The primary objective of this study was to determine the prevalence of neurocognitive impairment among HIV-positive individuals in Botswana, using the International HIV Dementia Scale (IHDS). We also compared performance on the IHDS with performance on tests of verbal learning/memory and processing speed, and investigated the association between performance on the IHDS and such variables as depression, age, level of education and CD4 count.We conducted a cross-sectional study of 120 HIV-positive individuals randomly selected from an outpatient HIV clinic in Gaborone, Botswana. Patients provided a detailed clinical history and underwent neuropsychological testing; measures of depression, daily activities and subjective cognitive complaints were recorded.Despite the fact that 97.5% of subjects were receiving highly active antiretroviral therapy (HAART), 38% met criteria for dementia on the IHDS, and 24% were diagnosed with major depressive disorder. There was a significant association between neurocognitive impairment as measured by the IHDS and performance on the other two cognitive measures of verbal learning/memory and processing speed. Level of education significantly affected performance on all three cognitive measures, and age affected processing speed and performance on the IHDS. Depression and current CD4 count did not affect performance on any of the cognitive measures.3 or below. The contribution of other factors should also be considered, such as poor central nervous system penetration of some antiretrovirals, drug resistance, potential neurotoxicity, and co-morbidities. Memory impairment and poor judgment may be underlying causes for behaviours that contribute to the spread of HIV and to poor adherence. It is important to identify these neurobehavioural complications of HIV so that effective treatments can be developed.The prevalence of neurocognitive impairment in HIV-positive individuals in Botswana is higher than expected, especially since almost all of the subjects in this study were prescribed HAART. This suggests the need to reconsider the timing of introduction of antiretroviral therapy in developing countries where HAART is generally not administered until the CD4 cell count has dropped to 200/mm Individuals with HIV infection often experience neurological complications, including cognitive deficits, referred to as HIV-associated neurocognitive disorders (HAND). The specific pattern of neuropsychological deficits is attributed to damage to the fronto-striatal circuitry, which is commonly observed in HIV infection -3.In recent years, HIV-related neurological disease has been increasingly recognized in resource-limited settings. One of the first multi-centre international studies was carried out with HIV-positive symptomatic and asymptomatic patients and HIV-negative subjects by the World Health Organization (WHO) in five countries: Thailand, Zaire, Kenya, Brazil, and Germany. Researchers found that neuropsychological impairment of symptomatic HIV-positive individuals ranged from 13% in Brazil to 19% in Zaire .Since this study, there has been growing concern about the impact of HIV subtypes on disease progression. In Uganda, where clades A and D are the predominant subtypes, 31% of HIV-positive subjects met criteria for dementia, 47% mild cognitive impairment, and 22% no impairment on neuropsychological testing. The authors concluded that HIV dementia in the Uganda sample might be similar to the frequency of HIV dementia in the US in the pre-highly active antiretroviral therapy (HAART) era . A folloHowever, some researchers have suggested that the incidence of HIV dementia may vary due to different viral clades . For exaSimilarly, a study of HIV-positive subjects in southern India, where clade C is most common, found 60.5% had mild to moderate cognitive deficits in the domains of verbal fluency, working memory, and learning/memory in subjects who did not have clinically identifiable functional impairment . Most reCognitive impairment and depression frequently coexist in HIV . DespiteTo address these questions, this preliminary study focused on three objectives. The first was to explore the use of the IHDS in HIV-positive individuals in Botswana to measure the prevalence of neurocognitive impairment. Recent guidelines  support Participants were 120 randomly selected HIV-positive subjects (60 women and 60 men). They were asked to participate in this study during routine follow-up visits from March to May 2008 at the Infectious Disease Care Clinic at Princess Marina Hospital, a 550-bed tertiary referral hospital in Gaborone, which serves southern Botswana. Control subjects were not included because this pilot study was focused on determining the feasibility of adapting and validating neuropsychological tests and depression inventories used in developed countries for HIV-infected individuals in Botswana. No reimbursement for participation was offered.3 or below. Thus, most patients in this study had a history of very low CD4 counts since 97.5% were prescribed HAART.Inclusion criteria were: (1) documented HIV-positive status; (2) age of 21-50; (3) ambulatory status; (4) the ability to comprehend study procedures; and (5) provision of informed consent. Consistent with current treatment practices in Botswana at the time of this study, subjects started treatment with HAART when their CD4 cell count dropped to 200/mmExclusion criteria eliminated individuals with cognitive impairment unrelated to HIV, such as: (1) neurological conditions ; (2) medical illness unrelated to HIV  or severe HIV-related disease (current opportunistic infection); (3) current fever; (4) severe psychiatric disorder ; (5) a history of substance abuse; and (6) inability to function independently.Participants underwent a standardized neuropsychological examination and assessment of depression, activities of daily functioning, and subjective cognitive complaints. Details of the depression findings are reported elsewhere . AssessmStructured interviews and chart reviews were performed to obtain information about medical and psychiatric history, pattern of substance use, marital status, education, current medications, recent CD4 counts and viral loads. Viral load testing was performed using the Amplicor HIV-1 Monitor Test . Viral load measurements have been categorized dichotomously (detectable vs. undetectable) using a threshold of 400 copies/ml. The investigators evaluated neuropathy using a targeted history composed of structured validated questions designed to elicit symptoms of distal sensory loss or pain.This research study was approved by the Institutional Review Boards of the Botswana Ministry of Health, Princess Marina Hospital, and the University of Pennsylvania. All consent forms, questionnaires, and tests were translated into Setswana and back translated. For patients with limited reading ability, the examiners orally administered the Activities of Daily Living (ADL) Scale.The test battery was selected to assess multiple cognitive-motor ability domains that have repeatedly been shown to be affected by HIV-associated brain disease in both developed and developing countries.The IHDS is a screening measure of neurocognitive impairment that includes: memory registration for four common objects; motor speed involving the rapid tapping of the thumb and first digit of the non-dominant hand; speed of information processing and/or executive functioning measured by repetition of a three-position alternating hand sequence; and memory recall of the four objects . A priorThe World Health Organization-University of California Auditory Verbal Learning Test (AVLT) is a list-learning task that assesses verbal learning and memory with 15 words carefully selected to be familiar in most cultures . Scores The Digit Symbol (DS) Coding is a paper-pencil measure of processing speed in which subjects use a key of digit-symbol pairs at the top of the test page and are required to fill in the correct symbol for each number as quickly as possible within a 120-second time limit. The score is the number of correct items completed within the time limit [The Mood Module (MM)  is a focThis questionnaire was adapted from the original measure and selected for its wide use and demonstrated validity in studies of medically ill and dementia populations, including HIV . It is aThis is a brief self-report questionnaire of subjective cognitive complaints with specific items for four cognitive domains: memory, concentration, speech and thinking. This simple questionnaire asked patients to rate the amount of difficulty they were experiencing on a scale of 0 to 2, as follows: 0 no problem; 1 yes, but currently absent; 2, yes, currently present. Scores for each cognitive domain were added together for a total subjective cognitive score, with a maximum possible score of 8. This questionnaire was previously used in a cross-cultural study that assessed cognitive complaints in HIV-positive individuals in five different countries .We performed descriptive analyses and comparisons to examine the demographic and clinical characteristics of patients. All analyses were conducted using StataMP 10.0 . Correlation coefficients were used to assess relationships between continuous variables. Student's t-tests and analysis of variance were used for comparisons of normally distributed variables. Kruskal-Wallis rank was used for non-normal variables, and Fisher's exact tests were used for categorical variables. The relative importance of dementia on cognitive outcomes, adjusted for demographic , and HIV  characteristics, was assessed using regression models. As this was an exploratory study, alpha was set at 0.05 to determine statistical significance.Demographic and clinical characteristics of the cohort are described in Table Using a cut-off score of 9.5 or less on the IHDS, 38% of the patients met criteria for neurocognitive impairment Table . SubjectCurrent CD4 count (p > 0.445), gender (p = 0.556), substance use (p = 0.194) and employment status (p = 0.419) were not associated with neurocognitive impairment. Language of test administration was not associated with performance on the IHDS (p = 0.613). There was no association between peripheral neuropathy and neurocognitive impairment (p = 0.389). In addition, there was no association between neurocognitive impairment and length of time since HIV diagnosis (p = 0.528). However, subjects with neurocognitive impairment had been prescribed HAART for longer than those without impairment (p = 0.026).Subjects classified with neurocognitive impairment were characterized by increased age (p = 0.008) and had fewer years of education (p = 0.035). Looking at the three subtests of the IHDS, increased age (p = 0.039) and those with less years of education (p = 0.002) had slower motor speed (finger tapping). Similarly, information processing speed  was adversely affected by increasing age , but level of education did not affect performance on this subtest (p = 0.195). Performance on the memory subtest was not associated with age (p = 0.381) or education (p = 0.778). Because of the effect of education, we re-analyzed the IHDS data excluding subjects with no education. With this adjustment, 35% of subjects met criteria for neurocognitive impairment.MDD was diagnosed with the MM in 24% of subjects, but depression was not significantly associated with the total score on the IHDS (p = 0.620) or scores on any of the subtests of the IHDS .Age was not significantly associated with verbal leaning/memory with the AVLT . Similarly, gender , substance use , employment status , CD4 count , time since beginning HAART , and depression  were not associated with verbal learning/memory.Level of education