Coronary heart disease (CHD) causes an estimated 7 million deaths worldwide each year. In the last few decades, mortality from CHD has been decreasing in many countries. The aim of this study was to analyze the trends of mortality from CHD and myocardial infarction (MI) in the population of Belgrade during the period 1990–2010.Mortality data for CHD and MI were obtained from the Municipal Institute of Statistics in Belgrade and used to calculate age- and sex-specific and age-adjusted mortality rates. Joinpoint regression analysis was used to estimate annual percent changes (APCs) in mortality and to identify points in time where significant changes in trend occur.Trends in CHD mortality rates showed significant decline in men during the period studied , but no significant change among women . While we observed significant declines in CHD mortality in men aged 35–44, 55–64 and 65–74 and women aged 55–64, there was a significant increase in mortality in men aged ≥85 and women aged 75–84 and ≥85. Trends in MI mortality rates showed similar patterns in both genders, with a significant decline from the mid-1990s. Significant decline in MI mortality was observed in almost all age groups, except the two oldest (75–84 and ≥85) in women population.Given that CHD and MI mortality trends showed different patterns during the period studied, especially in women, our results imply that further observation of trend is needed. Cardiovascular diseases (CVDs) are the leading cause of death worldwide, accounting for 17.3 million deaths in 2008 and 30% of total global mortality[Mortality rates from CHDs have been declining in the United States of America, Australia and Northern and Western Europe, with approximately two thirds of the decline attributable to changes in risk factors and one third due to evidence-based treatments-5. MeanwMortality data for CHD  and MI (ICD-9 code 410 & ICD-10 codes I21–I22) were obtained from the Municipal Institute of Statistics in Belgrade. Deaths from MI were included in the numerator for deaths from CHD. We used projected Belgrade population figures, which were extracted from unpublished data from the Statistical Office of the Republic of Serbia, as the denominator for each year. We calculated age- and sex-specific mortality rates for CHD and MI, and then standardize them using the direct method, according to the European Standard population. Age-speDuring the observed period of twenty-one-years 1048 men and 765 women died from coronary heart diseases annually. The average age-adjusted CHD mortality rate was higher in men  than in women . For MI, average age-adjusted mortality rates were 72.6 per 100,000 in men and 45.9 per 100,000 in women. Proportional contribution of MI to CHD mortality was approximately 75% in men, and 68% in women, although this had been declining in recent years, especially in women , and non-significant change in women, with a small, steady increase   and a significant decrease from 1996–2010 . In women, one joinpoint was also found, showing a significant increase from 1990 to 1996  and a significant decrease from 1996 to 2010  , and a non-significant increase in men aged 75–84. In all other age groups, however, we observed a decline in CHD mortality over the entire period studied, which was significant for men aged 35–44, 55–64 and 65–74  and non-significant for men aged 45–54 Table . For womDeclines in mortality rates from MI were identified in almost all age groups, except in two oldest age groups in women where a non-significant increase was present. Significant declining trends in MI mortality were observed for men aged 35–44 and 75–84 for the entire period. This was also the case for: those aged 45–54 years from 1993, those aged 55–64 from 1995, and those aged 65–74 from 1996. For women, significant decrease in MI mortality started in 1998 and 1996 for those aged 55–64 and 65–74 years respectively, while for those aged 45–54 years it was present over the entire period studied , and started later in countries such as Denmark and Germany. In deveOur results, which show a significant decrease in MI mortality rates and no change in CHD mortality trend in women, may suggest that mortality rates from other CHDs such as angina pectoris, complications following acute MI and other acute CHDs and chronic CHDs may have increased. According to data from the Serbian Registry for acute coronary disease for the year 2011, CHD was the third-most frequent cause of death from CVDs. While oIn developed countries, CHD mortality rates decreased in all age groups, including the oldest,25. ThisThe significant rising trend in MI until the mid-1990s can be explained by the difficult political and economic situation faced by the people of Serbia, including the most severe hyperinflation recorded globally during the mid-1990s. This situation resulted from the collapse of the Socialist Federal Republic of Yugoslavia and the refugee crisis precipitated by the outbreak of war in the region.Subsequent decreasing trend of MI mortality, present in almost all age groups in both genders, may be the result of reduced exposure to risk factors  includinRates of mortality for CHD and MI showed different patterns of change during the period 1990–2010, especially in women, implying that further analysis of trend is needed.CVD: Cardiovascular disease; CHD: Coronary heart disease; MI: Myocardial infarction; APC: Annual percentage change; AAR: Age-adjusted rate; CI: Confidence interval.The authors declare that they have no competing of interests.IV - conceived and designed study, acquired, analyzed and interpreted data, and wrote manuscript. SS – participated in plan of the study and revised manuscript for important intellectual content. ED - participated in the design and helped to draft the manuscript and wrote some parts. HV – revised manuscript critically for important intellectual content and gave final approval of the version to be published. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2261/13/112/prepubAge-adjusted coronary heart diseases and myocardial infarction mortality rates for men and women aged 45-75 years, Belgrade, 1990-2010.Click here for file


Plasmodium falciparum malaria and the progression of MQ resistance.Studies in South-East Asia have suggested that early diagnosis and treatment with artesunate (AS) and mefloquine (MQ) combination therapy may reduce the transmission of The effectiveness of a fixed-dose combination of AS and MQ (ASMQ) in reducing malaria transmission was tested in isolated communities of the Juruá valley in the Amazon region.P. falciparum malaria incidence rates, P. falciparum/Plasmodium vivax infection ratio, and malaria hospital admissions rates. All the time series ranged from January 2004 to December 2008, whilst the intervention period span from July 2006 to December 2008.Priority municipalities within the Brazilian Legal Amazon area were selected according to pre-specified criteria. Routine national malaria control programmatic procedures were followed. Existing health structures were reinforced and health care workers were trained to treat with ASMQ all confirmed falciparum malaria cases that match inclusion criteria. A local pharmacovigilance structure was implemented. Incidence of malaria and hospitalizations were recorded two years before, during, and after the fixed-dose ASMQ intervention. In total, between July 2006 and December 2008, 23,845 patients received ASMQ. Two statistical modelling approaches were applied to monthly time series of falciparum malaria incidence rates, 0.67 (95%CI 0.50 – 0.89) for the P. falciparum/P. vivax infection ratio, and 0.53 (95%CI 0.41 – 0.69) for the hospital admission rates. There was also a significant change in the seasonal (or monthly) pattern of the time series before and after intervention, with the elimination of the malaria seasonal peak in the rainy months of the years following the introduction of ASMQ. No serious adverse events relating to the use of fixed-dose ASMQ were reported.The ASMQ intervention had a highly significant impact on the mean level of each time series, adjusted for trend and season, of 0.34 (95%CI 0.20 – 0.58) for the P. P. falciparum malaria.In the remote region of the Juruá valley, the early detection of malaria by health care workers and treatment with fixed-dose ASMQ was feasible and efficacious, and significantly reduced the incidence and morbidity of  Plasmodium falciparum malaria, and is at the heart of the global strategy for malaria control [Artemisinin-based combination therapy (ACT) is considered the best available treatment for uncomplicated  control . One of  control .The use of ASMQ was considered in Asia as a strategy to mitigate resurgence of malaria and the intensifying spread of anti-malarial drug resistance well before the World Health Organization (WHO) recommended using ACT. Over the last 20 years, in the low transmission areas of the Thai Burmese border, the combination of AS and MQ has shown high efficacy and has been of great benefit in considerably reducing the transmission of multidrug resistant malaria, as well as reversing the trend of increasing MQ resistance. .P. falciparum malaria. Fixed-dose combinations eliminate the possibility of patients taking only one component of the combination and are expected to improve patient compliance[Safe, rapid, and reliably effective, the combination of AS and MQ is one of five forms of ACT currently recommended by the WHO as a first-line anti-malarial treatment. The WHO also recommends that fixed-dose combinations (FDC) be used whenever possible  to increompliance. With spP. falciparum malaria were reported in Latin America, where Brazil has the highest malaria burden [P. falciparum malaria treatment, greater investments, capacity building on prevention and control, and epidemiologic data analyses, which allow a focus on malaria burdens in real time, both from decision makers and the populations involved [In 2008, approximately 320,000 cases of a burden . In Braza burden . With a a burden . A decreinvolved . MalariaPlasmodium resistance, showed cure rates below 90 % for quinine sulphate and doxycycline  for P. falciparum treatment, and prompted the introduction of ACT in 2006 by the National Malaria Control Programme (PNCM). Chloroquine and primaquine 0,50 mg base/kg during seven days remain the treatment for P. vivax malaria [Efficacy studies initiated by the Amazon Network for the Surveillance of Antimalarial Drug Resistance (RAVREDA), created in 2001 to monitor  malaria .P. falciparum incidence, P. vivax and P. falciparum ratio, and on the number of hospital