cilag, Novartis, and Shire. The other authors declare that they have no competing interests.All authors but LAR contributed equally to the design and conduct of the study, interpretation of the results, and writing of the manuscript. LAR was responsible for the methodology and statistical analysis of the data. All authors read and approved the final manuscript.


The purpose of this study was to investigate the association between Attention Deficit/Hyperactivity Disorder (ADHD) and various factors using a representative sample of US children in a comprehensive manner. This includes variables that have not been previously studied such as watching TV/playing video games, computer usage, family member’s smoking, and participation in sports.This was a cross-sectional study of 68,634 children, 5–17 years old, from the National Survey of Children’s Health . We performed bivariate and multivariate logistic regression analyses with ADHD classification as the response variable and the following explanatory variables: sex, race, depression, anxiety, body mass index, healthcare coverage, family structure, socio-economic status, family members’ smoking status, education, computer usage, watching television (TV)/playing video games, participation in sports, and participation in clubs/organizations.Approximately 10% of the sample was classified as having ADHD. We found depression, anxiety, healthcare coverage, and male sex of child to have increased odds of being diagnosed with ADHD. One of the salient features of this study was observing a significant association between ADHD and variables such as TV usage, participation in sports, two-parent family structure, and family members’ smoking status. Obesity was not found to be significantly associated with ADHD, contrary to some previous studies.The current study uncovered several factors associated with ADHD at the national level, including some that have not been studied earlier in such a setting. However, we caution that due to the cross-sectional and observational nature of the data, a cause and effect relationship between ADHD and the associated factors can not be deduced from this study. Future research on ADHD should take into consideration these factors, preferably through a longitudinal study design. The diagnosis of psychiatric, behavioral, and learning disorders has increased over the past decade. Attention Deficit/Hyperactivity Disorder (ADHD) is one of the most common childhood neurobehavioral disorders in the U.S,2. AccorIn the past 15 years, studies on uncovering the etiology of ADHD focused mainly on the association between ADHD and genetic factors, specifically DRD2 and DRD4 genes. Some othttp://www.nschdata.org.NSCH is a population-based, cross-sectional, random-digital-dialing survey using a complex, multi-cluster, probability sampling design. This suAccording to the DSM-IV, the diagnosis of ADHD requires the child to experience ADHD characteristics such as lack of attention, impulsive behavior, and hyperactivity in at least two different settings, namely home and school; while previous studies also show that the earliest onset age for a child to be diagnosed and treated for ADHD can be below 7 years. As one th percentile), normal weight (≥5th and <85th percentile), at risk of overweight (≥85th and <95th percentile) and overweight (≥95th percentile). To explicate, we refer to the last two categories as overweight and obese, respectively. Other independent variables included in the analysis were sex, race/ethnicity , highest level of education (EDUC) attained by anyone in the household (More than high school vs. High school graduate or less), family structure , dichotomized poverty level based on DHHS Federal Poverty guidelines , healthcare coverage  or Medicaid vs. Not having healthcare coverage), participation in sports , participation in clubs , daily average computer usage (COMP) for purposes other than school work (≥ 1 hour vs. < 1 hour), daily average television watching/playing video games , family member’s smoking status ; this may have resulted in diagnostic misclassification. In other words, as this is not a clinical study, it is unclear how many children who met the ADHD criteria were undiagnosed and/or untreated. Further, the survey question on SMOKE  does not specify whether the child or someone else in the household including parent/guardian was a smoker; results may alter if the smoker in the household was the child him/herself. Also, some bias is expected due to the cross-sectional nature of the study. For example, the survey fails to capture whether the ADHD, DEP, and ANX diagnosis were concurrent or at different time points in the lifetime of the child. Due to these and the observational nature of the study design, the association found in our study cannot be interpreted as causation for ADHD. For example, the association observed between ADHD and the factors SPORTS and CLUBS could be due to the fact that ADHD diagnosed children are just not welcomed on a sport/club teams because of their behavioral problems rather than lack of sporting/physical activity being a risk factor for ADHD. That is, some of the associated factors could be consequences of having ADHD.The results show that the ADHD diagnosed children were most likely from a household having insurance. It is not known how many of the children from the uninsured households may have met the ADHD criteria but were undiagnosed. We performed a sensitivity analysis by analyzing only the insured 5–17 years old. The results were similar as before Table  except fWith the inclusion of the medication effect, the significance of the association of ADHD with TV and SPORTS was lost. This suggests that these associations could be due to a behavior related factor that could be monitored. However, the results from the model utilizing medication effect may not be totally reliable due to limitations in the medication variable as collected in the NCSH. First, the survey question does not collect information about past medication use for ADHD because of which a child who was diagnosed with ADHD in the past and hence took medication in the past would be categorized into the ADHD-NCM group. While this group is supposed to include only those children who satisfy the conditions of having ADHD and not taking medication for ADHD concurrently. This limitation is similar in essence to the one elucidated earlier due to the cross-sectional nature of the survey. Secondly, the unweighted sample