was not associated with total learning (TL) on the AVLT (p = 0.758), but was significant for delayed recall (DR) (p = 0.027) and did not quite reach statistical significance for recognition (R) (p = 0.053). Language of test administration was not associated with AVLT TL (p = 0.065), but those tested in Setswana had higher DR scores (p < 0.001), and subjects tested with a combination of both languages had lower R scores than those tested in either English or Setswana alone (R p = 0.009).In fully adjusted analyses  for AVLT TL, neurocognitive impairment did not reach statistical significance . However, due to the limited sample size in this pilot study, we also reduced the final model down to only significant effects. In this final model, neurocognitive impairment and language of test administration were significantly associated with AVTL TL. Subjects with neurocognitive impairment tended to have four-point lower TL scores , while subjects tested in English had four-point lower TL scores than subjects tested in Setswana .In fully adjusted models for AVLT DR, both neurocognitive impairment and language of test administration still significantly impacted the scores. Subjects with neurocognitive impairment tended to have 1.5-point lower DR scores , while subjects tested in English had two-point lower DR scores than subjects tested in Setswana .There was an inverse relationship between a subject's age and processing speed ; as the subjects' ages increased, they performed significantly slower than their younger counterparts (p < 0.001). Similarly, subjects with less years of education were also slower (p < 0.001); for each additional year of education, subjects completed an average of 2.3 additional items. Males were significantly slower than females (p = 0.032), and subjects tested in Setswana were slower than subjects tested in either English (p < 0.001) or both English and Setswana (p < 0.001).Processing speed was not affected by substance use (p = 0.194), employment status (p = 0.470), CD4 count (p = 0.197), or time since beginning HAART (p = 0.656). In addition, there was no observed association between processing speed and depression (p = 0.345). Twenty-six percent of subjects reported symptoms of peripheral neuropathy, but there was no association between those with and without these symptoms for processing speed on the DS (p = 0.865).In fully adjusted models, neurocognitive impairment and education were the only characteristics associated with DS. Subjects with fewer years of education were slower, with a decrease of two points for each year of missed education . Subjects with neurocognitive impairment had DS scores that were seven points lower on average than subjects without neurocognitive impairment .Individuals who met criteria for a MDD on the MM reported greater levels of impairment (p = 0.044) on the ADL Scale, but classification of neurocognitive impairment with the IHDS was not related to ADL scores (p = 0.334). Similarly, speed of information processing (DS p = 0.395) and learning/memory  were not associated with ADL scores. There was no association between the ADL score and CD4 level (p = 0.949), age (p = 0.482) or education (p = 0.081). Similarly, there was no significant association between neurocognitive impairment and subjective cognitive complaints .Eighty percent of subjects in our sample did not have a detectable viral load . Thus, it was not feasible to analyze the data as a continuous outcome. We did look at viral load as a dichotomous outcome of yes/no detectable viral load. However, there were no significant associations with this variable, even at a bivariate level.The prevalence of neurocognitive impairment in our study was higher than anticipated. Most of our subjects were receiving treatment with HAART, thus we did not expect so many to meet criteria for neurocognitive impairment on a screening measure, such as the IHDS. Yet it is important to note that the high prevalence of neurocognitive impairment in this study may reflect our use of a tool, such as the IHDS, which requires further refinement. Given the association between demographic variables and performance on the IHDS, future studies in Botswana should establish demographically adjusted normative standards for the IHDS, as has been done for a similar screening measure, the HIV Dementia Scale, in the United States .A study in Uganda using the IHDS found HIV dementia in 31% of subjects, but in that study, less subjects were receiving antiretroviral treatment . Furtheret al hypothesized that clade C virus, which is most prevalent in Ethiopia, may be less neurotropic than clades A and D, which are predominant in Uganda [For example, in Ethiopia, the IHDS did not detect impairment in untreated HIV-positive subjects, and Clifford n Uganda . HoweverFurthermore, the relatively high prevalence of neurocognitive impairment in HIV-positive subjects found in our Botswana study, a region where clade C HIV predominates, is consistent with several Pacific Rim studies. Authors of one Pacific Rim study concluded that that clade C HIV is no less neurotropic than other clades . In addiNeurocognitive impairment and depression are independent complications of HIV. For example, although 24% of our subjects were diagnosed with MDD, depression did not affect the total score or any of the three subtest scores of the IHDS. Similarly, depression was not significantly associated with verbal learning/memory (the AVLT) or processing speed (DS Coding), which is consistent with prior studies showing that depression and cognitive impairment are independent in HIV ,30. FurtThe results of our study may have implications for when it is best to start HIV therapy. The number of years since HIV diagnosis was not a significant predictor for neurocognitive impairment. However, the longer a subject was prescribed HAART, the more likely he or she was to be diagnosed with neurocognitive impairment. Persistent cognitive deficits in HAART-treated subjects may be a result of pre-treatment neurological damage, poor central nervous system penetration of some antiretroviral agents, drug resistance, poor adherence, potential neurotoxicity, and co-morbidities ; future Continuous viral loads should generally be log-transformed in order to meet normal assumptions, but in our sample, 80% did not have a detectable viral load . Thus, it was not feasible to analyze the data as a continuous outcome. We did look at viral load as a dichotomous outcome of yes/no detectable viral load, but there were no significant associations with this variable even at a bivariate level.The finding that current CD4 count was not associated with performance on the IHDS or the other two cognitive tests was expected, since most of the subjects were receiving HAART. Studies in the HAART era have shown that biomarkers, such as current CD4 count, plasma viral load, CSF viral load and CSF immune markers, are not correlated with, or predictive of, neurocognitive impairment . Recent The IHDS has the potential to be a useful screening instrument for HIV-associated neurocognitive disorders (HAND). As anticipated, subjects who were classified with neurocognitive impairment on the IHDS also performed significantly lower on other tests of cognitive function, specifically processing speed (DS Coding) and verbal learning/memory (the AVLT). On a cautionary note, it is sobering that several variables also affected performance on the IHDS and the two other cognitive measures. For example, language of test administration was related to performance on tests of both verbal memory (AVLT) and processing speed (DS), which may be related to cultural factors or socio-economic status, and requires further investigation.Even though there was a relatively small range of ages in our sample, age adversely affected performance on the IHDS. Similarly, education was associated with performance on the IHDS. Older and less educated subjects obtained significantly lower scores on the IHDS. Performance on most mental status examinations does not depend on processing or motor speed, but speed is a major component of two of the three IHDS subtests; thus performance is particularly vulnerable to the effects of age and education. This highlights the need to take these factors into account when determining local normative cut-off scores for screening tests of HIV dementia .Our results support the hypothesis that memory impairment in HIV infection is caused by disruption of subcortical processes. This is reflected in impaired free recall due to retrieval inefficiencies, but relatively preserved recognition. Subjects meeting criteria for neurocognitive impairment on the IHDS performed poorly on the learning/memory test (AVLT) for both total learning (TL) and delayed free recall (DR), but not recognition (R). This is consistent with the neuropsychological pattern of memory deficits documented in prior studies of HAND, characterized by impaired learning and retrieval, but intact recognition -37.This pilot study has several limitations: the absence of an HIV-negative control group for comparison; symptoms of peripheral neuropathy being elicited by a targeted clinical history and not by examination; and the study being conducted among HIV-positive patients in an urban outpatient HIV clinic in Botswana, which may not be representative of HIV-positive individuals in community and rural settings. In addition, appropriate demographically corrected norms and determination of possible learning effects with repeated screenings are needed for the IHDS before it can be used confidently to classify HAND in individuals in Botswana.More than one-third of HIV-positive subjects met criteria for neurocognitive impairment with the IHDS, despite the fact that almost all were treated with HAART for an average of two years and were asymptomatic from the perspective of subjective complaints and everyday activities. This finding, combined with recent research indicating that cognitive impairment may become more likely the lower the value of the nadir CD4 cell count, gives further weight to the importance of initiating treatment with HAART at earlier stages of the disease process.In addition, there may be more than one explanation for the persistence of neurocognitive deficits, with different policy implications. These include: treatment regimen, particularly poor central nervous system penetration of some antiretrovirals; drug resistance; poor adherence; potential neurotoxicity; and co-morbidities, such as long-term cardiovascular disease side effects of HAART, and chronic HIV brain infection. Our results, while admittedly tentative, indicate that neurocognitive impairment is likely to be an important component of HIV infection in developing countries, and highlight the need to develop effective treatments.The authors declare that they have no competing interests.KL conceived the study design, supervised and participated in the data collection and psychological evaluation of subjects, participated in the statistical analysis, and drafted the manuscript. AS participated in the study design, data collection, and revisions of the manuscript. ES contributed to the study design and revisions of the manuscript. MM participated in the study design, provided supervision for the overall collection of data in the Infectious Disease Care Clinic, and undertook revisions of the manuscript. SR participated in the study design and provided statistical analysis and interpretation of data. KS participated in the data collection, psychological evaluation of subjects, and revisions of the manuscript. RN participated in the data collection and psychological evaluation of subjects. All authors read and approved the final manuscript.


