The training of health workers in the use of malaria rapid diagnostic tests (RDTs) is an important component of a wider strategy to improve parasite-based malaria diagnosis at lower level health care facilities (LLHFs) where microscopy is not readily available for all patients with suspected malaria. This study describes the process and cost of training to attain competence of lower level health workers to perform malaria RDTs in a public health system setting in eastern Uganda.Health workers from 21 health facilities in Uganda were given a one-day central training on the use of RDTs in malaria case management, including practical skills on how to perform read and interpret the test results. Successful trainees subsequently integrated the use of RDTs into their routine care for febrile patients at their LLHFs and transferred their acquired skills to colleagues (cascade training model). A cross-sectional evaluation of the health workers’ competence in performing RDTs was conducted six weeks following the training, incorporating observation, in-depth interviews with health workers and the review of health facility records relating to tests offered and antimalarial drug (AMD) prescriptions pre and post training. The direct costs relating to the training processes were also documented.Overall, 135 health workers were trained including 63 (47%) nursing assistants, a group of care providers without formal medical training. All trainees passed the post-training concordance test with ≥ 80% except 12 that required re-training. Six weeks after the one-day training, 51/64 (80%) of the health workers accurately performed the critical steps in performing the RDT. The performance was similar among the 10 (16%) participants who were peer-trained by their trained colleagues. Only 9 (14%) did not draw the appropriate amount of blood using pipette. The average cost of the one-day training was US$ 101 (range $92-$112), with the main cost drivers being trainee travel and per-diems. Health workers offered RDTs to 76% of febrile patients and AMD prescriptions reduced by 37% six weeks post-training.One-day training on the use of RDTs successfully provided adequate skill and competency among health workers to perform RDTs in fever case management at LLHF in a Uganda setting. The cost averaged at US$101 per health worker trained, with the main cost drivers being trainee travel and per diems. Given the good peer training noted in this study, there is need to explore the cost-effectiveness of a cascade training model for large scale implementation of RDTs. With the widespread implementation of the use of artemisinin-based combination therapies (ACTs) as first-line anti-malarials in many African countries , World HTask shifting is becoming a popular strategy to improve health care within the constraints of limited resources in sub Saharan Africa -7 and maThis implementation research was conducted to train and document the process of training health workers to perform and use RDTs in a public health care setting. Observations from this study should guide the National Malaria Control Programme (NMCP) in the training and scale up of RDTs use as one of the strategies to implement the policy of parasite-based diagnosis and treatment of malaria in the public sector.In a cross-sectional study, one-day training on the use of RDTs in malaria case management was conducted for health workers at LLHFs, typically primary health facilities that serve 1,000-25,000 people , within five eastern districts in Uganda. Each district was a representative of the different malaria epidemiological settings in Uganda; that is; Kapchorwa - a hypo-endemic, Mudende - meso-endemic and Iganga – a hyper endemic setting. Jinja and Mbale were included to represent the fourth epidemiological stratum (urban setting), which potentially differs from the rural settings in terms of knowledge, health seeking behaviour and general health services delivery. The one-day training was done within a larger implementation trial to determine the utility of RDTs at LLHFs .One week following the one-day training, RDTs  were distributed to all the study health care facilities together with accessories, including gloves, sharps disposal bins, lancets and timers. The facilities were eligible for training on RDTs if they met the following criteria: 1) current lack of functional parasite-based diagnostic services, 2) no previous involvement in a similar research, 3) over 200 suspected malaria cases managed at the facility per week and 4) availability of a recording system that comprised outpatient registers, health management information system (HMIS) and drug consumption data. Of the eligible facilities , four were randomly selected from each district. In each of the five districts, one health facility did not receive training and was used as a comparison group. Baseline data was extracted from out-patient department (OPD) registers to document antimalarial prescriptions practices before the training and introduction of RDTs.All health workers from the selected facilities were invited to a district-based training in the use of RDTs for malaria in a staggered fashion to ensure minimal interruption of the service delivery at the health care facilities. Two one-day trainings were conducted, each consisting of half the trainees with a ratio of 5:1 trainees for one facilitator. The training and assessment tools were piloted and standardized in accordance with the Ministry of Health. A total of 135 health workers received training. A pre-training exercise was conducted to assess the health workers’ knowledge in malaria case management. The training was based on the WHO curriculum for the use of RDTs  and the Participants were considered competent if