admissions in three municipalities in the Amazon Basin (Brazil), characterized by high malaria incidence and concerns of increasing anti-malarial resistance to quinine-doxycycline. The study aimed to assess the suitability of replacing quinine sulphate and doxycycline as a national first-line treatment policy for children and adults with uncomplicated P. falciparum malaria in Latin America. This study is the largest to date on the programmatic use of ASMQ in Latin America.This article presents the results of a phase IV deployment study investigating the impact of fixed-dose ASMQ combination on P. falciparum malaria cases per month between 2003 and 2004, and had a stable population (defined by < 15 % proportion of imported cases), as well as supportive and cooperative local health authorities.The study included data collected between July 2004 and December 2008. The ASMQ intervention was carried out between July 2006 and December 2008 in three municipalities in the Juruá valley, within the state of Acre in Brazil: Cruzeiro do Sul, Mancio Lima and Rodrigues Alves Figure . The munAcre state, situated in the most occidental region of Legal Amazon Figure  has a poP. falciparum malaria differed from those used in Brazil [Anti-malarial drug resistance has not been investigated in this region for almost two decades ,15. Acren Brazil ,16,17. An Brazil ,15.Malaria incidence in some areas of the Amazon Basin rose between 2003 and 2005, which was attributed to several environmental and socio-economic factors, including climatic changes, new urban growth, cattle ranching and agricultural practices associated with deforestation) ,18. PublP. vivax predominates in urban populations, but in 2009, P. falciparum still accounted for more than half of the malaria burden in isolated river communities [In the Juruá Valley of Acre, Brazil, munities .Anopheles darlingi, and the most vulnerable group consists of low-income workers (fishermen and gold miners) [Anopheles darlingi is the main species captured outside and inside the homes of inhabitants of the Juruá valley, in a proportion of 4:1, respectively. The mean rate of An. darlingi per person/hour was 3.2 indoors and 11.5 outdoors. Both inside and outside feeding preferences are from ~8:00 p.m. until ~0:00 a.m. .The most important malaria vector in Brazilian endemic areas is  miners) -22. AnopThe control strategy of the Brazilian National Malaria Control Programme  is based on early diagnosis and treatment, selective vector control interventions, early epidemic detection and involvement of local government and concerned populations. Malaria is diagnosed by rapid tests based on the immunochromatographic method or Giemsa-stained thick blood smears, according to PNCM training recommendations and quality control guidelines. Malaria treatment in Brazil is free of charge, as per government policy.P. falciparum vs. P. vivax). The Brazilian Ministry of Health regulated drug distribution and provided epidemiological data by mapping drugs received, distributed, and consumed.In 2000, malaria control programmes were decentralized to individual states of Brazil. In Acre state, as 90 % of total cases of malaria occur in the Juruá valley, a regional coordination body for malaria control was created. This organization includes support teams , information systems as well as administrative and pharmaceutical assistance. In November 2005, intensified malaria control activities included active systematic screening of the population as routine practice, with weekly screenings performed by healthcare workers. Health surveillance workers in each locality collected thick blood smears, administered therapy and provided health education. These agents also prevented prescription mistakes and sought to improve treatment compliance. Local health facilities supported diagnosis and drug distribution. Drug supply was planned according to the number of cases reported for each type of malaria infection . Epidemiological and service indicators were evaluated on a weekly basis by the management team and all supervisors, ensuring that all information was disseminated widely.P. falciparum infections, population age range, continuous access to treatment and diagnosis, good management of information systems, evidence of transmission inside the homes, and acceptability of bed nets to the population. These localities also had the least reduction in malaria incidence in previous years compared with other localities in the Juruá valley. Adequate anti-malarial drug control and supplies were available through the cooperation of health authorities and malaria control programs.Vector control strategies included outdoor thermo-nebulization and indoor chemical residual spraying with cipermetrine, and environmental management of larvae control until 2006. Biolarvicide was used in water tanks from December 2005 to July 2007. As of October 2006, the strategy focused mainly on vector control, with the goal of using indoor chemical spraying in 80 % of the highest risk area homes. In December 2007, an intervention with long lasting impregnated bed nets was initiated, where 7,000 insecticide-treated bed nets were distributed to cover 100 % of the population in 13 localities selected on the basis of epidemiological criteria: high disease burden, high proportion of The study was designed as an open-labelled, community-based intervention study in three municipalities of Acre state. These municipalities contained 73 localities, as notification units with laboratory and drug supply capabilities. The study included data collected routinely through the national surveillance system, SIVEP-malaria, between July 2004 and December 2008. The ASMQ intervention was initiated on July 2006, with completion on December 2008. The population within these municipalities was compared pre- and post-ASMQ intervention.P. falciparum infection diagnosis, confirmed by thick smear enrolled in the public health system of the intervention municipalities were eligible for the study. The exclusion criteria were pregnancy and presence of clinical danger signs and other clinical features of severe malaria [All patients aged ≥6 months with an uncomplicated  malaria .There were no restrictions to the use of other medications, except for other anti-malarials and drugs with known interaction. Prior to study initiation, malaria health workers were trained in order to prevent uncontrolled use of other anti-malarials in the study areas. Special attention was paid to MQ single tablet therapy, which is no longer recommended and has been withdrawn from Acre state. Standard treatment with quinine, doxycycline or primaquine was available in case patients declined trial participation.Participating subjects who met the inclusion criteria were treated with FDC ASMQ (manufactured by Farmaguinhos), according to age, using either a low dose (LD) tablet 25 + 50 mg  or a high dose (HD) tablet: 100 + 200 mg .Dosing was daily for 3 days and stratified by age: (i) 6–11 months of age: 1 ASMQ LD tablet, (ii) 1–6 years of age: 2 LD ASMQ tablets, (iii) 7–13 years of age: 1 HD tablet, and iv) 14 years of age and older: 2 HD tablets. Most of the time, treatment was directly observed, but for patients living in remote areas, only the first dose was directly observed and the remainder of the treatment was taken home. At study entry, diagnoses were performed by rapid test or thick smears, and patients were enrolled on the basis of a positive result. During the study period, rapid tests were performed in remote areas, but slides were collected to confirm the result by thick smear.P. falciparum slide was revised, identified by a SIVEP number and filed under the month and notification area. As a routine procedure, 10 % of negative slides were re-examined. When conflicting results occurred, which was less than 1 % of cases, the microscopy technician was advised to attend a training programme.During the first six months of the study, samples of positive and negative malaria smears from every microscope technician were reread in blinded conditions for quality control, according to PNCM standard operational procedures. From November 2006, every positive only The procedure to identify double entries and cure-verifying slides used the RecLink software duplicity routine ,25 that Adverse events monitoring was done by spontaneous reporting through the local pharmacovigilance system. In order to record these events, standard national regulatory agency forms were made available at the health facilities, diagnosis posts, and to every health surveillance agent.The study was approved by the São Paulo University Ethics Committee. Participation in the study was entirely voluntary. Patients were informed of the risks and benefits relating to the study, and participants could withdraw from the study at any time. Written informed consent was obtained from all literate patients or guardians. Illiterate patients or guardians recorded their consent with a thumbprint. A witness confirmed both types of consent. Before starting the study, a state health team visited the communities involved to describe study objectives and methods. Communities and local healthcare providers also gave approval.falciparum malaria, the ratio between monthly incidences of P. falciparum and P. vivax malaria, and the monthly hospital admission rates due to malaria (ICD-10: B58 to B54). The period of analysis ranged from January 2004 to December 2008 (before and after introduction of FDC ASMQ).In order to address the impact of the introduction of ASMQ in the Juruá valley region, three outcome variables were analysed: monthly incidence rates of P. Because the time series spanned five years, and the outcome variables were based on monthly incidence of the disease, the model had to take into account the effects of trend and seasonality.The effect of ASMQ on the mean level of each outcome time series was evaluated in terms of main effects and interactions. Three main effects were evaluated, years, months and intervention, whilst the interaction involved intervention and months. The model, therefore, contemplated the time trend based on indicator variables for years and seasonality, with indicator variables for months, and the intervention impact with an indicator variable for the whole period after the introduction of ASMQ.For each outcome variable two models were fitted: (a) only main effects of years, months and the intervention; and (b) the same main effects with the addition of an interaction between months and intervention. Hence, regarding the intervention, the first model exploits its effect adjusted for trend and seasonality, whereas the second model exploits its effect on if and how the intervention affected the seasonal pattern of the time series. The two models therefore evaluate different aspects of the intervention. The full model specification is presented in the expression below:β0 is a common intercept or the baseline (here July 2004), α is the intervention effect, and P. vivax malaria for the P. falciparum/P. vivax ratio.Where The above is the expression for the second modelling approach. To obtain the expression for the first approach the interaction terms should be dropped from the formula.As July 2006 was the first month after the start of the intervention, the month of July was chosen as the reference for the monthly pattern. 