The characteristics and clinicopathological factors were evaluated retrospectively.Six of the 11 patients with ADSQ were male and five were female. The mean age was 67.3 years' olds. Three patients had pathological stage IA, one patient each had stage IB and IIA, five patients had stage IIIA, and one patient stage IIIB. Five patients had coexistent double cancer including 2 gastric, 1 rectal, 1 prostate and 1 bladder cancer. ADSQ was found less frequently in males than squamous cell carcinoma (SQ). ADSQ was found more frequently in older patients, with advanced stage, advanced T status, and lymph node metastases than adenocarcinoma (AD). The proportion with coexistent double cancer of AD, SQ, and ADSQ were 21.1, 17.6, and 45.5%, respectively. ADSQ had a significantly correlation with double cancer (ADSQ vs. non- ADSQ p = 0.03). A multivariate analysis showed no significant prognostic difference between the patients with ADSQ and non- ADSQ.In this study, cases with ADSQ showed no significantly prognostic difference in comparison to AD and SQ. However, surgeons must be cautious of any coexistent double cancer because approximately half of all patients with ADSQ of the lung have double cancer. Lung cancer remains the most common cause of cancer death worldwide . DespiteThe characteristics and clinicopathological factors were evaluated retrospectively. Seven hundred and seventy-nine patients with primary lung cancer underwent a surgical resection between April 1994 to March 2006 at the Second Department of Surgery at University of Occupational and Environmental Health, Kitakyushu, Japan. Of these, 403, 187, 39, 24, 11, 125, and 10 patients were diagnosed as AD, SQ, large cell carcinoma, small cell carcinoma, ADSQ, multiple primary lung cancer (MPLC), and others, respectively. According to the established criteria, on routine microscopic examination using hematoxylin and eosin staining, a sample from a tumor containing at least 20% each of AD and SQ is classified as ADSQ [Statistical analyses were performed using the Stat view 5.0 software program for Windows . The chi-square test was used to assess the relationship between ADSQ and each of the clinicopathological features. In the prognostic analysis, the Kaplan-Meier method was used to estimate the probability of survival, and survival differences were analyzed by the log-rank test. A multivariate analysis was performed according to Cox's proportional hazards model. A p-value less than 0.05 was considered to be significant.The clinical features of the patients with ADSQ are summarized in Table The prevalent histological differentiation of AD was moderately differentiated , while the prevalent SQ was moderately differentiated in 82%  and NSCLC is highly reproducible. However, there are few studies about ADSQ with anecdotal reports on the subtype of ADSQ. ADSQ of the lung is a subset of pulmonary carcinomas, and comprises less than 4% of lung carcinomas -8. It isThis study identified no significant prognostic difference was identified between the patients with ADSQ and non-ADSQ. These findings are consistent with Hsia's data . HoweverThis is the first report to address the problem of coexistent double cancer with ADSQ. In general, the prevalence of coexistent double cancer for the patients with lung cancer is about 10% of lung carcinomas . SurprisCarcinogenesis is a multi-step gene abnormality and requires a certain amount of time to progress. ADSQ was found more frequently in old age than AD in this study suggesting that age is the one of the main risk factors for this type of cancer. Heterogeneity is frequent in cancer and ADSQ is an atypical heterogeneous tumor containing two distinct components of AD and SQ. Therefore, the current findings, which show a high prevalence of coexistent double cancer, suggest the cancer cells not only consist of mixture of AD and SQ, but also show progression of carcinogenesis with specific mechanisms in separate organs. The original histogenesis remains unclear. However, the biological impact is very interesting because the squamous cell component does not exist in the peripheral lung. Lymph node metastases were more frequent in cases with a larger SQ component in the primary lesion in this study. Therefore characterization of ADSQ may be a first step based on carcinogenesis to resolve the mystery of the heterogeneity. The histogenesis of ADSQ includes some possibilities including AD with squamous metaplasia, collision tumor, high-grade mucoepidermoid carcinoma, and bi-potential undifferentiated cell origin . Niho etIn conclusion, this current study demonstrated that ADSQ is an unusual histological subtype of NSCLC and approximately half of the patients with ADSQ had coexistent double cancer, thus suggesting that cancer cells in ADSQ demonstrate progressive carcinogenesis with specific mechanisms.ADSQ: adenosquamous carcinoma; SQ: squamous cell carcinoma; AD: adenocarcinoma; NSCLC: non small cell lung cancer; SCLC: small cell lung carcinoma; MPLC: multiple primary lung cancer; 95% CI: 95% confidence interval.The authors declare that they have no competing interests.This report reflects the opinion of the authors and does not represent the official position of any institution or sponsor. The contributions of each of the authors were as follows:HU were responsible for reviewing previous research, journal handsearching, drafting report. SY was responsible for pathological data. TH was responsible for quality checking and data processing. HU was responsible for project coordination. All authors have read and approved the final manuscript.


High blood pressure (HBP) is a major risk factor for cardiovascular disease (CVD). European hypertension and cardiology societies as well as expert committees on CVD prevention recommend stratifying cardiovascular risk using the SCORE method, the modification of lifestyles to prevent CVD, and achieving good control over risk factors. The EDUCORE  project aims to determine whether the use of a cardiovascular risk visual learning method - the EDUCORE method - is more effective than normal clinical practice in improving the control of blood pressure within one year in patients with poorly controlled hypertension but no background of CVD;This work describes a protocol for a clinical trial, randomised by clusters and involving 22 primary healthcare clinics, to test the effectiveness of the EDUCORE method. The number of patients required was 736, all between 40 and 65 years of age (n = 368 in the EDUCORE and control groups), all of whom had been diagnosed with HBP at least one year ago, and all of whom had poorly controlled hypertension (systolic blood pressure ≥ 140 mmHg and/or diastolic ≥ 90 mmHg). All personnel taking part were explained the trial and trained in its methodology. The EDUCORE method contemplates the visualisation of low risk SCORE scores using images embodying different stages of a high risk action, plus the receipt of a pamphlet explaining how to better maintain cardiac health. The main outcome variable was the control of blood pressure; secondary outcome variables included the SCORE score, therapeutic compliance, quality of life, and total cholesterol level. All outcome variables were measured at the beginning of the experimental period and again at 6 and 12 months. Information on sex, age, educational level, physical activity, body mass index, consumption of medications, change of treatment and blood analysis results was also recorded;The EDUCORE method could provide a simple, inexpensive means of improving blood pressure control, and perhaps other health problems, in the primary healthcare setting;http://ClinicalTrials.gov].The trial was registered with ClinicalTrials.gov, number NCT01155973 [ In the year 2000, the worldwide prevalence of high blood pressure (HBP) among the adult population was 26.4% . The higThe control of HBP in Europe is far from optimum -8, and iComité Español Interdisciplinario para la Prevención Cardiovascular: CEIPC) .In recent years, many studies have investigated the use of tables based on the Framingham study -19 (e.g.: CEIPC) , recomme: CEIPC) ,23. The on 2009)  recommenIn randomised clinical trials, several strategies aimed at health professionals and patients have been shown capable of improving compliance with antihypertension therapy  and of rAs a strategy, improving the perception of risk is closely related to making behavioural changes that affect health . Leal etIt is unclear which communication format  is the best to use in educational interventions, although each type has its own advantages . Some auAffective communication can improve the perception of risk in high risk patients, and alleviate concerns in those whose risk is low . The EDUThe aim of the present work is to determine whether the EDUCORE method for communicating cardiovascular risk in patients with poorly controlled HBP, but no history of CVD, is more effective than normal practice in terms of improving the control of blood pressure at one year.Comunidad de Madrid) took part. The recruitment of these centres was undertaken between March and May 2009. The research group was comprised of 166 persons: 148 general practitioners and nurses , and 19 scientific researchers . A member of the clinical assistance team at each participating centre was appointed as liaison officer between the centre and the technical group.The present work involves a two-year community, parallel clinical trial, randomised by clusters. Twenty two primary healthcare centres (PHCC) in the Madrid Region (Comité de Ética de Investigación Clínica del Área 3) (2009/24/06), and met all good clinical practice demands. The trial was registered with ClinicalTrials.gov, number NCT01155973 [http://ClinicalTrials.gov].The study protocol was approved by the Area 3 Research Ethics Committee  at least one year ago, with analytical  and ECG results available, whose blood pressure was poorly controlled (last determination systolic ≥ 140 mmHg and/or diastolic ≥ 90 mmHg), and for whom information on organ involvement was available.b. Aged 40-65 years.c. To be able to meet the demands of the trial, i.e., to have no intention of moving, to be localisable for the next year, and to have the capacity to understand the questionnaires presented.d. To have given signed, informed consent to be included in the study, and to meet no exclusion criterion.Exclusion criteria. Patients were required not to have suffered a major cardiovascular event , or to be suffering