sizes for ADHD-NCM 1,690) and ADHD-CM  groups do not add up to the total number of ADHD-diagnosed children (7,137 from Table ,690 and ADHD diagnosis and management has been an important feature of child healthcare over the past few decades. Our study uncovered some new factors associated with ADHD at a national level such as TV, SPORTS, SMOKE, and INS after accounting for many other factors. Our findings suggest that children with ADHD are to be monitored for the above factors in addition to the other known factors. This may help pediatricians diagnose and manage ADHD. Further, after accounting for the ADHD related factors, obesity was not found to be significant, contrary to some previous studies. Future research should be directed towards a longitudinal study designed to examine the association between pharmacological factors, ADHD, and related factors in a concurrent manner.ADD: Attention Deficit Disorder; ADHD: Attention Deficit/Hyperactivity Disorder; ADHD-CM: Attention Deficit/Hyperactivity Disorder and currently taking medication; ADHD-NCM: Attention Deficit/Hyperactivity Disorder and not currently taking medication; ANX: Anxiety; BMI: Body Mass Index; CI: Confidence Interval; CLUBS: Participation in Clubs; COMP: Computer usage; DEP: Depression; DHHS: Department of Health and Human Services; DSM: Diagnostic and Statistical Manual of Mental Disorders; EDUC: Education; FAMILY: Family structure; HMOs: Health Maintenance Organizations; INS: Healthcare coverage; POVERTY: Poverty Level; IQR: Inter-Quartile Range; NSCH: National Survey of Children’s Health; OR: Odds Ratio; POVERTY: Department of Health and Human Services Poverty guideline; S.C: Selected Child; SE: Standard Error; SES: Socio-Economic Status; SMOKE: Family member’s smoking status; SPORTS: Participation in Sports; TV: Watching TV/playing Video games.The authors do not have any financial interests related to this study.None of the authors have any conflict of interest.Ravi K. Lingineni reviewed literature, carried out all data analyses, and drafted the manuscript. Swati Biswas supervised Ravi Lingineni in data analysis, interpretation, and critical re-drafting of the manuscript. Naveed Ahmad helped in the conception and design of the study and revision of the manuscript. Bradford E. Jackson helped in data analysis and drafting of the manuscript. Sejong Bae supervised Ravi Lingineni in analysis using complex survey procedures in SAS and revision of the manuscript. Karan P. Singh supervised the whole group, helped in the conception and design of the study, and revision of the manuscript. All authors have approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2431/12/50/prepub


Attention-deficit/hyperactivity disorder (ADHD) is a common neurobehavioral psychiatric disorder that afflicts children, with a reported prevalence of 2.4% to 19.8% worldwide. Stimulants (methylphenidate [MPH] and amphetamine) are considered first-line ADHD pharmacotherapy. MPH is a catecholamine reuptake inhibitor, whereas amphetamines have additional presynaptic activity. Although MPH and amphetamine can effectively manage ADHD symptoms in most pediatric patients, many still fail to respond optimally to either. After administration, the prodrug stimulant lisdexamfetamine dimesylate (LDX) is converted to l-lysine and therapeutically active d-amphetamine in the blood. The objective of this study was to evaluate the clinical efficacy of LDX in children with ADHD who remained symptomatic  on MPH therapy prior to enrollment in a 4-week placebo-controlled LDX trial, compared with the overall population.In this post hoc analysis of data from a multicenter, randomized, double-blind, forced-dose titration study, we evaluated the clinical efficacy of LDX in children aged 6-12 years with and without prior MPH treatment at screening. ADHD symptoms were assessed using the ADHD-RS-IV scale, Conners' Parent Rating Scale-Revised short form (CPRS-R), and Clinical Global Impressions-Improvement scale, at screening, baseline, and endpoint. ADHD-RS-IV total and CPRS-R ADHD Index scores were summarized as mean (SD). Clinical response for the subgroup analysis was defined as a ≥ 30% reduction from baseline in ADHD-RS-IV score and a CGI-I score of 1 or 2. Dunnett test was used to compare change from baseline in all groups. Number needed to treat to achieve one clinical responder or one symptomatic remitter was calculated as the reciprocal of the difference in their proportions on active treatment and placebo at endpoint.Of 290 randomized participants enrolled, 28 received MPH therapy at screening, of which 26 remained symptomatic (ADHD-RS-IV > 18). ADHD-RS-IV total scores, changes from baseline, clinical responsiveness, and rates of symptomatic remission in this subgroup were comparable to the overall population. The safety and tolerability profiles for LDX were comparable to other stimulants currently available.In this analysis, children with significant clinical ADHD symptoms despite MPH treatment improved during treatment with LDX and experienced similar improvements in their symptoms as the overall study population.NCT00556296ClinicalTrials.gov:  Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) from the American Psychiatric Association ). Mean change in ADHD-RS-IV total score for the overall population was assessed using 2-way analysis of covariance. Dunnett test for multiple mean comparisons with least-squares adjustment was used to compare change from baseline in the 3 active treatment groups versus placebo. NNT to achieve 1 clinical responder or 1 symptomatic remitter was calculated as the reciprocal of the difference in proportions of clinical responders or symptomatic remitters on active treatment and placebo at treatment endpoint.In total, 297 children were enrolled at 40 study sites in the United States, of which 7 children discontinued prior to randomization, and 290 were randomized to receive LDX (n = 218) or placebo (n = 72). Of these, 285 had a postrandomization symptom assessment and were included in the efficacy population. Full demographic data for this population have been previously reported .Of the 290 randomized participants, 28 were receiving MPH treatment at screening and 26 of these were classified as nonremitters on prior MPH at the screening visit, prior to randomization Table . Median Mean (SD) screening, baseline, and endpoint ADHD-RS-IV total scores for nonremitters during prior MPH treatment, nonremitters stratified according to prior