2004 was selected as the baseline year, as it represents pre-epidemic and pre-intervention levels of the time series. The intervention was implemented in June 2006, so the intervention period in the statistical analysis was regarded as beginning in July 2006 and ending in December 2008. The impact of the ASMQ intervention was assessed in comparison with the baseline, adjusting for the effects of other years and months. Thus, the coefficients of the remaining months and years represent the variation on the log-incidence rates in comparison to July 2004. The monthly counts were analysed and the model parameters estimated according to a quasi-Poisson estimation procedure and took into account an offset variable. Further, residual diagnostics were performed for each of the six adjusted models.The two modelling approaches are presented as they address different questions;- the main effect model establishes that the intervention had an effect on the mean level of the time series,- whereas the main effect + interaction model establishes if the intervention had an effect on the monthly pattern  of the time series, hence altering the mean level of the time series, albeit indirectly.Data were analysed using the following software: Tableau 3.5, Microsoft Office Excel 2003, RecLink, and the R (R Development Core Team 2011) version 2.11.The total population who received ASMQ between July 2006 and December 2008 amounted to 23,845 subjects in the Cruzeiro do Sul, Mancio Lima and Rodrigues Alves districts. The total population at risk in the three municipalities ranged from 96,496 to 103,799 between 2006 and 2008. The proportion of female participants was 43 %. There was no significant change in the total population of the different study municipalities in the Juruá valley during the observed period (January 2004 to December 2008). Over the intervention period, the yearly distribution of ASMQ-treated subjects living in the Juruá valley stratified by age showed that the majority (62.6 %) were aged 14 and over  over one year were stratified by age range in the Juruá valley  on the mean levels of the outcome are shown for a given month, year or intervention period. The different columns represent the adjusted model according to what was described in the methods section, thus six models altogether, two for each outcome. The two first columns correspond to incidence rates, the next two to the P. falciparum/P. vivax ratio, and the last two to hospital admission rates. Under each outcome, the first column shows results based on the first approach (main effects) whilst the second column presents results of the second approach (main effects and interactions). In order to address the difference between both approaches, the number of parameters fitted under each model and its quasi-deviance statistics are shown at the bottom of the table.Model parameter estimates transformed to the original scale are presented in Additional file P. falciparum, the P. falciparum/P. vivax ratio, and for malaria hospital admission rates, respectively. These results are adjusted for trend and seasonality (i.e. under the first modelling approach). It is important to notice the yearly effects pattern: for P. falciparum malaria incidence rates, 2005, 2006, and 2007 had significantly higher levels than 2004, whereas in 2008 there was no significant increase or decrease compared to 2004; for the P. falciparum/P. vivax ratio, 2006 had a significantly higher level than 2004; and for malaria hospital admission rates, 2005 and 2006 had significantly higher levels than 2004, but 2008 had significantly lower levels than 2004. In all outcomes, there was no statistically significant effect for months.There was a significant decrease in the mean level of the time series for the three outcomes after the introduction of ASMQ, namely 0.34 (95%CI 0.20 – 0.58), 0.67 (95%CI 0.50 – 0.89), and 0.53 (95%CI 0.41 – 0.69) for incidence rates of P. falciparum/P. vivax ratio regarding the months of October, November, and December. It is important to notice that usually October marks the beginning of the rainy season in the Juruá valley region . The incidence rates in the months of November  and December  before the intervention showed increases compared to July, whereas there were decreases of 0.74 (95%CI 0.42 – 1.32) for November and of 0.56 (95%CI 0.30 – 1.05) for December, after the intervention. A similar picture emerged for the P. falciparum is statistically significant for every year except 2008, suggesting a return to pre-epidemic levels . The study strengthened the local health management system in Acre state and showed that it is feasible to extend early malaria diagnosis and provide adequate treatment to remote communities. The positive effect of ASMQ was seen both in terms of malaria incidence and malaria-related hospital admission rates.P. falciparum malaria in Brazil. Indeed, prior to the intervention, the P. falciparum malaria incidence rate had increased dramatically during the second half of 2005, with an epidemic peak at the beginning of 2006 and the beginnings of a decline when ASMQ was introduced in June 2006. Various reasons were considered for the increase in P. falciparum malaria incidence in the Juruá valley during this period, including issues related to local malaria control management, vector control activities and decreased efficacy of the standard anti-malarial regimen (quinine and doxycycline).FDC ASMQ was assessed in the Juruá valley, an area with one of the highest incidences of P. falciparum malaria incidence rates returned to pre-epidemic levels by December 2008, preceded in 2007 by the P. falciparum/P. vivax ratio and hospital admission rates. ASMQ yielded a significant difference in the seasonal pattern of P. falciparum incidence rates, by eliminating the rainy season increase.When using 2004 as the baseline, pre-epidemic reference year, our results show P. falciparum/P. vivax ratio returned to pre-epidemic levels (2004) in the year immediately following the intervention (2007), before a fall in the cumulative incidence of P. falciparum cases, suggesting that the P. falciparum/P. vivax ratio may be more sensitive and specific than P. falciparum incidence alone as an epidemiological marker in areas of mixed transmission.The Data analyses were clouded because ASMQ was introduced in the declining phase of a steady malaria epidemic, after some 18 months, and in areas where vector control activities had been increased. Nevertheless, elimination of the seasonal peak and a significant decline in hospital admission rates are important and our data suggest ASMQ was the major contributor to these observations. A significant decline in severe malaria hospitalizations and malaria incidence were also documented in South Africa and on the Thai Burmese border following the deployment of artemether-lumefantrine and non-fixed ASMQ, respectively ,26-28.P. falciparum/P. vivax ratio is considered a key epidemiological indicator, as a signal of ACT activity and its potential impact on malaria mortality and morbidity. As the MQ dose is divided in three equal parts in the fixed-dose combination, it is expected that the fixed dose would have a better tolerability than non-fixed AS + MQ. A decrease in early vomiting with the new fixed-dose formulation has been previously reported [The inversion of the reported . Our stude novo resistance. Moreover, the fixed-dose formulation may translate clinically into better compliance, thereby further contributing to limiting the development of drug resistance.Studies have also shown that a fixed-dose ASMQ combination has improved bioavailability, compared to split-dose regimens , a benefIn areas with low levels of MQ resistance, initial recommendations by the WHO quoted lower doses of MQ in combination with an artemisinin drug, but based on pharmacokinetic and pharmacodynamic modelling data, it is now believed that MQ should be used at a dose of 25 mg/kg. Indeed, it was shown that initial use of a 15 mg/kg dose provided a greater opportunity for selection of resistant mutations and could thus lead more rapidly to resistance than using a dose of 25 mg/kg .versus the current standard of care. However, our results show that FDC ASMQ represents an advance over the poorly tolerated and lengthy 7-day regimen of quinine and doxycycline.This phase IV deployment study had the intrinsic limitation of not being a randomized comparative study P. falciparum malaria in Latin America. The impact of this intervention was seen rapidly. The study strengthened the local health management system in the state of Acre and showed that extending early malaria diagnosis and providing adequate treatment to remote communities is feasible.The fixed-dose ASMQ formulation was developed with the aim of addressing the treatment needs of those at risk of malaria, with ease of administration and a potential for improved treatment compliance. Based on convincing efficacy and safety data, this new product may have a considerable impact in the treatment of uncomplicated P. falciparum drug resistance and to reduce the impact of malaria [These results further support large-scale use of fixed-dose ASMQ and of ACT in general, a strategy that has shown similar results in other regions ,27 and i malaria .P. vivax malaria.Based on the data presented here, fixed-dose ASMQ was launched on a national scale. Ongoing investigations include continued pharmacovigilance monitoring, studies of ASMQ in pregnancy and an evaluation of ACT for the treatment of P: falciparum; Plasmodium: falciparum; P: vivax; Plasmodium: vivax; PAHO: Pan-American Health Organisation; PNCM: Plano Nacional de Controle da Malária – National Malaria Control Programme; WHO: World Health Organization.ACT: Artemisinin-based Combination Therapy; API: Annual Parasite Index; AS: Artesunate; ASMQ: Artesunate-Mefloquine; FDC: Fixed-dose combination; MQ: Mefloquine; The authors declare that they have no competing interests.ACS coordinated the study and contributed to the writing of the manuscript; IR designed and coordinated the study and contributed to the writing of the manuscript; AD participated in coordinating and conducting the study and contributed to the writing of the manuscript, MB contributed to the study conception and design; PM participated in the study design and coordination and helped draft the manuscript; RLC participated in study design and coordination; MBFL conducted the study and helped draft the manuscript; IM conducted the study, and helped to draft the manuscript; APL conducted the statistical analysis and drafted the manuscript; WJ conducted the statistical analysis and drafted the manuscript, and discussed the study results; JLBL coordinated the study and helped draft the manuscript. All authors read and approved the final manuscript.Effect estimates and 95%CI for the indicator variables of years (trend), months , and intervention (ASMQ) on three outcomes: P. falciparum Malaria Incidence Rates, P. falciparum-P.vivax Ratio, and Malaria Hospital Admission Rates in Vale do Juruá, Acre, Brazil, from January 2004 to December 2008. Interaction effects between months and intervention are presented on every second column. Significant effects are highlighted. Click here for file