diabetes mellitus, a serious chronic disease with a notable impact on physical or psychological status , or to be unable to collaborate (patients with dementia or other psychopathologies).2. Sampling was performed by clusters, the PHCCs being the randomisation units. This minimised possible contamination effects between centres. An independent statistician randomly assigned the 22 PHCCs (each of which were randomly assigned a code) to either the EDUCORE method group or a control  group (11 centres in each group) using Epidat 3.1 software.Consecutive patients were chosen to minimise the risk of bias in their selection. Figure Method of calculation. For an alpha of 0.05, a power of 80%, and in order to detect an increase in good control of blood pressure of 15% in the EDUCORE group, the overall sample size required was 354 patients (177 in each arm of the study). Since randomisation was by clusters, the sample size had to be larger than if simple randomisation had been performed, in order to take into account the design effect (DE). The DE was calculated as follows: DE = 1 + (nc - 1) * ICC   [The criteria followed for the assessment and control of CVR were those published by the Madrid Regional Health Service .b. Calculation of the projected CVR at 60 years of age for all patients under this age.Use of visual impact images. Patients were shown images embodying different stages of a high risk action using the consultation room's computer with the aim of increasing consciousness regarding risk-entailing habits.2. Handing the patient a pamphlet. This pamphlet contained advice on how to maintain cardiovascular health plus the low risk SCORE table with the patient's current score marked.3. The main outcome variable measured in both groups was the control of blood pressure (systolic blood pressure < 140 mmHg and dyastolic blood pressure < 90 mmHg). The blood pressure was determined using the Riva-Rocci method and listening for Korotkoff sounds, using validated and calibrated sphygmomanometers). The secondary outcome variables recorded were: CVR (measured using the low risk SCORE table), total cholesterol level, the use of tobacco, therapeutic compliance (measured using the Morisky-Green treatment compliance questionnaire) , and quaThese included sex, age and level of education . Patient physical activity was measured using a questionnaire  .2), use of tobacco , total cholesterol and LDL-cholesterol (mg/dl), serum creatinine (mg/dl) and microalbuminuria/creatinine (mg/g). The consumption of cardiovascular medication  at the beginning and end of the study was also recorded. Changes in medication were also recorded, as were the reasons for such change, including secondary effects and ineffectiveness. The type of change made to antihypertension treatment was also recorded, including increasing/reducing dose, drug associations, suspensions and substitutions.These included body mass index (weight in kg/height in cmAll data (excluding patient identification data) were recorded in an electronic database  searchable only by the EDUCORE group via a corporative intranet system. This centralised the collection of data, facilitating later analysis.These included sex, age, professional category , the date of qualification, and the PHCC where services were rendered. This facilitated the description of the clinical assistance personnel associated with each cluster.The number of patients who declined to participate was recorded, as required by the CONSORT statement .The causes of all losses and withdrawals  were recorded. When patients failed to attend an appointment, two attempts were made to enter into contact by telephone.http://www.educore.es.A web page was used to facilitate the monitoring of the work performed and to make the project more visible The use of randomisation by clusters conditions the statistical analysis that can be employed, especially in the calculation of confidence intervals and the testing of hypotheses. The following analyses were undertaken:Descriptive analysis and the description of the profile of patients who abandoned the study plus their reason for withdrawal.1. Baseline comparison of the two intervention groups in terms of the analytical variables measured, prognostic factors and descriptive variables, using appropriate bivariate statistical tests.2. Effect of the EDUCORE method on blood pressure control (main outcome variable): i.e., a comparison of the proportion of patients with good/bad control in the two study arms using the Chi-squared test, and the calculation of confidence intervals at 6 months and 1 year after patient inclusion. Logistic regression with random effects was used to adjust for prognostic factors; the dependent variable was good/bad control of patient blood pressure, and the independent variable the intervention group to which each patient belonged. Confounding factors or factors that might alter the effect recorded were taken into account in this analysis.3. Effect of the EDUCORE method on secondary outcome variables  using appropriate statistical tests.4. © v.18 software.All statistical tests were performed with intention to treat. The 'last observation carried forward' was used for missing data . SignifiEducational intervention strategies aimed at either patients or health professionals can provide benefits in the control of HBP ,31,33. TThe study design used in this work is, however, subject to certain limitations. Firstly, the intervention cannot be masked, which could influence the assessment of its effect. Certainly, the possibility of contamination exists if the patients involved communicate with one another . For thiIn practice, the percentage of patients with good control of their HBP that can be reached, though improvable, is limited. Thus, achieving a very large impact with the EDUCORE method might be difficult. For this very reason, any positive effect achieved - even though small in magnitude - could be understood to represent success, especially in this disease of such great pubic health importance.The conclusions drawn from this study might influence clinical practice and research into blood pressure control. In the clinical setting, the reduction of CVR in patients with HBP could have an important effect on health and quality of life.Comité Español Interdisciplinario para la Prevención Cardiovascular; DE: Design effect; EDUCORE: Education and Coronary Risk Evaluation; ECG: Electrocardiogram; GP: General practitioner; HBP: High blood pressure; ICC: Intracluster correlation coefficient; nc: Number of individuals in a cluster; SCORE: Systematic Coronary Risk Evaluation;CVD: Cardiovascular disease; CVR: Cardiovascular risk; CEIPC: The authors declare that they have no competing interests.IRS conceived of the study and participated in its design. EEM participated in the design and coordination of the study. IRS and EEM carried out the EDUCORE method. MRB, RRF, AAS, ICG, LCB, SGE and RRB (senior authors) participated in the design of the study. EEM, IRS, MRB, RRF and AAS directed the writing of the manuscript. Contributions were made by the remaining authors. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/10/449/prepub


Enterobacteriaceae bacteremia.There is limited information about the effect of diabetes on the prognosis of patients with bacterial infections. We performed a retrospective cohort study to investigate possible correlations between diabetes and prognosis in patients with Enterobacteriaceae bacteremia from January 1997 through June 2007. Factors associated with in-hospital mortality were analyzed by logistic regression analysis.We reviewed the medical charts of 1112 patients who were treated at a community teaching hospital for Enterobacteriaceae bacteremia, 181 (16.3%) were diabetic patients; 90 patients (8.1%) died while in the hospital. Compared to non-diabetic patients, diabetic patients were older  and had more comorbidities. However, mortality among diabetic and non-diabetic patients was not different . In a multivariate analysis, the variables associated with in-hospital mortality were age, the origin of the bacteremia, and the presence of immunosuppression. Diabetes was not associated with outcome.Among the 1112 patients with Enterobacteriaceae bacteremia, diabetes was not associated with a poorer prognosis.In this cohort of patients with  Enterobacteriaceae are the dominant causal agents of Gram-negative bacteremia and are a major cause of morbidity and mortality [Enterobacteriaceae bacteremia [Enterobacteriaceae is likely to increase as well. However, information about the effect of diabetes on the prognosis of patients with infections in general, and with Enterobacteriaceae bacteremia in particular, is limited. In addition, some data are contradictory [Enterobacteriaceae bacteremia so this would support the convenience of a more aggressive approach for these patients. We performed a retrospective cohort study to identify possible correlations between diabetes and prognosis in patients with Enterobacteriaceae bacteremia.ortality . The preortality ; considecteremia , the numadictory -6. It isEnterobacteriaceae bacteremia who were treated at Hospital Sierrallana, from January 1997 through June 2007. Only the first episode of bacteremia in a particular patient, accounted at our hospital during the period of study, was included. Enterobacteriaceae strains recovered from blood samples were identified and tested for antimicrobial susceptibility by the automated testing systems MicroScan  or BD Phoenix .The Sierrallana Hospital is an adult acute-care center in Torrelavega, a city in the province of Cantabria, Spain that forms part of a health district of 160,000 inhabitants. It is a teaching institution with 250 beds that has approximately 8000 admissions and 65000 assistances at the emergency service annually. It includes most major departments and specialties, except transplantation, thoracic, cardiovascular surgery and neurosurgery units. Using the microbiology laboratory database, we identified patients with 3 at the bacteremia onset. Immunosuppression was defined as the presence of neutropenia, infection with the human immunodeficiency virus or current treatment with immunosuppressive agents. Sepsis, severe sepsis and septic shock were defined according to the guidelines of Bone et al [A standardized data collection form was used to review the hospital records of the included patients. Diabetes was considered present if there was a previous diagnosis of type 