they scored ≥ 80% in the post-training practical concordance test .All trainees were provided with a transport refund, per diem and one night’s accommodation on the day of the training. The data on direct training costs were collected retrospectively from financial and operational records, in particular expenditure and salary records. Additional costing information was acquired from interviews with key personnel; in particular the project’s Logistic Manager Data extraction was based on an itemised menu approach (Drummond MF 2005 Oxford press) and was undertaken between July and December 2008 at the time of the parent study. The results are presented from the implementer’s perspective. Where possible both financial and economic costs were collected in order to estimate financial requirements for programme implementation. The financial costs included monetary costs of the training while economic costs represent the value (or opportunity cost) of the resources used to implement the training including the trainees’ time as estimated from government pay scale. Research costs were not included except support supervision after the training which we considered as a recurrent cost in large scale implementation of RDTs. Operational costs included venue hire, meals and transportation of trainees to the practical training sites, transport and per diem, lodging and incidental expenses for both trainers and trainees. Training materials were not included in this estimate given they are readily available from the NMCP and capital costs were not included given the short term nature of the training. Programme implementation costs were classified into a) set-up costs which included a three-day trainer of trainers (TOT) based on a pre-developed protocol/guidelines and b) recurrent costs such as the one-day training  as well as the national and local supervision costs. The cost of training each health worker including the follow up supervision was computed based on expense documents. The ingredient approach was used, whereby inputs were identified valued and classified into activities. Where this approach was deemed too sensitive , aggregated expenditure was used. All costs were converted to United States dollars (USD) based on official yearly average exchange from OANDA (http://www.cioms.ch/frame_guidelines_nov_2002.htm). Ethical approval was granted by the Uganda National Council for Science and Technology (UNCST). Support for the study was acquired from the ‘in-charge’ of each of the selected health facility and each participant provided written informed consent to participate.The study was conducted according to the principles of the declaration of Helsinki and the international guidelines of biomedical research involving human subjects . The two data files were compared to check for completeness and conflicting entries. One copy was then cleaned and exported to SPSS 12 (SPSS USA 2005) for further checks on completeness of the data and subsequent analysis.Demographic data were recorded for the health workers that attended the training and the subsequent evaluation of competences. The skills and competences attained were compared among the nursing assistants  and the formally trained health workers  to evaluate the feasibility of transferring the skills of performing RDTs to the lowest cadre of health care providers at LLHFs. Qualitative data from in-depth interviews of health workers was analysed manually using pre-set themes of ease of use of the test, competence and confidence to perform the test and feasibility of using RDTs in routine management of suspected malaria. Costing information was documented and analysed.A total of 135 health workers were trained within the 5 districts of Iganga, Jinja, Kapchorwa, Mbale and Mubende. Each district was represented by health workers from at least four health care facilities. The health workers included clinical officers, nurses, laboratory technicians and nursing assistants ; see Table A total of 64(47%) health workers were available for the six weeks post training evaluation. During the evaluation, 51/64 (80%) of all health workers accurately performed the steps critical for performing a RDT, and interpreted and recorded the results correctly in the books and registers (see Table The average cost of the one-day training per person trained was $101 (range $92-$112). Over half of the total cost of training $7,861/15,160 (52%) was spent on participants’ travel and per-diem . Five (20%) nursing assistants reported difficulty in pricking a finger and filling a pipette with blood as the most challenging steps. All the other steps were well performed considering that over 85% reported them as very easy to perform tasks.During the follow up evaluation, the health workers reported that training materials were easy to use and many of them felt they were able to train other health workers. In addition, majority (98%) of the health workers felt that they had acquired adequate knowledge and skills to integrate RDTs into their routine patient care. However some health workers (50%) felt that there is need to increase the numbers of health workers if RDTs are to be performed regularly.Our study showed that health workers across different cadres and with different levels of pre-service training were able to learn how to effectively perform the ICT RDT, from a one day training session. Nursing assistants experienced some difficulties in pricking the finger with a lancet and filling a pipette with blood. Similar difficulties have been reported in Tanzania , South AThe one-day training given at a central location seems sufficient to transfer the skills necessary to roll-out the use of RDTs for malaria diagnosis at peripheral health facilities in Uganda. Immediate