Photodynamic inactivation (PDI) has been used to inactivate microorganisms through the use of photosensitizers. The inactivation of mammalian viruses and bacteriophages by photosensitization has been applied with success since the first decades of the last century. Due to the fact that mammalian viruses are known to pose a threat to public health and that bacteriophages are frequently used as models of mammalian viruses, it is important to know and understand the mechanisms and photodynamic procedures involved in their photoinactivation. The aim of this review is to (i) summarize the main approaches developed until now for the photodynamic inactivation of bacteriophages and mammalian viruses and, (ii) discuss and compare the present state of the art of mammalian viruses PDI with phage photoinactivation, with special focus on the most relevant mechanisms, molecular targets and factors affecting the viral inactivation process. Consequently, the use of antivirals has largely increased in the last years and resistance to antiviral drugs is now well documented for several pathogenic viruses ,7,8,9,10etc.), have photodynamic techniques for the inactivation of viruses received growing attention Serratia ge kappa , T7   . These radicals can react with oxygen to form active oxygen species, such as the superoxide radical anion [Equation (3)]. Superoxide is not particularly reactive in biological systems but, when protonated, can lead to the production of hydrogen peroxide and oxygen [Equations (4) and (5)] or highly reactive hydroxyl radicals [Equations (6)–(8)] . Type IIion (9)] . 1O2 is on (10)] ,100. Likemselves . Therefoemselves . 3PS* determines whether the reaction pathway is type I or type II and the predominant mechanism can be changed during the course of the PDI process [Both type I and type II mechanisms can occur simultaneously or exclusively, and the ratio between these processes depends on the PS used and on the concentrations of substrate and oxygen . The com process . i.e., if singlet oxygen is absent, it cannot be the reactive species responsible for the photodynamic effect [i.e., compounds that can intercept these ROS at high rates and in a putatively selective manner [An important goal in the investigation of viral PDI is to identify the type of mechanism involved (type I or type II) in the presence of a selected PS . The simc effect . The sime manner ,101,104.A first line of defence against ROS is, of course, the protection against their formation. However, the interception of the damaging species once formed, to prevent it from further deleterious reactions, is also a deactivation strategy of defence. In general, free radical scavengers neutralize the radical species by donating one of their own electrons. The quenching agents themselves are not particularly toxic before and after the electron donation .Three different types of quenching are possible, which include the transfer of the radical character with the formation of a reactive scavenger-derived radical; trapping of free radicals with the formation of a stable or inert free radical trap; and molecules which mimic quenching enzyme activities. In general, scavenger molecules either prevent free radicals from being formed or remove them before they can damage vital molecular components .Several free radical scavengers have been used to evaluate the specific involvement of type I mechanism during mammalian viruses and bacteriophages PDI with different PS .Free radical species had, in general, little or no effect on the photoinactivation of the studied mammalian viruses . In factmeso-tetraarylporphyrins varied according to the structure of the PS and the concentration of dimethylthiourea (DMTU) [meso-tetrakis(4-β-D-glucosylphenyl)porphyrin [D-galactosylphenyl)-20-(pentafluorophenyl)porphyrin [D-glucosylphenyl)-20-phenylporphyrin, which revealed a significantly smaller contribution from type I mechanism. The highest inhibition was reached at about 1.0 mM of DMTU; further increase in scavenger concentration did not decrease the slope of photoinduced inactivation of phages. However, in spite of inhibiting the efficacy of the PS, DMTU did not completely inhibit T7 phage PDI [The photoinactivation rate of some bacteriophages can be reduced in the presence of free radical scavengers, suggesting a contribution of radical species in the inactivation process . In parta (DMTU) ,87. In forphyrin  and 5,10hage PDI ,87. Simihage PDI . T4-likehage PDI .3O2) by physical quenching, achieved by either energy or charge transfer, without consumption of oxygen or product formation [In general, the action of chemical singlet oxygen quenchers involves the reaction of singlet oxygen with the quenching agent, producing an oxidized product. Another possibility is the deactivation of singlet oxygen to ground state porphyrin, the rate of inactivation decreased 42% in the presence of 1,3-diphenylisobenzofuran. When 5,10,15-(4-β-D-glucosylphenyl)-20-phenylporphyrin was used, the rate of protection substantially increased (74%). It can then be concluded that the type of PDI mechanism depends on the PS structure, with the symmetric derivative exerting its toxic effect mainly via the generation of free radicals, whether the asymmetric derivative proceeds mainly by singlet production [et al. [The efficiency of 5,10,15--20-(pentafluorophenyl)porphyrin to photoinactivate T7 phage decreased in 38% in the presence of sodium azide . This reet al.  in the poduction . However [et al.  using th [et al. ,87.A recent study showed that irradiation of polyhydroxylated fullerene suspensions (40 μM) in the presence of β-carotene reduced the photoinactivation rate of PRD1 and T7 phages, demonstrating singlet oxygen involvement . Also, wAlthough some data about the importance of the type I and II mechanisms in PDI of bacteriophages are discrepant, in general, it seems that the type II pathway is more important than the type I mechanism in phage PDI. On the other hand, there are only a few studies focusing on the simultaneous effect of singlet oxygen and free radicals scavengers under the same protocol of viral PDI ,106,107.The short-lived ROS generated by photodynamic mechanisms are responsible for the damage induced to critical molecular targets . DiffereDepending upon the viruses, the nucleic acid can be either DNA or RNA (single or double stranded). The size of the nucleic acid also varies depending on the viruses. Several studies have shown that both DNA and RNA mammalian viruses and phages are efficiently inactivated by PDI. There is now considerable information that PS like MB can bind to and penetrate viral membranes, whereupon they intercalate with nucleic acids. Upon activation by light, the generated ROS can cause the destruction of the nucleic acids, particularly at guanine residues, preventing viral replication . HoweverFrom the four DNA bases, guanine is the most susceptible component to suffer a type I photosensitization reaction, due to the fact that it exhibits the lowest oxidation potential among DNA bases and it is the only base that can be oxidized by singlet oxygen (type II process) .The treatment of viruses with MB and other heterocyclic dyes resulted in the damage of viral DNA ,65,75,76The binding of cationic porphyrins to DNA is presumably due to the electrostatic interaction between the positively-charged substituents in the porphyrin macrocycle and the negatively charged phosphate oxygen atoms of DNA . HoweverViral DNA is thought to be a critical target structure for PDI by MB and light . DNA iso4) caused strand breaks and piperidine-labile bonds in DNA, which is correlated with the loss of infectivity. This is in agreement with the proposal that lesions of the viral genome might be responsible for the lethality induced by sensitization [in vitro [An internal component of T4 phage has been suggested as an important target because MB needs to cross the outer barrier made by its protein capsids in order to produce a significant effect . In facttization . DNA strtization . The obstization . Concernin vitro .RNA has been suggested to be a key factor in viral PDI with many PS, but direct evidence of a correlation between RNA damage and infectivity loss has not been reported yet, as is the case of VSV when treated with phthalocyanine derivatives . In RNA,−1 of a chlorophyll derivative and red light illumination which caused a decrease of as much as 85% in RNA polymerase activity, which can be due to damage in the viral RNA polymerase complex, and 98% inhibition of viral RNA synthesis in 6 hours [et al. [4 and MB. MB and phthalocyanine derivatives inactivated VSV and inhibited fusion of the virus envelope with Vero cells. The degree of inhibition was small compared to the extent of virus inactivation, suggesting that non-membrane targets, like the viral RNA, might be involved in VSV photoinactivation. However, there is no report of a correlation between RNA damage and loss of infectivity [VSV genome was damaged by 30 μg mL 6 hours . Accordi [et al. , the RNAectivity . Photoinectivity .et al. [per se. The results demonstrated that Qβ RNA infectious activity is significantly more affected by photoinactivation in its protein-associated virion state as compared with its purified isolated polymer state [Following MB plus light exposure, the Qβ RNA genome exhibited sufficient lethal lesions to account for phage inactivation . Howeveret al.  suggesteet al. . 