1 or type 2 diabetes, or if diabetes was diagnosed during the patient's treatment for bacteremia. For the diagnosis of diabetes the American Diabetes Association diagnostic criteria were used . Renal ine et al . The inip-value <0.1 in the univariate analysis. Diabetes was included as a variable in the regression model independent of the p-value. The statistical analyses were performed using SPSS software, version 14.0. The study was approved by the Comité Etico de Investigación Clínica de Cantabria.A significance level of 0.05 (2-sided) was used for all tests. Relative risk (RR) was calculated as the ratio of the incidence rate in patients exposed to a risk factor to the incidence rate in those not exposed. Univariate analyses and logistic regression were performed to identify factors associated with patient death. Factors were added to the model using variables with a Enterobacteriaceae bacteremia were identified; of these, full medical records were available for 1112 (98%). We identified 181 (16.3%) patients with a diagnosis of diabetes, and 90 (8.1%) patients died while undergoing treatment for bacteremia. The following Enterobacteriaceae were isolated from the blood cultures: Escherichia coli in 862 (77.5%) patients, Klebsiella spp. in 82 (7.4%) patients, Proteus mirabilis in 51 (4.6%) patients, Salmonella spp. in 42 (3.8%) patients, Enterobacter spp. in 36 (3.2%) patients, Morganella morgagnii in 12 (1.1%) patients, Citrobacter spp. in 11 (1%) patients, Serratia marcescens in 9 (0.8%) patients, Pantoea agglomerans in 5 (0.4%) patients and Leclercia adecarboxylata and Yersinia enterocolitica in one patient each. Comparing the characteristics of diabetic and non-diabetic patients, significant differences in sex, age, comorbidities and initial blood glucose concentration were detected  for diabetic patients and 8.2% (76/927) for non-diabetic patients (p = 0.39).During the study period, 1134 cases of Variables significantly associated with mortality in the univariate analysis are shown in Table Variables that were associated with in-hospital mortality in the multivariate analysis in the whole cohort, in nosocomial cases and in community acquired cases are reflected in Table Enterobacteriaceae bacteremia [Enterobacteriaceae bacteremia conducted from 1992 through 2001. Other explanations could be related to differences in the population analyzed, comorbidities and the adequacy of antibiotic empirical treatment. We included some variables that were not analyzed by Thomsen et al, which could also contribute to the difference in results. In addition, we only evaluated the effect of diabetes on in-hospital mortality, not on 30- and 90-day mortality [Diabetes has traditionally been associated with a worse prognosis for patients with infectious diseases such as cteremia , liver acteremia  and tubecteremia . Howevercteremia , and forcteremia . This imcteremia . Improvecteremia ; this grEnterobacteriaceae bacteraemia may contribute to the low mortality [The mortality in our series is lower that previous similar studies . Severalortality . The incortality ,13; thisOur study has several potential limitations. The differences in mortality could be lower that the sample size would permit to detect. As the data, including the diagnosis of diabetes has been obtained retrospectively, this could lead to information bias between exposure and outcome, so the diagnosis of diabetes may actually have been missed in patients dying precipitously, and conversely there is a risk that a diagnosis of diabetes has been wrongly inferred from hyperglycemia in some surviving patients. We did not routinely collect measures of long-term glucose control, such as HbA1c, which would have allowed us to distinguish the effects of long-term poorly-controlled diabetes on patient prognosis. Finally, our population was mainly elderly people with type II diabetes, thus, these results may not be applicable to patients diagnosed with type I diabetes.Enterobacteriaceae bacteremia. Improved management of diabetic patients with acute illnesses in recent years may account for the absence of an effect of diabetes on outcomes in these patients.Our data suggest that the presence of diabetes is not related to in-hospital mortality in patients with Enterobacteriaceae bacteremia, diabetes was not associated with a poorer prognosis. Improved management of diabetic patients with acute illnesses in recent years may account for the absence of an effect of diabetes on outcomes in these patients.In conclusion, in our cohort of patients with Galo Peralta has been a consultant for Janssen-Cilag, Wyeth, Bristol-Myers Squibb, and Boehringer Ingelheim, and has also served as a speaker for Wyeth and GlaxoSmithKline.GP, MPR, MBS, and JCG were involved in the study conception, RT and FT were involved in the coordination, and data acquisition, GP performed the data analyses, all authors were involved in the interpretation and validation of the results, GP, MPR and MBS were involved in the drafting of the manuscript and all authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/9/94/prepub


P=0.001) was comparable in prognostic value to that based on stage and performance status  in patients with inoperable non-small-cell lung cancer. The former is simple to measure and well standardised.A score based on the combination of the systemic inflammatory response and albumin hazards ratio (HR) 1.70, 95% CI 1.23 – 2.35,  Non-small-cell lung cancer is the most common cause of cancer death in North America and Western Europe. Most patients present with advanced inoperable disease and consequently the prognosis of these patients is extremely poor. Conventionally, the selection of patients for active or supportive treatment has been based on clinicopathological criteria, including age, stage, performance status, weight-loss or hypoalbuminaemia   to a single multidisciplinary oncology clinic in Glasgow Royal Infirmary between January 1997 and October 2002 were included in the study. Information was abstracted from the case notes by a specially trained research nurse (LMF). Data for 1997–1999 were collected retrospectively and that for 2000–2002 prospectively. All patients had cytologically or histologically confirmed disease and were staged on the basis of clinical findings, chest X-ray, and where appropriate, bronchoscopy, liver ultrasound, isotope bone scan and computerised tomography of the thorax, according to the American Thoracic Society TNM classification .Clinical stage, tumour type and performance status  were recorded at the time of diagnosis. A blood sample was also obtained for measurement of haemoglobin, white cell count, albumin and C-reactive protein concentrations. Patients were considered to have undergone active treatment if they received chemotherapy (mainly cisplatin based) and/or radical radiotherapy. Patients receiving palliative radiotherapy and/or palliative care (symptom control) were considered to have had supportive treatment.The study was approved by the Research Ethics Committee of Glasgow Royal Infirmary.Routine laboratory measurements of haemoglobin, white cell count, albumin and C-reactive protein concentration were carried out. The coefficient of variation for these methods, over the range of measurement, was less than 5% as established by routine quality control procedures.et al, 1997; −l) as previously described , haemoglobin, white cell count and albumin was carried out using standard thresholds  were performed using the Cox regression analysis with prognostic scores as covariates. Deaths up to 31 January 2003 were included in the analysis. Analysis was performed using SPSS software .n=161) are shown in Table 1The characteristics of patients with inoperable NSCLC (P<0.05), tumour type (P<0.01), performance status (P<0.001), white cell count (P<0.01), albumin (P<0.001) and C-reactive protein (P<0.01) were significant predictors of survival. On multivariate survival analysis all these variables, with the exception of white cell count, remained independent significant predictors of survival. There was a significant correlation between C-reactive protein and albumin concentrations .In all, 118 (73%) of patients died during the follow-up period. On univariate survival analysis, stage  and the score based on stage and performance status  retained independent significance.On multivariate analysis, when the three scores based on the combinations of the systemic inflammatory response and stage, performance status and albumin were compared with the combination of stage and performance status, only the score based on the combination of the systemic inflammatory response and albumin  and 14% of those with poor performance status (ECOG 3–4) received active treatment. Treatment was not included in the prognostic model since the selection of patients for active treatment is based primarily on performance status.These results are consistent with the evidence that chronic activation of the systemic inflammatory response is detrimental to the outcome of patients with NSCLC, being associated with an increase in weight loss (r2 less than 15%). This would suggest that infection was not the main stimulus to the increased C-reactive protein concentrations.The mechanism by which a systemic inflammatory response is evoked in patients with NSCLC is not clear. One possibility is that since many patients have coexisting pulmonary infection, the resultant increase in white blood cells would lead to increased proinflammatory cytokine release and a subsequent increase in circulating C-reactive protein concentrations. However, in this study, although the white cell count was significantly correlated with C-reactive protein concentrations, the magnitude of the relationship was small (An alternative explanation would be that proinflammatory cytokines are produced by the tumour. Indeed, there is evidence that proinflammatory cytokines are produced locally by tumours in patients with NSCLC (In conclusion, the results of the present study suggest that a cumulative score based on C-reactive protein and albumin may have prognostic value similar to that provided by more conventional measures. However, the cumulative score based on C-reactive protein and albumin has the advantage that of being simple to measure, routinely available and well standardised. Further large-scale prospective studies are required to confirm these findings and establish its value as a guide to the selection of patients for treatment.