utilisation of RDTs (within 6 weeks of training) appears to be recommended as evidenced by the finding that 75% of febrile patients who presented to the health facilities thereafter were offered a diagnostic test. It is important to design studies to evaluate the long-term retention of knowledge and skills attained as well as to assess the need and timing of re-fresher training to maintain the high utilization and quality in performance of RDTs for parasite-based malaria diagnosis at lower level health care facilities.It was interesting to demonstrate that health workers who received the one-day training in RDT use were able to multiply this effect by adequately training their counterparts at their respective health facilities (cascade model training). This finding is consistent with reports from the malaria case-management team at the Infectious Diseases Institute in Uganda, which showed that cascade training by trainees after a trainer of trainers (TOT) course was comparable to the initial first-hand training . HoweverThis is one of the first studies to document the cost of the training process on the use of RDTs in a resource-limited setting. The one-day training cost up to US $101 to train one health worker to use RDTs. The documented costs included the cost of facilitating the core trainers from the centre  to the districts, transport and operational costs. This study did not document capital costs including preparation of training materials since this is an intervention that should be integrated into the current health care system to improve the efficiency of service delivery. We recommend cost-effectiveness studies to understand the utility and long-term impact of one-day on the scale up of parasite-based malaria case management in resource-limited settings.Lessons learnt: Given the limited numbers of health workers, there is need for strategic phasing of the training sessions to avoid interruption of services at the health facilities. It is for this reason that we performed two one-day trainings to have half the trainees attend independently while the other half offered patient care and vise-versa. Within the context of the health system where the majority of health care providers are not formally trained, the training content should be tailored to suit the lower academic levels and should include more on background knowledge in addition to the practical skills.This study did not cost the preparation of the training materials since these materials are readily available from the NMCP. Generic training materials have also been made available through collaborations with international agencies like WHO . It is lOne-day training cost $101 per health worker and successfully delivered adequate skills and competences among health workers to perform and use RDTs in fever-case management at LLHFs in Uganda. The cascade training model should be explored as a potential strategy for affordable implementation and scale up of parasite-based diagnosis of fever at LLHCFs in Uganda.The authors declare that they have no competing interests.All authors read and approved the final manuscript. DJK CA and JKT participated in the conception and design of the study, protocol development and the training, and participated in data analysis and interpretation of results. DJK and DN wrote the first draft of the manuscript and were responsible for finalising this article. CA coordinated the study. CS participated in manuscript review. JN and MB participated in the training of health workers and acquisition of data. JKT and JPV provided technical oversight, critical review of the manuscript and its revisions.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/12/291/prepubFigure S1. Scatter plot showing the concordance test results plotted against post test written results for 126 trainees that participated in the study to introduce RDTs at lower level health centres in Uganda 2007.Click here for fileTable S1. Date collection instrument used to evaluate Health worker performing RDT with instructions to the supervisor. During the post - RDT training supervisory visit, observe and assess the performance of each step for a maximum of five RDTs performed by each user. All problems encountered by the user which should be addressed during subsequent discussions.Click here for file


A mechanistic codon substitution model, in which each codon substitution rate is proportional to the product of a codon mutation rate and the average fixation probability depending on the type of amino acid replacement, has advantages over nucleotide, amino acid, and empirical codon substitution models in evolutionary analysis of protein-coding sequences. It can approximate a wide range of codon substitution processes. If no selection pressure on amino acids is taken into account, it will become equivalent to a nucleotide substitution model. If mutation rates are assumed not to depend on the codon type, then it will become essentially equivalent to an amino acid substitution model. Mutation at the nucleotide level and selection at the amino acid level can be separately evaluated.The present scheme for single nucleotide mutations is equivalent to the general time-reversible model, but multiple nucleotide changes in infinitesimal time are allowed. Selective constraints on the respective types of amino acid replacements are tailored to each gene in a linear function of a given estimate of selective constraints. Their good estimates are those calculated by maximizing the respective likelihoods of empirical amino acid or codon substitution frequency matrices. Akaike and Bayesian information criteria indicate that the present model performs far better than the other substitution models for all five phylogenetic