8-oxodger state ,122. InaIn spite of the reduced number of reports focusing on the damage induced by PDI in the nucleic acids of mammalian viruses and bacteriophages, it can be concluded that both DNA and RNA are potential targets of viral PDI. However, there are no studies specifically focusing on the damages induced to DNA and RNA of both mammalian viruses and bacteriophages under the same PDI protocol.Enveloped viruses are inactivated more rapidly than non-enveloped viruses because the destruction of the envelope structure is generally accompanied by loss of virus infectivity ,123,124.The higher susceptibility to PDI of enveloped viruses, relatively to non-enveloped viruses, indicates that the viral envelope may be a more important target than nucleic acids for photosensitization. It also indicates that the unsaturated lipids present in the envelope, as well as the major envelope proteins, are important PDI targets. However, as far as it is known, no studies focus on the degradation of viral envelope lipids after PDI or even on other viral internal lipids. There are, however, many studies about the effects of PDI on viral envelope proteins as well as on other core proteins.et al. [The statement that enveloped viruses are more easily inactivated than non-enveloped ones is only based in indirect studies which compare the inactivation results of enveloped and non-enveloped viruses. The enveloped viruses used in PDI protocols ,81,82,83et al.  with theRelative to proteins degradation by PDI, the results of different studies showed that the main damage is the formation of protein cross-links, followed by other types of damage, which include loss of proteins, alterations in protein molecular conformation, mass and charge, and alterations in protein band intensity .When proteins are irradiated with UV or visible light in the presence of a PS, photooxidation of sensitive amino acid residues such as cysteine, L-histidine, tyrosine, methionine and tryptophan, and covalent cross-linking of peptide chains can be observed, leading to the formation of molecular aggregates ,126, disper se can induce alterations in the folding of some enzymes, leading to the exposure of some amino acid residues normally shielded in the protein, and to the shielding of others usually exposed in the molecule. These protein modifications lead to changes in properties such as solubility, proteolytic susceptibility, absorbance, and fluorescence emission of several of their amino acids. These alterations are mainly mediated by hydrogen peroxide and hydroxyl radical generation, although singlet oxygen mediated reactions could also occur [The PS so occur . The amiso occur ,130.et al. [It has been shown that enveloped viruses can be inactivated due to protein damage ,83,131. et al.  showed tThe proteins in the viral envelope of HSV-1 were considered to be major targets of merocyanine 540 photosensitization . Some ph−1 of chlorophyll derivatives and light, the M protein band was not detected, which was accompanied by a decrease in the intensity of the G protein band [4 caused a decrease in the intensity of the G-protein  band and a slight decrease in the intensity of M protein (matrix protein) band and protein cross-links. However, the observed damage in viral proteins could not account for VSV PDI [vs. 4.7 log or 99.998% inactivation, for MB) [4 and MB, no changes in protein patterns after SDS-PAGE of the viral proteins were observed, under conditions that caused complete VSV inactivation [In VSV treated with 3.75–30 μL mLein band . Large cein band . Using a VSV PDI . VSV was for MB) . Abe and for MB)  also fou for MB) . It was  for MB) . Howevertivation .et al. [The results from Vzorov et al.  indicateet al. .PDI of influenza virus by rose bengal altered the HA fusion protein and led to protein cross-links .Photoinactivation of vaccinia virus with rose bengal significantly altered the concentration and oxidized histidine in vaccinia virus protein, suggesting that inactivation was attributed to alterations in viral proteins, as opposed to nucleic acids .Treatment of of influenza and Sindbis viruses by hypericin , lead toThe protein profile of the non-enveloped enterovirus 71 was considerably altered after a low dose PDI and a MB concentration ≥0.5 μM, as revealed by a smearing and the disappearance of several protein bands . Howeveret al. [et al. [Escherichia coli since it contains highly reactive amino acids such as methionine, cysteine, histidine, and tyrosine and not to damages to the nucleic acid [In spite of the limited available data for enveloped bacteriophages, substantially higher photoinactivation rates compared with other non-enveloped phages were described . The phoet al. . The sur [et al.  for a ph [et al. . Phage p [et al. . The hig [et al. . The higeic acid . Glycosyeic acid . Besideseic acid ,88. The eic acid . The dameic acid . The RNAeic acid . The proeic acid . The cloeic acid . 4 photoinactivation was observed by Abe and Wagner [et al. [meso-tetrakis(1-methylpyridinium-4-yl)porphyrin did not interact with capsid proteins and did not disturb protein-DNA interaction, even if it has a strong stabilization effect on the intraphage DNA.Little alteration of M13 phage proteins on SDS-PAGE after MB and AlPcSd Wagner . The res [et al. , suggestThe development of increasing numbers of antiviral agents over the past decades, in the same way as with antibiotics, has provided the clinician with therapeutic options previously unavailable. With the increasing utilization of antiviral drugs, however, has come an enhanced appreciation of the development of antiviral resistance ,139,140.There is now an urgent need for the development of novel, convenient and inexpensive measures for combating antimicrobial-untreatable infections and limiting the development of additional antimicrobial resistant microorganisms. Photodynamic technology may provide one approach to meet this need, both in terms of therapy and in terms of sterilization, by a mechanism that is markedly different from that typical of most antimicrobials ,141,142.As mentioned before, photosensitization involves the generation of singlet oxygen and free radical species, which cause molecular damage. Whether microorganisms could develop resistance to these active oxygen species is still questionable  and, conAntimicrobial PDI, when compared to standard treatments which may require application for several weeks to achieve an effective killing of the microorganism, shortly after initiation of light exposure, exhibits serious and irreversible damage of microorganisms ,68. Thiset al. [Data from North et al.  show thaet al. .Studies focusing on the possible development of viral resistance are extremely scarce and little is known about the recovery of viral viability after consecutive photodynamic treatments.+-Me-PF) at 5.0 μM under white light irradiation, exhibited no changes in the rate of photoinactivation during the course of the experiments, meaning that no resistance was observed. If phage resistance would occur, important reductions on phage photoinactivation efficiency would be detected between experiments. Besides that, T4-like phage did not recover its viability after exposure to Tri-Py+-Me-PF during 120 min of irradiation [Staphylococcus phage by MB were unsuccessful. Concerning bacteriophages, there is only one study focusing on the possible development of viral resistance after photosensitization . After 1adiation . In a prIt has been shown that the location and binding site of the PS, which is highly dependent on the structure and intramolecular charge distribution, is an important factor in microbial PDI ,147.In terms of molecular structure, molecular charge is important in determining antimicrobial activity. Positively charged PS are generally more efficient and can act at lower concentrations than neutral and anionic PS molecules . The posCationic PS photodamage can be induced in nucleic acid or viral outer structures by PS binding or by PS localized in its vicinity . For insmeso substituent groups also affect the photodynamic effect. PS with opposite charged groups are more symmetrical than PS with adjacent charged groups. The adjacent positive charges in the PS macrocycle should result in a molecular distortion due to electrostatic repulsion [The symmetry and the size of the chain of epulsion . The toxepulsion . The amphiphilic nature of a PS is another important feature affecting PDI efficiency and can be modulated by the introduction of adequate functionalities in the macrocycle periphery, such as different numbers of positive charges, an asymmetrical charge distribution, or introduction of aromatic hydrocarbon side chains ,151. PS concentration is also an important parameter that must be taken into account since viral PDI was shown to be strongly influenced by PS concentration. Increasing the PS concentration reduces the time needed to achieve complete viral inactivation, thus increasing the efficiency of a particular PDI protocol .4 and 5 min illumination with red light. For the neutral phthalocyanine derivative (Pc4), complete inactivation (4 log) was achieved using a much lower amount of PS (4.5 nM) in combination with 10 min illumination [meso-tetrakis(1-methylpyridinium-4-yl)porphyrin. In contrast, inactivation of HAV to 3.6 log with the anionic symmetric porphyrin meso-tetrakis(4-sulfonatephenyl)porphyrin required 90 min [meso substituent groups [Complete inactivation of VSV (4.2 log) can be obtained by treating it with 1.0 μM of the anionic phthalocyanine derivative AlPcSmination . The inamination . Inactivmination . HAV in d 90 min . The ratt groups . HIV andt groups .meso-tetrakis(1-methylpyridinium-4-yl)porphyrin and 5-(pentafluorophenyl)-10,15,20-tris(1-methylpyridinium-4-yl)porphyrin, respectively) lead to complete T4-like phage inactivation (~7 log) after 270 min of irradiation with 40 W m−2 [meso-tetrakisporphyrin derivatives with different alkyl substituent groups were tested for MS2 phage inactivation but, with the exception of 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin, showed toxicity even in the absence of light [MS2 phage inactivation has been observed with neutral porphyrin derivatives. However, this required higher irradiation periods (30 min) than for the cationic ones (1 min) . Neutral40 W m−2 . This te40 W m−2 . Increas40 W m−2 . A conce40 W m−2 , but ove40 W m−2 . Cationiof