Plasmodium sp., including the most serious agent Plasmodium falciparum, depends on the local epidemiological and demographic situations, such as the incidence of infected people, the vector transmission intensity and migration of inhabitants (i.e. exchange between sites). Analysing the structure of P. falciparum populations at a large scale, such as continents, or with markers that are subject to non-neutral selection, can lead to a masking and misunderstanding of the effective process of transmission. Thus, knowledge of the genetic structure and organization of P. falciparum populations in a particular area with neutral genetic markers is needed to understand which epidemiological factors should be targeted for disease control. Limited reports are available on the population genetic diversity and structure of P. falciparum in Thailand, and this is of particular concern at the Thai-Myanmar and Thai-Cambodian borders, where there is a reported high resistance to anti-malarial drugs, for example mefloquine, with little understanding of its potential gene flow.The population structure of the causative agents of human malaria, P. falciparum populations were analysed using 12 polymorphic apparently neutral microsatellite loci distributed on eight of the 14 different chromosomes. Samples were collected from seven provinces in the western, eastern and southern parts of Thailand.The diversity and genetic differentiation of P. falciparum transmission areas (average HS = 0.65 ± 0.17), where the lowest is observed in South America and the highest in Africa. However, uniquely the Yala province, had only a single multilocus genotype present in all samples, leading to a strong geographic differentiation when compared to the other Thai populations during this study. Comparison of the genetic structure of P. falciparum populations in Thailand with those in the French Guyana, Congo and Cameroon revealed a significant genetic differentiation between all of them, except the two African countries, whilst the genetic variability of P. falciparum amongst countries showed overlapping distributions.A strong difference in the nuclear genetic structure was observed between most of the assayed populations. The genetic diversity was comparable to the intermediate level observed in low Plasmodium falciparum shows genetically structured populations across local areas of Thailand. Although Thailand is considered to be a low transmission area, a relatively high level of genetic diversity and no linkage disequilibrium was found in five of the studied areas, the exception being the Yala province (Southern peninsular Thailand), where a clonal population structure was revealed and in Kanchanaburi province (Western Thailand). This finding is particularly relevant in the context of malaria control, because it could help in understanding the special dynamics of parasite populations in areas with different histories of, and exposure to, drug regimens. Plasmodium falciparum, the agent of the most malignant form of malaria that accounts globally for some 300–600 million cases of clinical malaria and 1.5 – 2.7 million deaths each year. Although P. falciparum can cause the most severe of the four human forms of malaria [P. falciparum drug resistance [P. falciparum strains have become more resistant, in terms of both resistance level and frequency in the population, to mefloquine, in addition to the widespread resistance to chloroquine, and sulphadoxine/pyrimethamine observed in many endemic areas, especially within those provinces located along the Thai-Myanmar border , or the Thai-Cambodian border . As a result of declining mefloquine efficacy, a combination of mefloquine and artesunate is now administered in those areas [Plasmodium falciparum gene flow between transmission areas and the existent level of P. falciparum population genetic diversity are directly implicated in the spread of drug resistance. Understanding the genetic complexity and organization (structure) of P. falciparum populations is a crucial aspect for the control of this disease, since the genetic diversity and population structure of P. falciparum in each location will have profound impacts on clonal diversity [Malaria is still one of the most important infectious diseases, endemic in the tropical and sub-tropical parts of about 102 countries, especially in the African continent. In Thailand, although the total number of malaria cases has been decreasing annually [se areas . The Plaiversity , dynamiciversity , persistiversity  and malaiversity .P. falciparum is known to exhibit a diverse and patchy array of population genetic characteristics, which are apparently correlated with local levels of endemicity and transmission intensity [Globally, ntensity . Howeverntensity . It thusP. falciparum transmission rate [P. falciparum in different locations have been reported [P. falciparum populations in Kenya was also observed where this form of malaria is also transmitted at a relatively high frequency [The ion rate ,15, and ion rate , which dion rate -18. At areported ,20, whilreported . Moreoverequency . Geograprequency ,23. Howerequency ,24.P. falciparum, as well as other malaria parasites such as Plasmodium vivax, is essential for predicting how fast given phenotypes, such as drug or host resistance or novel antigenic variants, will originate and spread within and between populations [P. falciparum populations, and to lead to an efficient management of this disease agent in particular areas. Indeed, the genetic composition and evolutionary change of each P. falciparum population is of great importance to ascertain the ecophysiology of the parasite and host-parasite interactions, as well as the evolution of resistance in the pathogen. Studies using the extensive polymorphism in antigen coding loci provide little valid information on the population structure of P. falciparum since, being under strong but varying selection, they reflect the combined effects of population history and natural selection [Understanding of the genetic structure of ulations . Studieselection .P. falciparum population genetic studies have focused on the boundary with Myanmar in the western part using different genetic markers [P. falciparum genetic structure relies on the epidemiological and demographical situations encountered in different areas and may change within a relatively short period of time. In this report, the population genetic structure of P. falciparum, is evaluated in seven provinces of Thailand, using 12 polymorphic and apparently neutral nuclear microsatellite loci as genetic markers. These were also compared with samples from other endemic countries, while using the same loci described published literature for other populations. Such studies could later be extended to other endemic areas displaying different treatment policies and different uses of prophylactic anti-malarial drugs.In Thailand, most  markers ,27,28, bP. falciparum based malaria incidences in Thailand [The parasitized blood samples were collected from patients who attended for malarial diagnosis at clinics in seven provinces of Thailand from 2002 to 2007 Figure . All patThailand . In 2006Thailand . Four ofThailand . The samP. falciparum-infected human blood samples from two sources in Thailand were used in the study: 1) samples from in vitro culture, which had been preserved in liquid nitrogen at the Malaria Research Programme, College of Public Health Sciences, Chulalongkorn University, and 2) blood samples from P. falciparum malaria patients preserved on filter papers. Blood samples collected before 2004 were in vitro cultivated after blood collection from the malaria patient, and the cultivated samples were then cryopreserved in liquid nitrogen until being used in this study. The DNA was extracted from cultured parasites by standard phenol/chloroform extraction of whole blood. Thereafter, between 2004 and 2007, parasitized blood samples were collected from thick film-confirmed falciparum malaria patients, before drug administration. These patients were informed of the objectives of this study, agreed to participate in it, and signed a standard consent form. Blood samples were taken from finger-pricks of P. falciparum-infected  patients, and were absorbed onto 3 MM Whatman filter paper and air-dried. DNA from the dried blood sample was subsequently extracted using a DNeasy Tissue kit (Qiagen) according to the manufacturer's instructions. The blood collection protocol was approved by the ethical committee of the Institute of Health Research, Chulalongkorn University. Socio-demographic information of patients was collected by interview, especially the most likely place of infection, to help identify the parasite's origin. The provincial origins and year of collection of the infected blood samples, and the likely country of infection source where not Thailand, are summarized in Table The P. falciparum from each infected blood sample, the DNA was extracted and used as template for the PCR based amplification of 12 established polymorphic and apparently neutral microsatellite loci distributed on eight of the 14 different chromosomes [et al [For genotyping omosomes , as listomosomes , followis [et al . For comP. falciparum clone infection were analysed, because of the difficulty in defining multi-locus genotypes in samples with a polyclonal infection. To this end, given that these 12 loci are all single genome copy, not in linkage, and that P. falciparum from the blood (schizonts and early trophozoites) has a haploid genome, then isolates with more than one allele at more than one locus were deemed to be from multiple infections. Genetic diversity was measured by the allelic richness per locus and sample (RS) and Nei's unbiased expected heterozygosity (HS), after adaption to haploid data, using FSTAT, v2.9.4 [Only isolates that successfully amplified at all 12 loci within the Thai samples or at the selected seven loci for international samples were included in the respective analyses. Moreover, for population genetics, only those samples that showed a single , v2.9.4 . Differe® software [Linkage disequilibrium analyses between pairs of loci were performed with FSTAT v2.9.4. Genetic variation and differentiation were analyzed using FSTAT. Canonical correspondence analysis (CCA) was carried out to illustrate the measures of population structure, using CANOCOsoftware ,33. Onlysoftware , which aP. falciparum, i.e. schizonts and early trophozoites, present in human blood samples were analysed, the presence of multiple infections was assessed in a given isolate by the presence of more than one allele at any of the microsatellite locus surveyed. The minimal multiplicity of infection for each sample  was analysed locus by locus, and was estimated from the locus that exhibited the highest number of alleles in a given isolate. Therefore, a single P. falciparum isolate infection is deemed to correspond to an isolate exhibiting only one allele at each of the 12 loci investigated. Of course, multiple infections that are genetically distinct overall but identical at the 12 microsatellite loci, for example recombinants or new mutations at other sites, or whose differences lead to null alleles and so would not be detected, would not be distinguished. Although the former would still be likely to be closely related isolates all the same, the later maybe quite different and thus the data represents the minimal rather than the actual level of multiple infections. Accordingly, the minimal proportion of multiple infections in each sample group was assessed for each locus and between loci in a sample group , leaving 346 single isolate infected (72%) samples suitable for genotyping and population genetic analysis. The multiple infected samples were distributed across six of the seven provinces assayed, with frequencies of multiple infections as a ratio of infected individuals to the total number of samples ranging from 44% in Maehongson to 22% in the Ubonratchathani population  in each population was 0.68 ± 0.23, 0.63 ± 0.25, 0.60 ± 0.18, 0.70 ± 0.21, 0.56 ± 0.25, 0.62 ± 0.20 and 0 in the Maehongson, Tak, Kanchanaburi, Ubonratchathani, Trat, Ranong and Yala populations, respectively.For the population genetic analysis, based on 12 polymorphic nuclear microsatellite loci, only the 346 single Linkage disequilibrium (LD) was tested at three levels, (i) for the whole population (ii) for the three regional areas; and (iii) amongst sites.p-value = 0.00076) associations of loci were found.