MPH dosage received, and overall efficacy population are shown in Figure P = .0063).The mean (SD) change in ADHD-RS-IV total score from baseline with LDX treatment was -24.0 (12.56)  participants receiving LDX and 1 (14.3%) receiving placebo were classified as remitters during the study  treated with LDX and 3 42.9%) treated with placebo, respectively. In the overall efficacy population, 169 (79.3%) treated with LDX and 21 (29.2%) treated with placebo achieved clinical response  morning, afternoon, and evening CPRS-R ADHD index scores at baseline and endpoint in nonremitters on prior MPH are shown in Figure Full safety analyses have been reported previously . In the In this post hoc analysis, LDX showed efficacy when given to children with significant clinical ADHD symptoms despite prior MPH treatment. Efficacy outcomes were similar to the results of the overall population assessed in the clinical trial.Among participants previously treated with MPH, more than half were receiving doses (average daily dose ≥ 1 mg/kg) considered generally effective according to the regimens administered in randomized, controlled trials ,29. ConvDifferential responses to MPH and amphetamine may explain a successful clinical response to LDX in participants who had significant ADHD symptoms despite prior MPH therapy. In 2 separate crossover studies ,32 compaClinical trial design may have contributed to the observed clinical response to LDX treatment in nonremitters on prior MPH. LDX treatment was administered in a forced-dose titration, while prior MPH therapy was provided according to community standards and included potential suboptimal dosing. Use of different definitions of therapeutic response may have altered the rates observed.This post hoc analysis has limitations. The classification of participants as nonremitters on prior MPH considers only the ADHD-RS-IV total score at screening and may not reflect the participants' overall clinical response to MPH. It should be noted that switching from MPH formulations to LDX was done as part of the study protocol and not purely as a clinical practice decision.The 4-week study duration limits the ability to extrapolate the findings to the long-term treatment generally required in managing ADHD. This study was not prospectively designed or powered to detect differences between the treatment groups. A prospective study would be required to confirm these preliminary findings.In this post hoc analysis of children who had significant clinical ADHD symptoms despite previous MPH treatment, LDX demonstrated efficacy and clinical response in the subpopulation assessed. Efficacy outcomes in this population were similar to those in the overall study population.: Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision; ECG: electrocardiogram; ER: extended-release; IR: immediate-release; LDX: lisdexamfetamine dimesylate; MAS: mixed amphetamine salts; MPH: methylphenidate; NNT: number-needed-to-treat; OROS: osmotic release oral system; RCT: randomized controlled trial; SR: sustained-release; TEAE: treatment-emergent AEADHD: attention-deficit/hyperactivity disorder; ADHD-RS-IV: ADHD Rating Scale IV; AE: adverse event; BP: blood pressure; CADDRA: Canadian ADHD Resource Alliance; CD: controlled delivery; CGI-S: Clinical Global Impressions-Severity; CGI-I: Clinical Global Impressions-Improvement; CI: confidence interval; CPRS-R: Short Form: Conners' Parent Rating Scale-Revised; dMPH: dexmethylphenidate; DSM-IV-TRDr Jain or Saundra Jain receives or has received grant research support from Abbott, Addrenex, Aspect, Forest, Lilly, and Pfizer; served as a consultant for Addrenex, Impax, Lilly, and Shire; served on a speaker's bureau for Cyberonics, GlaxoSmithKline, Jazz, Pfizer, Shire, and Takeda; received honorarium from Cyberonics, Forest, Jazz, Lilly, Pfizer, Roche, Shire, and Takeda. Dr Babcock is formerly an employee of Shire and holds stock and/or stock options in Shire. Dr Burtea is an employee of Shire Canada Inc. and holds stock and/or stock options in Shire Canada Inc. Dr Dirks is an employee of Shire and holds stock and/or stock options in Johnson & Johnson and Shire. Mr Adeyi is an employee of Shire and holds stock and/or stock options in Shire. Dr Scheckner is an employee of Shire and holds stock and/or stock options in Shire. Dr Lasser is an employee of Shire and holds stock and/or stock options in Shire.RJwas an investigator on the parent study and participated in data acquisition, analysis, interpretation, and presentation. RJ was fully involved in drafting the manuscript and revising the intellectual content of this manuscript. He has given final approval of this version. TBabcockwas the associate director, Scientific Publications, Clinical Development, and Medical Affairs for this study, and made substantial contributions to the analysis and interpretation of the data. He was deeply involved in drafting the manuscript and revising the intellectual content. He has given final approval of this version. TBurteawas the medical director, Global Clinical Development and Medical Affairs for this study and made substantial contributions to the analysis, and interpretation of the data. He was deeply involved in drafting the manuscript and revising the intellectual content. He has given final approval of this version. BDwas the director, Clinical Development and Medical Affairs for this study, and made substantial contributions to the analysis and interpretation of the data. He was deeply involved in drafting the manuscript and revising the intellectual content. He has given final approval of this version. BAwas a statistician involved in all post hoc data analysis, interpretation, and presentation. Statistician BA was fully involved in drafting and revising the intellectual content of this manuscript. Statistician BA has given final approval to this version. BSwas the associate director, Scientific Publications, Clinical Development, and Medical Affairs for this study, and made substantial contributions to the analysis and interpretation of the data. He was deeply involved in drafting the manuscript and revising the intellectual content. He has given final approval of this version. RLwas the senior director, Clinical Development and Medical Affairs for this study, and made substantial contributions to the analysis and interpretation of the data. He was deeply involved in drafting the manuscript and revising the intellectual content. He has given final approval of this version.