light . et al. [meso-substituent groups. The presence of a lipophilic aryl group in one of the meso positions of the porphyrin core appears to have an important role in phage inactivation, affecting the rate and efficiency of T4-like phage [et al. [meso substituent groups.In a study conducted by Gábor et al. , the poret al. , the ratke phage . Casteel [et al.  have alsPDT requires a source of light to activate the PS by exposing it to visible or near-visible light at a specific wavelength . The ligIn parallel with the advances in chemistry (related with the discovery and synthesis of new and more efficient PS) there has also been much activity in developing new light sources, better suited for the photosensitization process. Briefly, these include user-friendly lasers frequently based on solid state laser diodes, as well as inexpensive light emitting diodes (LED) and filtered broad-band lamps . PS activation has been achieved via a variety of light sources, such as arc plasma discharge lamps, metal halogen lamps, slide projector illumination assemblies, and a variety of lasers. For treatment of larger areas, non-coherent light sources, such as tungsten filament, quartz halogen, xenon arc, metal halide, and phosphor-coated sodium lamps, are in use. Recently, non-laser light sources, such as LED, have also been applied in PDT. These light sources are much less expensive and small, lightweight and highly flexible, its lifetime can reach up to one hundred thousands hours, and can be manufactured to wavelengths that activate commercially available PS ,158,159.et al. [et al. [et al. [t is proportional to the fluence rate at that time and the local concentrations of PS and oxygen can be established. However, according to this model, relatively low fluence rates can be nearly as effective as high fluence rate sources if applied over the same period of time [At first glance, the available literature on fluence rate effects for PDT seems contradictory. Some studies indicate less damage at low fluence rate, others indicate more killing at lower, compared to higher, fluence rates for the same total fluence and some indicate no influence of fluence rate at all ,157,158.et al.  showed tet al. . Schindl [et al.  referred [et al.  also ind of time .vice versa. Lower doses of PS require higher activating light fluences, and higher fluence requires a longer duration of light application [There is also a direct correlation between the phototoxic effect and the PS concentration and light fluence. With a lowering of the PS concentration, more light has to be applied to achieve identical effects, and lication . −2) or green-yellow light (provided by low-pressure sodium lamps at a fluence rate of 165 W cm−2) but slower by white light (provided by a bank of fluorescent tubes at a fluence rate of 42 W cm−2) [et al. [−2, given at a total dose of 1.8 × 104 and 3.2 × 104 J m-2, inactivated MB-treated VSV by 6 and ≥7 log, respectively. VSV inactivation was linearly dependent on the fluence rate of red light illumination [The effects of dengue virus inactivation were increased with the increase of MB concentration, the enhancement of power density of the light source and the extension of illumination time, as well as the decrease of illumination distance. This enabled the narrow bandwidth light system to kill or inactivate the enveloped virus at much greater distance in much shorter time . VSV in  W cm−2) , showing [et al.  also shomeso-tetrakis(1-methylpyridinium-4-yl)porphyrin and 5-(pentafluorophenyl)-10,15,20-tris(1-methylpyridinium-4-yl)porphyrin), when irradiated with different sources of light  with fluence rates ranging from 40 W m−2 to 1690 W m−2, efficiently photoinactivated non-enveloped phages. All light sources tested lead to reductions of about 7 log for the somatic T4-like phage. However, the rate and the extent of inactivation were dependent on the light source, namely when low fluence rates were used (40 W m−2) and on the energy dose, being considerably more effective when light was delivered at a lower fluence rate. However, depending on the light source used, different irradiation periods were required to inactivate T4-like phage to the limits of detection. The results also showed that the efficacy of T4-like phage inactivation, using the same fluence rate, was dependent on the light source used, in particular when the light is delivered at a low fluence rate. M13 phage was phototreated with 5.0 μM MB and was inactivated in an irradiation dose-dependent manner [et al. [In terms of what is known about phage PDI, only one study focusing on the effect of different light sources and power densities  exists. t manner . Kasturyt manner  showed tt manner . At hight manner . In a si [et al. , the phaThe efficiency of different types of PS in viral PDI has been proved for different types of mammalian viruses and bacteriophages, whether they are enveloped or non-enveloped, for either DNA or RNA viruses. Even though enveloped viruses are more easily inactivated than non-enveloped ones, several studies confirm that non-enveloped mammalian viruses and phages can be efficiently inactivated by PDI. The type of viral nucleic acid has not been described as an important factor affecting viral photoinactivation but, as far as it is known, no studies specifically focus on the photoinactivation behaviour of DNA and RNA viruses. However, RNA phage MS2 was highly susceptible to photoinactivation when compared with DNA phages under the same conditions of photosensitization.The type of mechanisms involved in the process of viral photosensitization was already elucidated and singlet oxygen and free radical species were identified as important contributors for an effective viral PDI. However, the contribution of singlet oxygen seems to be more pronounced in mammalian viruses and bacteriophage PDI. There are, however, few studies simultaneously comparing the contribution of both types of mechanisms (type I and type II) involved in viral PDI. The primary targets for the photoinactivation of viruses, whether treating mammalian viruses or phages, are the outer structures. Although there are several studies about the specific effects of PDI on viral proteins, for different types of mammalian viruses and phages, there are no studies concerning the specific effects of PDI on viral lipids. However, it has been clearly shown that enveloped viruses are more easily inactivated than their non-enveloped counterparts, which imply that the lipids present on viral envelopes are important targets of viral PDI.PS are effective in inactivating the phages to the limits of detection in a way that they do not recover viability, avoiding the development of viral resistance. Nothing is known yet for the particular case of mammalian viruses but, as the viral targets are the same for mammalian viruses and phages, it is also expected that no resistance will be developed in the case of mammalian viruses. Besides that, antiviral PDI is equally effective whether the mammalian virus is sensitive or resistant to conventional antiviral agents. Taking into account all these advantages, PDI for viral inactivation can be regarded as a promising alternative therapy to conventional antiviral treatments, namely for the disinfection of blood and blood products, preventing viral contamination and for the treatment of wound and burn infections. Viral PDI has a fast mode of action and has also the additional benefits of being more economical and an environmental friendly technology, which might be successfully used also in the environmental field for wastewater, drinking water and fish-farming water disinfection.meso substituent groups and positive charges in order to facilitate their interactions with the viruses, making them more efficient for mammalian viruses and phage PDI.Different PS concentrations and different light sources and fluence rates were tested, showing that they are important PDI parameters that must inevitably be taken into account when a viral photosensitization protocol has to be elaborated. The inactivation of mammalian viruses and phages can be attained at micromolar-level PS concentrations and different light sources are equally effective, depending on the final dose at which the viruses are exposed to. Besides that, PS can also be modulated by the addition of different meso substituent groups, the fluence rate and energy dose. Consequently, it is important to persist in the development of more PDI phage studies to clarify some aspects of viral PDI, such as influence of viral nucleic acid type (DNA or RNA) in the photoinactivation efficiency and the possibility of viral resistance development and viability recovery after photosensitization. It will also be important to study the synergistic effect between viral PDI and antiviral classical methodologies using bacteriophages as models of mammalian virus’ photoinactivation.The similarity of the results obtained for mammalian viruses and bacteriophages show that they exhibit a similar behaviour when submitted to viral photoinactivation techniques: (i) the PS used for viral PDI were equally effective in the photoinactivation of mammalian viruses and bacteriophages; (ii) the mechanism of mammalian viruses and bacteriophage photosensitization involves the production of singlet oxygen (type II mechanism) with a slight contribution of free radical species (type I mechanism); (iii) singlet oxygen and free radicals were shown to affect viral nucleic acids and also the proteins and lipids present in the mammalian viruses and bacteriophage outer surfaces, with the latter being considerably more affected by PDI; and (iv) the rate and extent of mammalian viruses and phage PDI is also affected by the same factors, like the PS concentration and number of positive charges, the nature and position of