(i) For within the whole population , out of the 66 possible pairwise linkages, 34 significant , respectively.(ii) For within the three regions, we found that in the Eastern region , Western  and Southern area, , there were 0, 3 and 46 significant combinations of loci (out of 66) that were potentially in linkage  linkage disequilibrium was found in any combined pair of loci except for two combinations in Kanchanaburi (Polyα × TA81 and Ara2 × TA81), and, of course, a total linkage disequilibrium existed in the Yala population, since all samples have the same genotype.(iii) For within all the sites, no significant (FST) was estimated using the FSTAT software. A permutation test was applied  permuting alleles amongst all samples for all loci to test whether FST significantly differed from zero. A significant differentiation  of P. falciparum populations between sites was observed, with a very high differentiation between the Yala population and all the other populations  between the western, eastern and southern parts of Thailand with FST values equal to (i) 0.0478 for the western versus the eastern regions, (ii) 0.1321 for the western versus the southern regions and (iii) 0.1199 for the eastern versus the southern regions.Genetic differentiation  they can pass through several generations, and (ii) these genotypes could carry important traits, such as drug-resistance genes.P. falciparum populations with the already published reports for other populations, the observed patterns at seven common microsatellite loci , were compared with those observed from blood samples of two African countries, Congo (n = 15) and the Cameroon (n = 35), and one South American country, French Guyana (n = 137) (unpublished data). The gene diversity in the Thai populations (HS = 0.65 ± 0.17) was lower than those found in the two African countries , but higher than that of French Guyana (HS = 0.54 ± 0.08). Significant LD was observed in the two low transmission countries . The high LD value observed in Thailand is due to the Yala sample. The canonical correspondence analysis (CCA), searches for multivariate relationships between two data sets  [P. falciparum populations. A graphic representation of the results showed overlapping distributions . This suggests some degree of geographic differentiation. Indeed, all populations in different countries displayed significant genetic differentiation except between the two African countries, Congo and Cameroon  , was alsP. falciparum populations in different continents revealed that African countries with high transmission intensities had the highest population genetic diversity. The genetic diversity decreased with low transmission intensities, as in Thailand (Asia) and French Guyana (South America). The average genetic diversity of P. falciparum populations in Thailand (HS = 0.65 ± 0.17) was, however, close to that of the high transmission intensity level of Papua New Guinea [At the level of continents, the genetic structure of  HS = 0.6 ± 0.17 ww Guinea  and Brazw Guinea , also baP. falciparum in Thailand showed a deviation from random mating, in congruence with the French Guyana population, whilst, in contrast, the two African populations showed likely panmixia. The proportion of multiple isolate infections, and the number of clones in an individual host, which are related to the transmission intensity of malaria parasites in any endemic area, are important parameters that affect P. falciparum mating, because fertilization may occur between gametes of the same or different genotypes in mosquitoes. Therefore, an inverse correlation between the proportion of multiple clone infections and the degree of linkage disequilibrium is expected. Even though the genetic diversity of Thai populations was observed at an intermediate level, and multiple isolate infections occurred frequently, a significant linkage disequilibrium was found which could be caused by, (i) a higher chance of inbreeding (selfing) in this parasite population, or (ii) structure formation within populations into sub-populations of each foci in Thailand (i.e. the Wahlund effect). The latter reason would be more likely to explain this finding because samples from all the foci from across the country (the seven different provinces) were pooled in the analysis.The deduced mating patterns of P. falciparum analysis with 12 polymorphic nuclear microsatellite loci in isolates from multiple populations in seven provinces of Thailand.In Thailand, the malaria incidence rate shows a high spatial heterogeneity across the country, with high incidence regions found nearby the borders with Myanmar and Cambodia . This stP. falciparum populations in the areas bordering with Myanmar revealed a similar level of genetic diversity (0.60 <HS <0.68), whilst the populations bordering with Laos and Cambodia had slightly higher or lower levels of genetic diversity at 0.70 and 0.56 in Ubonratchathani and Trat, respectively. Although the multiple isolate infection rates in those areas bordering with Myanmar  were higher than that of Ubonratchanthani (22%) and comparable to that in Trat (36%), this could be explained by the notion that (i) the parasite mating in those areas is preferentially inbreeding, or (ii) the relatively small amount of sampled individuals in Trat and Ubonratchathani leading to a stochastic masking of the true population structure. The Yala population, however, showed an extreme difference in the gene diversity compared the other samples since it showed only a unique haplotype in all sampled individuals, but again may be a transient clonal expansion of an immigrant infection.The P. falciparum gene pool in each endemic area and affect the epidemiology of the P. falciparum caused malaria in these areas. Besides this migration rate of humans in each foci, there is additionally the factor of the geographic barrier between Tak and Kanchanaburi provinces which may be another cause of genetic differentiation by partial isolation by distance and breaking down of panmixis. Thus, the genetic differentiation found between populations within each province of Thailand may reflect both a variation of the transmission rate in each location and the migration rate of people from one location to another. Certainly seasonal migration has been suspected as a leading cause of malaria transmission in these areas [Anopheles vectors in local areas may also have an effect on the parasite structure. In Thailand, Anopheles aconitus sensu lato, Anopheles baimaii, Anopheles dirus, Anopheles maculatus, Anopheles minimus and Anopheles pseudowillmori have been incriminated as important vectors of P. falciparum in humans, and they are differentially distributed throughout the country [P. falciparum. Thus, although differences in the population structure of P. falciparum between two vectors (Anopheles gambiae and Anopheles funestus) have been preliminarily investigated [In contrast to the Yala population, a low but significant population structure was observed amongst the provinces that border with Myanmar, Laos and Cambodia. In the case of the Myanmar bounding provinces, the significant genetic structure detected between populations  was likely to be due to the fact that the pattern of human movement from neighbouring countries into each of these four provinces was different. In the Tak and Ranong provinces, the numerous movements in to Thailand from Myanmar from immigrant laborers, and from patients resident in Myanmar who cross the border to seek free malaria treatment, account for the high proportion (approximately 70%) of sampled individuals being likely to have been infected in Myanmar. Indeed, the same genotype was found in seven samples collected in 2007 in Kanchanaburi, from patients infected in Thailand and Myanmar. These, imported cases may play an important role on the se areas . However country -38, and stigated , no suchP. falciparum infected blood samples collected one year apart (2005 – 2006 and 2006 – 2007) was observed only in Ranong, where one case was found in a patient infected from Myanmar and the following year was found from a patient infected in Thailand. If a generation time is assumed to be two months, this parasite genotype has been transmitted through six generations without detectable change across the 12 loci (eight/14 chromosomes) due to recombination. This may imply that if this P. falciparum strain has an advantage (fitness) in that particular environment, it will propagate and expand in this population.Importantly, the same multilocus genotypes of P. falciparum haplotypes collected in the same year and over two years were detected. A unique spatial haplotype, distinct from the other populations, was found in all patients in the Yala population, which were obtained from individual residents in four different sub-districts in Bannang Sata and other three nearby districts during May – July 2007. Three possible explanations are available to explain this trend. Firstly, that a P. falciparum isolate with this genotype had a high infectivity in the mosquito's midgut or carried some genetic characteristics, such as drug resistance, which gave it a significant benefit over other haplotypes. Secondly, that there is no inhabitant movement into this area due to the political violence, and so as a result no new haplotype parasite was introduced into this area (no gene flow). Thirdly, there maybe a rare new gene influx by immigration but which was subsequently lost due to selection or stochastic failure to progress to a stable infection threshold. Finally, is the possibility that the decrease in sanitary cover in this area allowed this particular genotype to spread into the population rapidly. Thus, a longitudinal study in the population dynamics in this area is required. Nevertheless, this is the first study to reveal the potential clonal expansion of a single haplotype in a malaria endemic area.In this study, both a spatial and a temporal distribution of Plasmodium falciparum populations are highly structured in Thailand suggesting local and differential routes of genetic evolution for this parasite. Although Thailand is considered as a low transmission area, a high level of genetic diversity and weak linkage disequilibrium were found in most sites, except the transient infections in the Yala province in one year where only a single genotype was found. Moreover, (i) identical genotypes were encountered in the same site but from patients coming from different localities (i.e. Myanmar and Thailand), and (ii) two identical genotypes were observed from two successive years at the same site (i.e. Ranong); these findings are particularly relevant in the context of malaria control. They would help to understand the specific dynamics of malaria and parasite populations in areas displaying a different history of drug applications.The authors declare that they have no competing interests.TP carried out field work, the molecular genetic work, analysis, interpretation of data, and drafted the manuscript. CA carried out the molecular genetic work and data analysis. NK contributed to field work, DNA extraction of cultivated parasite. AS and NS were responsible for parasite cultivation. PD and FR carried out the statistic analysis, guidance for interpretation of data, participated in the manuscript preparation and revision. CS participated in the design of the study, the manuscript preparation and revision. PH participated in the design of the study, the manuscript preparation and revision and coordination. All authors read and approved the final manuscript.Allele frequencies. Allele frequencies at 12 microsatellite loci in P. falciparum from seven Thai populations.Click here for file