Plantar fibromatosis is a benign disease creating nodules on the medial plantar side of affected patients. While surgical removal is regarded as the therapeutic mainstay, recurrence rates and impairment of daily activities remains substantial. High-energy focussed extracorporeal shockwave therapy has been suggested to be potentially effective in plantar fibromatosis in terms of pain reduction.High-energy focussed extracorporeal shockwave therapy reduces pain in plantar fibromatosis.A total number of six patients  were included with plantar fibromatosis (Ledderhose’s disease) associated with pain. Three patients were operated on previously, one had concomitant Dupuytren’s contracture. High-energy focussed ESWT was applied using a Storz Duolith SD1  in two sessions with 7 days between. Pain was 6±2 at baseline, 2±1 after 14 days and 1±1 after 3 months. Softening of the nodules was noted by all patients. No adverse effects were noted.High-energy focussed extracorporeal shockwave energy reduces pain in painful plantar fibromatosis (Morbus Ledderhose). Further large-scale prospective trials are warranted to elucidate the value of high-energy focussed extracorporeal shockwave therapy (ESWT) in plantar fibromatosis in terms of recurrence and efficacy. Plantar fibromatosis is named after the German surgeon Prof. Dr. Georg Ledderhose following his report on a Dupyutren-like disease of the foot sole in 1897. As it is the case for Dupuytren’s disease of the palm, aetiology remains largely unknown to date[Similar to Dupuytren’s disease of the hand, where in early stages nodules are evident, plantar fibromatosis yields 1-2cm nodules located typically on the medial side of the foot arch. Therapeutically, radiotherapy as well as surgery have been applied in a number of cohort studies and case series with inconsistent results. A cohort study from the Netherlands reported on 27 patients with plantar fibromatosis who underwent 40 operations on 33 feet[Penile fibromatosis referred to as Peyronie’s disease affects middle-aged men between 40 and 60 years with penile pain, curvature during erection and potentially erectile dysfunction. Extracorporeal shockwave therapy (ESWT), which was introduced from urologists in order to destroy kidney stones, has been tested in penile fibromatosis (Evidence level Ib). AnotherGiven the similiarities of penile and plantar fibromatosis in a histological and clinical perspective, we sought to evaluate the effects of high-energy focussed ESWT on plantar fibromatosis in a pilot series, which is presented herein.Given the aforementioned effect in penile fibromatosis, we hypothesize that high-energy focussed extracorporeal shock wave therapy is able to reduce pain in plantar fibromatosis (Ledderhose’s disease).High-energy focussed ESWT was applied without anesthesia using a Storz Duolith SD1 device in two therepeutic sessions with a week interval in between with the following settings Figures:● Number of shockwaves: 2000 impulses● Frequency: 3 Hertz2● Energy flux density 1.24 mJ/mmPatients were allowed to walk or sport following ESWT therapy. All patients documented pain in the morning on a visual analogue scale (VAS 0–10) on a daily basis.A total number of six patients  were included with plantar fibromatosis associated with pain. Three patients were operated on before. High-energy focussed ESWT was applied using a Storz Duolith SD1  for 1–3 sessions. Pain was 6±2 at baseline for at mean 4±2 months and 2±1 after 14 days (p<0,05, FigureHigh-energy focussed extracorporeal shockwave energy reduces pain in painful plantar fibromatosis. Further large-scale prospective trials are warranted to elucidate the value of high-energy focussed extracorporeal shockwave therapy in plantar fibromatosis in terms of recurrence and efficacy.Written informed consent was obtained from the patient for publication of this report and any accompanying images.This pilot case series was in compliance with the Helsinki Declaration. This pilot case series was done with an approved CE-certified medical device and the local ethical committee gave approval as case series.KK has received speaker honoraries by Pfizer and Storz. PMV has nothing to disclose.KK has treated the patients, documented the cases and published it. PMV has co-composed the manuscript and discussed the case. All authors read and approved the final manuscript


Bartonella is a common parasite of voles and mice, giving rise to short-lived (4 weeks to 2 months) infections. Here, we report high sequence diversity in genes of the VirB/VirD type IV secretion system (T4SS), amongst Bartonella from natural rodent populations in NE Poland. The VirB5 protein is predicted to consist of three conserved alpha helices separated by loops of variable length which include numerous indels. The C-terminal domain includes repeat stretches of KEK residues, reflecting underlying homopolymeric stretches of adenine residues. A total of 16 variants of VirB5, associated with host identity, but not bacterial taxon, were identified from 22 Bartonella isolates. One was clearly a recombinant from two others, another included an insertion of two KEK repeats. The virB5 gene appears to evolve via both mutation and recombination, as well as slippage mediated insertion/deletion events. The recombinational units are thought to be relatively short, as there was no evidence of linkage disequilibrium between virB5 and the bepA locus only 5.5 kb distant. The diversity of virB5 is assumed to be related to immunological role of this protein in Bartonella infections; diversity of virB5 may assist persistence of Bartonella in the rodent population, despite the relatively short (3–4 weeks) duration of individual infections. It is clear from the distribution of virB5 and bepA alleles that recombination within and between clades is widespread, and frequently crosses the boundaries of conventionally recognised Bartonella species.The alpha-Proteobacterium The online version of this article (doi:10.1007/s00248-012-0033-y) contains supplementary material, which is available to authorized users. Bartonella includes such significant human pathogens as Bartonella quintana, Bartonella henselae and Bartonella bacilliformis. However the greatest diversity is found amongst rodent-infecting forms; of more than 1,400 citrate synthase (gltA) sequences within GenBank, over 530 are from