Objective. To examine general mental health in adult males and females, who in adolescence participated in a scoliosis-specific therapeutic exercise program or were under observation due to diagnosis of scoliosis. Design. Registry-based, cross-sectional study with retrospective data collection. Methods. Sixty-eight subjects (43 women) aged 30.10 (25–39) years, with mild or moderate scoliosis (11–36° Cobb angle), and 76 (38 women) nonscoliotic subjects, aged 30.11 (24–38) years, participated. The time period since the end of the exercise or observation regimes was 16.5 (12-26) years. Beck Depression Inventory (BDI) and General Health Questionnaire (GHQ-28) scores were analyzed with the χ2 and U tests. Multiple regression analyses for confounders were also performed. Results. Intergroup differences of demographic characteristics were nonsignificant. Scoliosis, gender, participation in the exercise program, employment, and marital status were associated with BDI scores. The presence of scoliosis and participation in the exercise program manifested association with the symptoms. Higher GHQ-28 “somatic symptoms” subscale scores interacted with the education level. Conclusions. Our findings correspond to the reports of a negative impact of the diagnosis of scoliosis and treatment on mental health. The decision to introduce a therapeutic program in children with mild deformities should be made with judgment of potential benefits, risks, and harm. In the coliosis , 3. It rcoliosis  and incrcoliosis . In contcoliosis –3.Spine deformity may significantly influence the general health and patient's self-reported health-related quality of life (HRQoL), with a poor correlation between the radiographic and clinical findings , 3, 7. LTo our best knowledge, there has been no investigation utilizing the Beck Depression Inventory (BDI) and the Goldberg General Health Questionnaire (GHQ) in patients with scoliosis, despite the fact that these instruments are highly popular and widely used in primary health care settings and general populations .Therefore, the aim of our study was to examine general mental health in young adult men and women, who in adolescence participated in a specific therapeutic exercise program or were under observation due to diagnosis of idiopathic scoliosis. We tested the null hypothesis that the participation in scoliosis-specific exercise regime in adolescence is not associated with mental health and well-being in adult life.To improve the quality of reporting, we followed the recommendations of Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement .We recruited participants by a cross-sectional evaluation from the population of subjects examined for scoliosis at the centre of Corrective and Compensatory Gymnastics, Bielsko-Biala, Poland. The Centre provides scoliosis screening for schoolchildren from the urban and suburban population of about 300 000. We analyzed the medical records of 5017 children examined between 1984 and 1995. We considered eligible registries of those children in whom observation or exercise program were recommended, while bracing and/or surgical treatment was exclusion criteria. Because of financial and technical limitations, we based our study on a sample of subjects. For that reason, we subsequently randomly selected 250 registries. We performed a simple random sampling and used a random numbers table for that purpose.n = 6), recent X-ray exposure (n = 1), mental condition (n = 1), history of treatment of depression or other psychological disorders (n = 2). Five people were excluded due to noncompliance with treatment regimen . Of 149 participants finally included in the study, 2 dropped out and 3 did not return the questionnaires. Thus, a total of 144 (57.6%) of the initially selected subjects completed the study. The flow of recruitment and the selection criteria are presented in detail in The largest of the previous observational studies in adults, who in adolescence were braced or treated surgically or observed, included 1476 patients and 1755 controls , while oMean age at diagnosis was 10.5 (range 9–16) years for the whole group, and treatment started for subjects aged 10.7 (9–16) years. The regime involved scoliosis-specific, symmetrical, strengthening, antigravity, and elongating exercises of the postural muscles, performed in group during 45-minute gym sessions twice a week and individually at home . Subjects who did not participate in the exercise program were observed for three to five years on the basis of scheduled follow-up orthopedic examinations. Follow-up period since the termination of treatment was 16.5 (12–26) years for the whole group, 17.1 (12–25) years for the exercising group, and 15.9 (12–23) years for the observed subjects. The differences in the distribution of variables were nonsignificant .Two specialists independently measured the magnitude of the curvature according to the Cobb method , on a fuThe BDI, in its long form, is a 21-item self-reported measure, with items rated 0 to 3 (most severe signs and symptoms) . We applThe assessment of depression alone can be oversimplified, as many depressive symptoms can be the expression of the condition itself . Thus, wThe GHQs are self-administered instruments designed to detect a probable psychiatric disorder. Different versions have been developed, with the most widely used 28-item GHQ-28 , 26, valU test was used for categorical data . To examine the interaction between the BDI scores  and a number of confounders  were within the category of mild depression [P < .01), with more scoliotic than nonscoliotic participants showing depressive symptoms . The differences between the subjects with mild and moderate deformities were also significant (P < .05), with a greater tendency for depressive symptoms in subjects with milder deformities. Only 11 (14%) nonscoliotic and 7 (10%) scoliotic participants exceeded the 10-point threshold. The results are shown in Median total BDI scores of both scoliotic and nonscoliotic groups  and in unaffected individuals . Differences between both groups and between subjects with mild and moderate scoliosis were nonsignificant .P < .05). In all comparisons, the overall mean and median values were far below the maximum possible results. None of the scores exceeded the figures of 11 for the subscales, which may be considered as achieving “caseness” [aseness” .For the multiple regression analysis for both BDI and GHQ-28—with the total score and subsequent domains analyzed—we included age, gender, presence of scoliosis (>10° Cobb), treatment type (exercise regime versus observation), marital status, employment, level of education, and presence of scoliosis in relatives as potential confounders. We only report findings with significant or nearly significant differences . TherefoP = .06), but taking into account a considerably low power of the study  and the risk for type II error, we cautiously considered these findings as indicating a possible tendency.Gender, employment, and marital status were associated with BDI results . Being mP < .05) between the level of education and somatic symptoms in GHQ-28 subscale A . Also, being male seemed to be negatively associated with the tendency towards somatization . In general, with lesser confidence (nonsignificant associations but with high odds ratios), the multiple regression for the GHQ-28 scores confirms the BDI findings.We found a significant negative interaction (P < .05). Depression can be an understandable response to loss and injuries to self-esteem associated with the diagnosis established in adolescence, which is the most vulnerable period for the formation of adult identity [There are no set thresholds for the interpretation of the GHQ-28 scores, but nonetheless both the total and domain scores in the scoliotic and nonscoliotic groups were below the 11-point threshold of possible “caseness” . Furtheridentity , 31. TheThere are no results available from similar studies regarding adults with a history of participation in scoliosis-specific exercise programs, but studies on adolescent patients treated with a brace indicate that scoliosis and therapy are significant risk factors for depression, suicidal thoughts, worry and concern over body development and appearance, and peer reactions, not only in girls, but also in boys . OutcomeWe observed a tendency for depressive symptoms associated with the participation in the exercise program. This finding is, on the surface, in opposition to the assumptions about the beneficial effect of exercising on mental health. Physical activity has been shown to be associated with decreased symptoms of depression and anxiety, life satisfaction, cognitive functioning, and psychological well-being. In contrast, physical inactivity is believed to be associated with the development of psychological disorders, and exercise and exercise training are considered as a treatment of depression , 36. WitP < .05) and somatization . The latter finding corresponds with the observation from intragroup comparisons: more women, irrespective of the group, had BDI scores greater than 10 points, considered in some interpretations as a threshold of depression [The multiple regression analysis revealed an adverse association of male gender with the tendency for depression (pression , and thepression . Howeverpression  and in apression . Transiepression .Lower level of education and being employed, unlike the family history of scoliosis, were shown to be associated with a lower tendency for depression. These observations confirm that we have to be cautious in drawing firm conclusions from this cross-sectional analysis, and that the reported associations are affected by confounding factors, which is typical for observational studies investigating real-life phenomena.Because it was an uncontrolled observational study with a long follow-up of 16.5 years, we cautiously analyzed the obtained data with respect to limitations of the method and a number of potential confounding factors. Also, to reduce selection bias, we identified the eligible participants by applying a registry-based procedure, comprising the records of subjects from the entire local population of children and adolescents, and used a systematic procedure of random selection of the records. Participants did not differ significantly in demographic  and clinIn conclusion, in this first outcome study of mental health of young adults with mild-to-moderate scoliosis, observed or treated with a program of scoliosis-specific exercises in adolescence, we have shown that in a long-term perspective people with mild scoliosis do not differ significantly from unaffected persons in terms of depression and other characteristics of general mental health: somatic symptoms, anxiety, insomnia, and social dysfunction. However, we found that participation in exercise treatment program in adolescence and spinal deformity even mild, or perhaps the awareness of the deformity, may have associations with depression symptoms. Thus, these findings correspond to the reports of a negative impact of scoliosis on mental health, regardless of its severity. Also, our observations may indicate that the decision to introduce a strenuous and demanding therapy should be made with caution, with proper evidence-based clinical judgment in terms of patients' needs and potential benefits, risks, and harm of an intervention.