In comparison with natural forests, plantations decreased aboveground net primary production, litterfall, and rate of soil respiration by 11, 34, and 32%, respectively. Fine root biomass, soil C concentration, and soil microbial C concentration decreased respectively by 66, 32, and 29% in plantations relative to natural forests. Soil available N, P and K concentrations were lower by 22, 20 and 26%, respectively, in plantations than in natural forests. The general pattern of decreased ecosystem C pools did not change between two different groups in relation to various factors: stand age (<25 years vs. ≥25 years), stand types (broadleaved vs. coniferous and deciduous vs. evergreen), tree species origin (native vs. exotic) of plantations, land-use history (afforestation vs. reforestation) and site preparation for plantations (unburnt vs. burnt), and study regions (tropic vs. temperate). The pattern also held true across geographic regions. Our findings argued against the replacement of natural forests by the plantations as a measure of climate change mitigation.Uncertainties remain in the potential of forest plantations to sequestrate carbon (C). We synthesized 86 experimental studies with paired-site design, using a meta-analysis approach, to quantify the differences in ecosystem C pools between plantations and their corresponding adjacent primary and secondary forests . Totaled ecosystem C stock in plant and soil pools was 284 Mg C ha Aboveground biomass is larger in plantations afforested in non-forested lands The inconsistent results may stem from the fact that individual studies do often not provide much information on ecosystem processes, which is helpful for our understanding of why plantations differ in ecosystem C stock from natural forests. For example, lower aboveground net primary production (ANPP), aboveground litterfall, and fine root biomass lead to lower C sequestration into ecosystems Several syntheses have been conducted to explore the effects of plantations on ecosystem C stock, but they focused on the comparison of soil C stock between plantations and non-forested lands e.g., . In thisCunninghamia lanceolata, Pinus caribaea, P. radiata and Picea abies, and consequently most of the plantations considered were pure coniferous stands. Mean age of plantations was 30 years with a range from 4 to 80 years , stand types (broadleaved vs. coniferous and deciduous vs. evergreen), tree species origin (native vs. exotic) of plantations , land-usThe results obtained from this synthesis suggested some mechanisms underlying the difference in ecosystem C stock between plantations and natural forests. There were consistent decreases in ecosystem C pools with decreasing ANPP, aboveground litterfall and rate of soil respiration in plantations relative to natural forests. The decrease in fine root biomass could also explain the decreased amount of C input into plantations observed RR++) might be sensitive to additions or deletions of published studies. Study sites were not randomly distributed in global forest ecosystems, and datasets compiled for this meta-analysis came from the regions where ecologists have extensively conducted relevant studies, while many other plantation regions have not attracted an attention from ecologists. These might cause biases in evaluation of the impacts of plantations. Thus, more experimental studies on ecosystem C cycle for plantations in comparison with natural forests are needed in the future. However, the general pattern of the decrease in ecosystem C pools in plantations relative to natural forests was independent of biomes, geographic regions or other factors . The decreased ANPP leads to less atmospheric C, via photosynthesis, into plants and soils, meaning decreased soil C stock This meta-analysis demonstrated that ecosystem C pools, including those in above- and belowground biomass, aboveground litter mass and soil, was 28% lower in plantations than in natural forests. Our results about the amount of ecosystem C pools were consistent with those from studies by Dixon et al RRs of soil C stock, for example, was not significantly correlated with stand age of plantations, latitude (north/south) and mean annual precipitation and temperature of the study sites . Thus, the differences in ecosystem C pools between plantations and natural forests were related to the interactions of these factors. Any differential effects resulting from the two different groups in one of these factors could be swamped by the others for such differences.Ecosystem C pools discussed above were statistically different between plantations and natural forests, such differences were affected by various factors –4. High 4, another important greenhouse gas, significantly decreased by 80% in plantations when compared with the natural forests based on 11 field cases Our findings had at least two implications. First, plantations, with reduced ecosystem C stock, failed to function as C sink as originally intended, in comparison with natural forests. Over the last two decades, C sequestration strategies might have overstated the role of plantations in climate change mitigation C. lanceolata plantations, and by 39% and 15% from second to the third rotation Second, our results on ecosystem C cycle provided an interpretation of ecosystem degradation associated with plantations e.g., . For exaThe shifts from natural forests to plantations can also generate other ecological problems. For example, soil bulk density, representing the degree of soil compaction, was 12.9% higher in plantations relative to natural forests 2 enrichment and warming) which did not belong to the normal range of silvicultural activities. Fifth, for soil variables, data were collected from the samples of soil surface layer. If data from the samples of different layers in a soil profile had been compiled into one, the compiled one was employed.To avoid bias in publication selection, the following five criteria were set for the inclusion of data related to ecosystem C stock and other related variables for plantations and natural forests. First, the reference ecosystems relative to plantations were primary and secondary forests which were naturally generated and free from disturbance . As a result, secondary forests were dominant in the reference ecosystems in this synthesis. Second, the trees in plantations were arboreal species, not including bamboos, shrubs, or fruit and non-timber species such as apple, coffee or rubber trees. Third, field studies were conducted by paired-site design in fields where there were both of plantations and natural forests Databases of Blackwell, CNKI, Elsevier, Kluwer, JSTOR, Springer and Web of Science, licensed to Fudan University library, were used to search for source data from inception to September 2009. Study sites were located in all continents except for Antarctic. All the data used here were extracted from figures and tables in published papers. For each variable, the mean (M), standard error (SE) or deviation (SD) or 95%CI, and sample size (n) in both plantations and natural forests were extracted. Information on the factors such as stand age and types of plantations, land-use history and site preparation for plantations, and geographical conditions of study sites was collected. To examine the effects of these factors on ecosystem C pools, plantations were categorized into two different groups in relation to stand age (<25 years vs. ≥25 years), stand types (broadleaved vs. coniferous and deciduous vs. evergreen), tree species origin (native vs. exotic), land-use history (afforestation vs. reforestation) and site preparation for plantations (unburnt vs. burnt), and study regions (tropic vs. temperate). The threshold value of 25 years was determined by the common practice that mature plantation stands with fast growth rate are generally considered to be of less than 25 years in age. In addition, study sites were grouped into different geographic regions such as Australia, China and USA, and then the differences in ecosystem C pools between plantations and natural forests were examined in each of the geographic regions.RR, the ratio of the mean value of a concerned variable in plantations to that in natural forests) was used here as an indicator of the difference in a variable between plantations and natural forests. To summarize the results from independent studies, weighted response ratio (RR++) was calculated from RRs to increase the precision of the combined estimate and the power of the tests. M, SE or SD or 95%CI, and n were used to compute RR, RR++ and 95%CI of RR++. Dixon's Q-test was performed to exclude outliers of RRs at α = 0.05. If the 95%CI value of RR++ for a variable did not overlap with zero, the variable was significantly different between plantations and natural forests. If the 95%CI value of RR++ for a variable did not overlap between the two different groups in relation to one of these factors: stand age, stand types and tree species origin of plantations, land-use history and site preparation for plantations, and study region, the RR++ was considered to be significantly different between the two groups and the factor has a significant effect on the variable. If the 95%CI value of RR++ overlapped, Student's-test was used to further examine the difference between the two different groups, which was considered to be significant at the level of P<0.05. The percent change in a variable from natural forests to plantations was calculated by [exp (RR++)−1] ×100%.The method of this meta-analysis followed previous studies e.g., . PlantatY = a [1-exp (−b/x)], was used to calculate the mean soil C stock within the depth of 100 cm in both plantations and natural forests, where x was the depth of sampled soil, Y was soil C stock, a and b were estimated parameters. For the regressions fitted here, correlation coefficient (R) was larger than 0.52, and statistical P value was less than 0.001 for both plantations and natural forests. Soil C stocks within a depth of 100 cm and their standard errors were derived from the fitted equations.A simple model: Table S1RR (N = 1) or RR++ (N>1) and the number of cases  for thirteen variables extracted from each of the 86 papers (Reference list follows in supplementary (0.26 MB DOC)Click here for additional data file.References S1List of 86 papers from which datasets of the thirteen variables were extracted for this meta-analysis.(0.10 MB DOC)Click here for additional data file.