rodents. Rodents are infected at high prevalence , encoded within pathogenicity islands in the Bartonella genome [Bartonella is similar to the type A T4SS of other gram-negative bacteria [Bartonella, but typically two or three systems are present [trw, responsible for red blood cell infection [virB/virD and vbh, involved in endothelial cell infection [trw system in Bartonella is unique in that several genes within the cluster are duplicated [B. grahamii genome [B. bacilliformis [fe cycle . An initfe cycle , althouga genome , 21. Thebacteria , 23, butbacteria . T4SS ge present ; trw, renfection  and virBnfection , 25. Theplicated , 26. Seqliformis , 21.Bartonella infections. Here, we present evidence of the molecular diversity and mode of evolution of two T4SS genes, virB5 (encoding a putative adhesin involved in host cell invasion) and bepA, located 5.5 kb from virB5  and virb5. The first pair of primers (virB5F: 5′-TGGAATGAATGATGAGATCGCC-3′ and virB5R: 5′-TAAAGTCGGACATCAGAWTTCYCAAG-3′) was used on all isolates but failed to consistently amplify virB5. The forward primer (virB4F: 5′-TACAGTCAGAATTAAGCAGAG-3′) was therefore designed to complement the 3′ end of the virB4 gene , to successfully amplify virB5 from a further 12 isolates. In the PCR reaction, after first denaturation in 94°C for 300 s, genes were amplified with 45 cycles of 94°C for 45 s, 51°C (using primer pair bepAF/bepAR) or 52°C (virB5F/virB5R and virB4F/virB5R) for 45 s, 72°C for 60s, and followed by a single 7-min extension step at 72°C. Sequencing used amplification primers on both strands of purified amplicons  with ABI technology . New sequences obtained in this study were deposited in GenBank under accession numbers: HM450047–HM450063 and JN797608-JN797610 (virB5), and HM450064–HM450072 (bepA); see The field study site represents a complex of forest and abandoned agricultural land in NE Poland, the focus of several previous studies of rodent-pathogen epidemiology , 29–36. lareolus , 36 fromlareolus , 38. Aft.Cs1137n . Further 10 days . Each is 10 days . The supvirB5 gene, it proved difficult to achieve an unambiguous alignment, and therefore three approaches to alignment were adopted. The first was to use standard and Expresso alignment algorithms within the T-Coffee package [rank algorithm via the WebPrank server (www.ebi.ac.uk/). The final approach was by homology modelling using the Escherichia coli TraC protein as a template [virB5 and bepA variation with clade identity or host identity. Analysis of recombination was carried out using the RDP-3 software package [Sequence outputs were checked visually for ambiguous peaks, which, where appropriate, were re-sequenced. Because of indels within the  package . The sectemplate , 44 via template –47). Thetemplate , 49, afttemplate ). Nestedtemplate –53, usin package .gltA sequences were recovered from 154 Bartonella strains isolated over 2 years from four rodent species [B. grahamii (five variants) or to Bartonella taylorii (26 variants), but one isolate of Bartonella birtlesii and three of Bartonella doshiae were also identified. Two variants had recombinant gltA genes [B. doshiae-like at other housekeeping gene loci. The characterisation of virB5 and bepA genes was attempted only for the 42 Bartonella isolates cultured on blood agar. VirB5 was amplified from 30 isolates, bepA from 17; both sequences were obtained from 12 isolates.A total of 37 variant  species . Most cotA genes ; one of virB5 was amplified, 20 were B. taylorii clades, six were B. grahamii, three were B. doshiae (including one recombinant) and one B. birtlesii [gltA gene identity. In all, the inferred VirB5 protein product was between 145- and 173-amino acids (aa) in length; amplicons from B. taylorii and B. birtlesii isolates were clearly shorter than those from non-recombined B. doshiae and B. grahamii isolates . Because of apparent similarities of sequence, the Bartonella elizabethae sequence from GenBank (AF195504) was included in the alignment. The B. grahamii isolate from Uppsala (GenBank accession number NC012846) proved identical to B. grahamii isolates collected in the present work . Homology modelling of Bartonella VirB5 molecules suggested that this protein is made up of three helices, a short helix at the carboxy terminus following the signal peptide, followed by two long helices separated by a loop. The most reliable structures generated were for B. elizabethae and B. taylorii  giving the best estimate . This alignment was therefore used in subsequent analyses. A phylogenetic reconstruction using this alignment divided the 16 basic variants into six clades .Phylogenetic reconstructions using alignments generated using different algorithms did not differ in the shape of resultant trees generated using maximum likelihood (PhyML), although the alignment did affect the maximum likelihood estimates of the trees, with Pte ML = −,769. Thite ML = −,769. Thite ML = −,769. ThiM. arvalis (see below). Subclade B2, from both Microtus species , varied by up to three bases. Subclade B3 isolates from Mi. oeconomus differed by up to two bases, and were sister group to B2, with a characteristic insertion of 6-amino acids  in the carboxy terminus of the protein generated by an imperfect repetition of two sections of DNA (TTAAAA and GAAAAA). Subclade B3 alleles were not recently derived from subclade B2, as there were 16 other mutational changes between these alleles. The fourth subclade B4, consisted only of the isolate Af6. By other phylogenetic approaches (Maximum Parsimony) this isolate is basal to the entire tree, and it is very different in sequence to the other clade B isolates. Its position within this clade is therefore uncertain.Differences between clades were much greater than those within clades, and were due to mutation, recombination and loss/gain of indels. Within clade A, isolates differed by up to two nucleotides; only Af1 (13–14 bases difference from the rest of clade A isolates) could not have been a result of clonal expansion. Clades C and F varied by, respectively, up to five and three bases while both clade G isolates were identical. Clade B was most diverse, with four subclades Fig. . SubcladApodemus and Microtus, respectively), with a breakpoint between positions 73 and 77 of the DNA sequence. Statistical support was high . Visual inspection showed 17 base changes, shared between