Individual health education is considered to be essential in the overall care of patients with type 2 diabetes (DM2), although there is some uncertainty regarding its metabolic control benefits. There have been very few randomized studies on the effects of individual education on normal care in DM2 patients with a control group, and none of these have assessed the long-term results. Therefore, this study aims to use this design to assess the effectiveness of the PRECEDE  education model in the metabolic control and the reduction of cardiovascular risk factors, in patients with type 2 diabetes.An open community effectiveness study was carried out in 8 urban community health centers in the North-East Madrid Urban Area (Spain). Six hundred patients with DM2 were randomized in two groups: PRECEDE or conventional model for health promotion education. The main outcome measures were glycated hemoglobin A1c, body mass index (BMI), blood pressure, lipids and control criteria during the 2-year follow-up period.Glycated hemoglobin A1c and systolic blood pressure (SBP) levels decreased significantly in the PRECEDE group . The decrease levels in diastolic blood pressure (DBP), triglycerides and LDL cholesterol were nonsignificant. PRECEDE increased compliance in all control criteria, except for LDL cholesterol. BMI did not change during the study in either of the two models analyzed.PRECEDE health education model is a useful method in the overall treatment in patients with type 2 diabetes, which contributes to decrease glycated hemoglobin A1c and SBP levels and increase the compliance in all the control criteria, except for LDL cholesterol.NCT01316367ClinicalTrials.gov  Type 2 diabetes mellitus (DM2) is one of the chronic diseases that have increased in prevalence and incidence rates in recent years , and somIndividual health education is considered to be essential in the overall care of patients with DM2, although there is some uncertainty regarding its metabolic control benefits. The PRECEDE  model developed by Green and Kreuter  is one oThe efficiency of the PRECEDE model has been proven in different studies in the health enviroment, such as improving care habits among asthmatic children and improving medication adherence in patients with a chronic disease ,8; howevThere have been very few long-term studies, with randomized controls, on the effects of individual education in normal care in DM2 . TherefoWe conducted an open community effectiveness study in which 21 Primary Care Centers (PCC), in the North-East Madrid Urban Area (Spain), were invited to participate; 13 refused, and 8 PCC were randomized in two arms: Precede Health Promotion Education (PHPE) and Conventional Health Promotion Education (CHPE). The participants at each PCC were randomized by selection from lists of patients with previously diagnosed DM2. Figure Eligibility criteria for patients were: older than 30 years, with previously diagnosed DM2  and the exclusion criteria were: gestational diabetes, patients involved in clinical trials, patients with life expectancy less than 1 year , patients who refused to participate, homebound patients. Patients meeting criteria for inclusion and not meeting any exclusion criteria were invited to participate, and were included after accepting and signing an informed consent form.Visits in both groups included the usual care and individual counseling, based on the CHPE or PHPE models, respectively.The CHPE model was defined according to the recommendations of the Spanish Ministry of Health National Conference on Diabetes Mellitus , which wThe PRECEDE HPE model is a diagnostic tool behavior and therefore the first step in its implementation is to identify the behavior to be analyzed. The model considers the influence of the following three factors on health-related behavior:-Predisposing: factors influencing the patient's motivation to undertake the behavior to be analyzed or encouraged.-Facilitators: factors influencing the level of easiness or difficulty the patient and his/her family have in undertaking a given behavior.-Reinforcing: factors arising after the patient has undertaken the behavior, and which reward or punish it.Nurses in the experimental group had to answer the following question: What does the patient need to change behavior? . After, two behaviors were selected for each patient. The nurses research-practitioners first looked at Predisposing factors that influence the analyzed behavior. Patient's responses and comments were written in two parallel lists: positive (+) and negative (-) factors in patients behaviors that need improvement. Predisposing factors are subjective . Subsequently, factors that facilitate the studied behavior were analyzed. These are objective factors such as patient's skills or availability of resources. Finally, subjective Reinforcing factors (what the patient says after his/her behavior) and objectives .Researchers in the experimental group received training in the PRECEDE model before patients were included in the study. This specific training involved two steps: first they were instructed about the basic, theoretical, and practical concepts involved in the application of the model. Second, they participated in a course on clinical interviews to improve their skills when dealing with patients.Researchers in both groups were subsequently trained in the procedure to be used in the study in three sessions. These covered the criteria for including/excluding patients, collecting variables, collecting biochemical/biological parameters, resolving doubts, and piloting the data collection process with the first histories.The study was carried out during a 2-year follow-up period (2003-2005) and the number of visits was identical for both groups: 10 visits .Baseline data were collected during visits 0 and 1, and during visits 2 to 9 assigned models were applied and the data collected. The PRECEDE model application for each behavior analyzed took 4 visits (sessions), as shown in Figure , associated morbidity , and cerebrovascular accident (CVA), diabetes mellitus complications , and the type of treatment prescribed . In follow-up visits data were collected on various biochemical-biological parameters , and diastolic blood pressure (DBP), total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides, and HbA1c). Blood pressure was measured according to the recommendations of the Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (2003) [The data gathered were sociodemographic variables , hygienic and dietary habits, female menopause, tobacco consumption (cigarettes/day), alcohol consumption , physical activity practice (measured in hours per week considering any exercise or activity outside the regular job), self-monitoring of capillary glycemia, foot self-care, medication adherence (was measured using Haynes-Sackett test : 'Most pe (2003) .The primary outcome was HbA1c, lipid levels, blood pressure, BMI after 24 months of follow-up.For an alpha of 0.05, a power of 80%, and in order to detect an reduction of HbA1c of 0.3 percentage units, with a common standard deviation (SD) of 1.15, in the PRECEDE group, the overall sample size required was 462 patients (231 in each arm of the study). Since randomization was by PCC, the sample size had to be larger than if simple randomization had been performed, in order to consider the design effect (DE). The DE was calculated as follows: DE = 1 + (nc - 1) * ICC . The ICC in the present work was deemed to be 0.01. The mean cluster size was assumed to be 30 patients. Given these assumptions, the final sample size required was 596 patients (298 in each arm).t-test or its nonparametric equivalent was used for paired data (Wilcoxon test). Furthermore, Pearson χ2 test was used for the qualitative variables, and McNemar's test was used for paired data.First, a descriptive analysis was carried out for each variable included in this study, involving the mean and SD for the quantitative variables and frequencies with confidence intervals of 95% (95% CI) for the qualitative variables. The Student's The change  was calculated in both diabetological education models, for the following variables: total cholesterol, LDL cholesterol, HDL cholesterol, HbA1c, SBP, and DBP. The effect of the PRECEDE HPE was determined for these variables using the formula: mean value of the change in HPE PRECEDE - mean value of the change in conventional HPE. The covariance analysis methodology (ANCOVA) proposed by Vickers was used to determine the adjusted effect of PRECEDE . The adjIn all instances, the accepted level of significance was 0.05 or less, with 95% CI. All the analyses were carried out using the intention-to-treat principle. Statistical analysis of the data was carried out with SPSS 15.0 .A total of 608 patients were included, of which 51.6% were women, with a mean age of 66.7 years (SD: 14.5), and a natural history of disease mean of 9.1 years (SD: 8.3). A total of 304 patients were assigned to the conventional model and 304 to the PRECEDE model. The two groups studied according to the type of health promotion model were observed to be homogeneous in terms of gender, age, and DM2 evolution time. The baseline clinical characteristics of the two groups, the distribution of morbidity, and chronic complications are shown in Table The PRECEDE model led to a favorable variation in all parameters studied, while the conventional model group failed to achieve an improvement in HbA1c, triglycerides, or SBP, which showed a slight increase Table .p < 0.01), and the decrease in DBP, triglycerides, and LDL cholesterol was nonsignificant. Furthermore, the total cholesterol remained unchanged  in the PRECEDE model. Furthermore, SBP decreased by 3 mmHg , metabolic control (HbA1c <7% and LDL cholesterol <100 mg/dl) (p = 0.02), SBP <130 mmHg (p = 0.02), DBP <80 mmHg (p = 0.01), BP control (<130/80 mmHg) (p < 0.01), and global control (metabolic and BP) (p = 0.02). Nevertheless, it was not significant for the criterion LDL <100 mg/dl and BMI <25 Kg/m2 , BP control (SBP <130 and DBP <80), or overall control (metabolic and BP <130/80 mmHg), indicating that our results cannot be compared.However, on comparing the increase in the proportion of patients with metabolic control in the PRECEDE model obtained in our study with those of transversal studies carried out in primary healthcare, such as the one by Spann et al , we wereThe increase obtained in the proportion of patients with metabolic, blood pressure, and overall control after the application of PRECEDE model is relevant and suggests that there is a need to complement pharmacological treatments with lifestyle modification strategies like the one proposed by the PRECEDE model.The most important limitation of this work is the nature of non-blind experimental studies, with the possibility of bias during response measurement, as researchers know which patients are members of the experimental group. Although, this bias is improbable because measurement of responses was objective, as it was based on results of analytical determinations. However, we believe that there could have been a possible Hawthorne bias effect, with a change in the behavior of the subjects owing to the knowledge that they are being studied. This would have had the same effect on both the groups, because the follow-up of the patients was stricter than normal in the two HPE arms, and they were all well aware of their participation in an experimental study when they signed the consent. A virtue of the study was that there were few losses, indicating that there was no selection bias owing to selective losses and that the analyses were carried out according to the intention-to-treat principle.As a result of all the above-mentioned factors, it can be concluded that the PRECEDE health promotion model is a useful method in the overall treatment of DM2 patients, because it contributes to significant decrease in HbA1c and SBP levels, as well as helps in increasing the compliance with all the control criteria, except for LDL cholesterol. Our findings indicate that further studies are necessary to substantiate these benefits. If they are confirmed, then the impact of the PRECEDE model should be evaluated in terms of cardiovascular morbimortality.The authors declare that they have no competing interests.MASF conceived of the study and participated in its design and perfomed the statistical analysis and drafted the manuscript. FJAB, JCAH, CBL drafted the manuscript and made substantial contributions to the analysis and interpretation. CMM participated in the design and coordinated the research group. ECSP, BRS helped in the statistical analysis and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/11/267/prepub