clade A and the Ma10 isolate but differing from subclade B2, up to position 73 and 73 changes from position 77 to the end of the gene, which were shared between Ma10 and subclade B2 but not with clade A. There were also indels of 18 bases between positions 223 and 240 and of 12 bases between positions 391 and 402, shared between Ma10 and subclade B2 but absent from clade A. Only a single base change (G436T) appeared to have occurred in Ma10 which was not shared with either clade A or subclade B2.The isolate Ma10 from subclade B1 Fig. , proved Differences between clades were due to both mutational change and to indels. Each clade had a characteristic pattern of indels, which could account for up to 100 bases difference between the shortest and the longest sequences  collected from M. arvalis (three isolates), M. glareolus (two isolates) and A. flavicollis (three isolates). The bepA from the fully sequenced Swedish B. grahamii isolate (NC012846), also belonged to this clade. Only two single nucleotide polymorphisms occurred within this group, one silent, the other substituting Q60P  and one from Mi. oeconomus (the B. doshiae with recombinant gltA). Six isolates within this clade had identical nucleotide sequences but one of the M. arvalis isolates (Ma1) differed by a single nucleotide causing an inferred amino acid change . Clade NC012846 ). CladesNC012846 ).Figure virB5 and bepA were successfully amplified, there was no consistent pattern of linkage between alleles  and Af5 (B. birtlesii) shared a bepA allele but exhibited the most disparate virB5 alleles, suggesting that recombination between these loci is common, despite their close proximity  according to housekeeping genes except for a recombinant gltA [virB5 allele.For the 12 isolates where both les Fig. . For exakb apart ). Alleleng genes . There want gltA , had an gltA demonstrated a complex of Bartonella species and clades which recombine with each other to varying degrees ; these two forms could not have resulted from clonal diversification alone. One virB clade C variant (Ma1) was also recovered from B. grahamii, indicating recombination of the whole virB5 gene between these species. VirB5 clade B had the most interesting distribution within the Bartonella network. Most isolates  came from a single gltA variant within B. taylorii clade B gltA demosee Fig. , and werBartonella genome is extensively reshaped by recombination, both at a genomic scale [virB5 analogue at the trw locus to demonstrate duplication and recombination within a single clade of B. grahamii, identifying potentially recombinant stretches derived interspecifically (B. henselae sequences within the B. grahamii and Bartonella tribocorum genes) or from an unknown donor. Here, we present the first account of virB5 evolution in a natural Bartonella population and show the relative importance of recombination and mutation in the evolution of this gene. Part of the pressure for recombination is thought to come from selection for optimal combinations of invasion genes, particularly through microevolution of adhesins such as BadA [virB5  is linkE. coli TraC protein is a prototype [Bartonella VirB5 protein is much shorter (145–177 aa), consisting of one short and two long helices. The second long helix, and the C terminal domain, both contain numerous charged residues and are the most variable parts of the molecule, including numerous indels. Although Backert et al. [Bartonella VirB5, as the fibronectin recognition domain of the three-helix VirB5 molecules is absent [Bartonella VirB5 protein does suggest a role in intra-molecular interactions. This could be related to the binding of the VirB5 molecule into the pilus formed by the T4SS [B. henselae VirB5 binds strongly to VirB3 [Agrobacterium this protein is an integral part of the pilus structure [Bartonella infections, being characterised as the 17 kDa antigen in clinical studies [Bartonella is exposed on the outer surface of the pilus, where it has a species-specific conformation. In its alternation of runs of charged lysine and glutamate residues, VirB5 in Bartonella shows interesting parallels with the immunologically relevant glutamate-rich merozoite surface proteins of Plasmodium . It. ItE. co evasion .virB5 in B. taylorii has clearly taken place through homologous recombination, and at least one (subclade B1) of the seven virB5 clades identified in this work is unambigiously derived by recombination from two others (B2 and A). The double duplication of 3-amino acids in subclade B3 is presumably due to misalignment of the repetitive (CTGAGAAA) motif between partner chromosomes during a recombinant event. The longest VirB5 molecules, those of B. grahamii and B. elizabethae have also evolved by duplication of short segments, with an insertion of 9-amino acids built up partly from a (GAAAAA) repeat. A second, imperfect (GCTTCAA) repeat (n = 3) gives a further insertion of 12-amino acids. Imperfect copying of partial repeat regions within virB5 appears to have been an important mechanism for the microevolution of this gene. The virB5 gene has numerous poly-A tracts, up to a maximum of A8, at which slippage during replication can occur [Evolution of an occur . This prvirB5 gene between B. taylorii and B. grahamii. This corresponded exactly with exchange of the housekeeping gene groEl between the same two clades of B. taylorii and B. grahamii [virB5 variants showed little or no correlation with the patterns of gltA alleles present in the Bartonella isolates, further suggesting widespread recombination between isolates. However, the distribution of virB5 variants correlated much more closely with host identity than did gltA. Recombination on a large scale in the Bartonella genome has been obvious since the comparisons of Frank et al. [B. bacilliformis [B. grahamii [virB5 homologue (trw), in this case inferred from a phylogenetic context. There was little evidence in the present work for linkage disequilibrium between virB5 and the bepA gene, and phylogenies based on the two genes are quite different, although these genes are only 5.5 kb apart [Bartonella gene transfer agent (GTA) [Bartonella species possess this GTA, as evidenced by the presence or absence of a 14-kb extra-chromosomal band in whole genomic DNA preparations. B. grahamii does possess this GTA  . Barbiannt (GTA)  indicated et al. ), but BaBartonella isolates in rodents and the epidemiological situation in Helicobacter, with a difference in the temporal scale of persistence of variants. In H. pylori, novel antigenic variants [H. pylori for perhaps 30 years. These variants allow the H. pylori infection to escape from host immunological surveillance [Bartonella in rodents, individual infections are transient, lasting only 4–11 weeks [virB5, a molecule known to be antigenically relevant, probably allows numerous Bartonella isolates to circulate within the wider rodent population; temporal persistence in this case is replaced by spatial persistence. Clearly, the role of the arthropod vectors in generating and maintaining allelic diversity through recombination in Bartonella is important, and requires further elucidation. This is particularly important given the reputation that rodent Bartonella isolates are acquiring as emerging human pathogens [The present work demonstrates a close parallel between variants , 67, 68 eillance . In the 11 weeks , 71, in athogens .ESM 1(DOC 51 kb)Below is the link to the electronic supplementary material.


If current trends continue, Ghana's aged population will increase in the coming decades. Currently, there is little knowledge on the health of the aged in Ghana. Research on vision problems among this group is virtually non-existent. This research gap needs to be filled immediately in order to promote the general health among older people in Ghana.The objective of the study was to analyse vision health and its determinants among the older adult population in a district in one of the poorest regions in Ghana – the Kassena-Nankana district.Data were obtained from the WHO multi-country studies unit (SAGE). A total of 4,294 people over the age of 50 responded to the survey. Data analysis was conducted using Stata statistical package. The aim of the analysis was to identify the prevalence of self-reported vision problems and assistive device use. Age, level of education, marital status, living arrangement, socio-economic status and proportion of people aged 50 and over in a household were used as determinants of vision health.p-value, 0.00) of men and women reported having far-sightedness, while 35% of men and 40.6% of women reported having near-sightedness . In total, 33.5% of men and 38.6% of women reported having both near-sightedness and far-sightedness . Of those who reported having either vision problems, 2.9% reported the use of visual assistive devices. Men had a higher assistive device use of 4.5% compared to 2.1% among women (p=0.002). Age and household socio-economic status was positively associated with reporting vision problems and assistive device use, respectively.In total, 54 and 63% (The results from this analysis showed that despite the high reporting of vision problems, only 2.9% reported using assistive devices. This outcome shows that there is a need to prevent vision problems and increase access to assistive devices among older people in the Kassena-Nankana district in Ghana. The United Nations observed 1999 as ‘the year of the older persons’ . OsteoarIn 2000, Africa, the ‘youngest continent’ had 4.3% of its northern population considered to be aged. This number was projected to increase to 5.3% in 2015 and 8.1% in 2030. Sub-Saharan Africa has some of the smallest proportions of aged person in the world. In 2005, there were 34 million people aged 60 and above, this number was projected to increase by 50% by 2030. The expected 50% increase in the ageing population makes it the most rapidly ageing region in the world  compounds, 1 mission health post, 1 private clinic, 2 nutrition centres, and several drug stores. Similar to many places in the country, there is a shortage of health professionals in this region. In 2009, It had a patient-to-doctor and patient-to-nurse ratio of 35,010:1, and 805:1 respectively .The data for this study were obtained from the WHO multi-country studies unit (SAGE) . This stThis study is an extension of the SAGE at the International Network for the Demographic Evaluation of Populations and Their Health in Developing Countries (INDEPTH) site in Ghana. Data on household and individuals aged 50 and above were collected from an already established health and demographic surveillance field site as part of (INDEPTH). Using the demographic information as a sampling framework, a single random sample of 6,074 people over the age of 50 was selected to be interviewed. A 75% response rate  resultedThe vision health questionnaire consisted of four questions and was divided into two groups of related questions. The first two questions focused on the use of vision assistive devices for far-sightedness and near-sightedness. Using a five-point scale, the last two questions measured far-sightedness and near-sightedness by asking respondents about difficulty experienced seeing from far and from near in the past 30 days.The five-point scales were then categorised into ‘No problem’ and ‘With problem’ for analysis. People who reported having no difficulty in seeing objects from far or near were grouped into the ‘No problem’ group. The remaining groups were categorised as ‘Have problem’. Similarly, the variable ‘Either problem’ represented any level of near-sightedness or far-sightedness. Analysis of assistive device usage was conducted among the ‘Have problem’ group only. Only significant results were discussed in the analysis.Age in this study referred to the chronological age of the respondent. It was further categorised into 50–59, 60–69 and 70 and above age groups for analysis. Sex and marital status were self-identified and categorised into male, female, married, unmarried, respectively. Education represented the number of years of formal education received. It was categorised into six years or less and six years or more. Household economic status represented the household wealth based on possessions and housing characteristics. It was categorised into: poorest, poorer, poor, less poor and least poor. The proportion of older people in the household represented the proportion of people in the house aged 50 years or above. This variable was divided into: ≥75, 50–74, 25–49, and <25%, respectively.The statistical package used for the analysis of this study was STATA. In descriptive studies, the distributions of study subjects across different socio-economic and demographic groups were presented. Each analysis was conducted with regard to near-sightedness and far-sightedness and sex. Results and discussions focused on significant findings.Women represented 62% of the respondents, while men represented only 38%. The average age of men, women, and the total populations were 63.8, 61.8, and 62.6, respectively. There were more men aged over 70 years compared to women of the same age group (27% vs. 17.8%). About 95% of the population lived with someone. Compared to women, a higher proportion of men were more educated, in current relationship and living with another. About 58% of men belonged to 40% of the poorest households compared to 50% of women in the same category. The majority of men and women 77.7 and 73.2%, respectively resided in households, where the proportion of people over the age of 50 was below 50% .More women reported experiencing vision problems. About the same proportion of men and women reported having experienced severe to extreme problems with far-sightedness and near-sightedness. Among those who were classified under ‘have problem’, there was a higher proportion in the mild and moderate groups. This is true for both men and women in both vision problems. A higher proportion of men compared to women reported having experienced ‘no problem’ with both far-sightedness and near-sightedness 66.5% vs.61.4% .Compared to the lowest age group 50–59, the oldest males and females (80 and above) had about 9 and 10 times higher odds of reporting far-sightedness. Age was positively associated with reporting far-sightedness and near-sightedness in both men and women although women had a slightly higher odds ratio in most stratums. The oldest age groups were about five times more likely to report near-sightedness. There was a steady increase in the odds ratios with age at a similar rate for both men and women. Single people had increased odds of reporting near-sightedness compared to those in a current relationship. Men had higher odds of reporting near-sightedness compared to their female counterparts .Nearly 2.9% of those who reported having vision problems used an assistive device. The proportion of men using visual aids was about double that of women 4.5% vs. 2.1%, respectively. Visual aids usage was higher among those who reported having experienced near-sightedness. Men with near-sightedness had the highest usage while women with far-sightedness had the lowest usage .Socio-economic status was significantly related to the use of an assistive device for both near- and far-sightedness and amongst both sexes. Women in the ‘less poor’ category had some of the lowest use of assistive device for near-sightedness (0.9%) and far-sightedness (1.1%). Among men, the lowest prevalence of use of 1.2 and 0.7% was observed among men in the poorer groups in both near-sightedness and far-sightedness. The highest percentage of use was observed among the ‘least poor’ category. Within this group, men had a higher percentage of use compared to women for far-sightedness (16.7% vs. 7.5%) and near-sightedness (18.8% vs. 11.3%) .This study observed that 33.5% and 38.6% of men and women, respectively reported having either near-sightedness or far-sightedness. Among those who reported having vision problem, 2.7% reported using assistive device. Men generally reported less visual problem but higher use of assistive device compared to women. The highest rates of assistive device usage were found among men, those with more higher education and high socio-economic status. The main explanatory variable for reporting vision problem was age. Assistive device usage was best explained by the household socio-economic status. The above results appear larger than the 22.3% and 18.4% far- and near-sightedness found in a similar study in Nigeria   study, cThe study found that 2.9% of those who reported having any vision problem used an assistive device. In total, 2.5 and 3.5% of those who reported far-sightedness and near-sightedness, respectively, reported using assistive devices. These numbers are low compared to the Nigerian study, which reported 31.5% and 28.7% assistive use for far-sightedness and near-sightedness, respectively (This finding suggests that the average person in the study did not have access to assistive devices or could not afford the cost of 10–20 USD for assistive device. Given that the study was conducted in the poorest region of Ghana could explain why so few of the visually impaired used assistive devices.The study may be generalised when you consider that declining to respond to the survey occurred at random. As a result of the categorisation of variables such as age, socio-economic status, questionnaire response and proportion of 50+ in a household, there were very few people in some groups, that is a small increase in absolute numbers resulted in higher percentages. Prevalence could have been different if data from the five-point scale questions were categorised differently. Recall bias can be a risk to the quality of the data as the event of interest was not severe and the time frame of 30 days might be long for some respondents.Similar to many developing countries, there is a steady increase in the number of aged person in Ghana. However, there is very little available data about the health of the aged in this country. To prepare the country for the challenges associated with an ageing population, Ghana must have an on-going surveillance system that will monitor the changing demography and its impact on health. The National health insurance scheme will need such information in order to budget for the coverage of the cost of health care for the elderly. As a member of the WHO multi-country SAGE, Ghana is able to have an idea of the ageing situation in this INDEPTH site. Visual impairment can inhibit the functionality and independence of the aged, thus requiring health care and assistance from the government and family. If vision care is provided for older persons, they can remain functional and independent for a long time. This study has highlighted age, sex, and socio-economic status as sources of health inequities in Ghana. Health care in Ghana is the responsibility of the government. Therefore, it is important to not only provide its people with health care but also to introduce social programmes that reduce inequitable disparities in health.