trees of highly-divergent to highly-homologous sequences of chloroplast, mitochondrial, and nuclear genes. It is also shown that multiple nucleotide changes in infinitesimal time are significant in long branches, although they may be caused by compensatory substitutions or other mechanisms. The variation of selective constraint over sites fits the datasets significantly better than variable mutation rates, except for 10 slow-evolving nuclear genes of 10 mammals. An critical finding for phylogenetic analysis is that assuming variable mutation rates over sites lead to the overestimation of branch lengths. Growing DNA and protein sequence data is now a valuable source of knowledge in many fields of science, especially in evolutionary biology. Evolutionary history of DNA sequences is a key to understand the diversity of homologous sequences. Any method for inferring molecular phylogeny is implicitly or explicitly based on the evolutionary model of nucleotide or amino acid substitutions, and the reliability of phylogenetic analyses strongly depends on models designed to approximate the substitution processes of nucleotide and amino acid. For the evolutionary analysis of protein-coding sequences, three types of models can be used: nucleotide, amino acid, and codon substitution models. Which type of model fits any sequence data better than the others? Mutational events occur at the nucleotide level, but selective pressure primarily operates at the amino acid level. Thus, a codon substitution model has a potential to outperform both nucleotide substitution models Codon substitution models are classified into either an empirical codon substitution model or a mechanistic codon substitution model. In empirical codon substitution models In mechanistic codon substitution models, a mutational mechanism at the nucleotide level and selection at the amino acid level are distinguished in various levels of separation. If no selection pressure on amino acids is taken into account, the codon substitution model will become essentially equivalent to a nucleotide substitution model. If mutation rates are assumed not to depend on the codon type, then the model will become essentially equivalent to an amino acid substitution model. Such a codon model with the infinitely large synonymous substitution rate, was proved There are two type of models for the mutational scheme of codon, depending on whether multiple nucleotide changes in infinitesimal time are allowed In the present models, mutational tendencies at the nucleotide level are tailored to each gene by the general time-reversible (GTR) model, but multiple nucleotide changes in infinitesimal time are allowed. In the Singlet-Doublet-Triplet (SDT) mutation model There are a variety of models for selection pressure on amino acid replacements in mechanistic codon substitution models; (1) models In the models In the present model, selective constraints on the respective types of amino acid replacements are tailored to each gene in a linear function of a given estimate of selective constraints in the same way with the physico-chemical-constraint models. The simplest model for the selective constraints is to assume equal constraint on amino acid replacements and equivalent to the second category of model; it is named here the Equal-Constraint model. Of course, physico-chemical estimates of the selective constraints can also be used A property of codon substitution models in which synonymous substitutions can be identified is an advantage over nucleotide and amino acid substitution models. Significance of rate variation over sites in proteins has been demonstrated mostly in nucleotide substitution models and empirical amino acid substitution models Besides the variation of substitution rate over sites, the variation of substitution rate over time at each site is also possible. The site-specific variation of substitution rate over time was first discussed as a covarion model by Fitch and Markowitz The estimation of branch lengths is critical on the estimation of phylogeny and divergence times. We examine how differently branch lengths are estimated between models. The present mechanistic codon substitution model can simulate a wide range of codon substitution processes by changing parameters, and can provide biologically meaningful information at both nucleotide and amino acid levels such as transition/transversion rate bias, the ratio of multiple nucleotide changes, the strength of selective constraints on amino acids, the variation of mutation rate or selective constraints over sites, and also the variation of mutation rate over time in branches. Here, the present codon substitution models with the various sets of parameters are extensively studied, and the advantages of the present model over other models are demonstrated.When substitutions independently occur at each site with a constant substitution rate In a given phylogeny of molecular sequences, a substitution process of codon or amino acid is assumed to be in equilibrium. In other words, the substitution process is assumed to be time-reversible. Also, exchangeabilities Amino acid exchangeabilities These amino acid substitution models can be converted into codon substitution models by defining codon exchangeabilities on the basis of amino acid exchangeabilities between encoded amino acids n, WAG-n, LG-n, mtREV-n, cpREV10-n, and cpREV64-n.The codon exchangeability n, where the suffix n means the number of ML parameters.Kosiol et al. In the present mechanistic codon substitution model The frequency-dependent term Selective constraints n, EI-n, JTT/WAG/LG-ML91+-n, and KHG-ML200-n.The Equal-Constraint model with The mutation rate matrix If multiple nucleotide changes were completely ignored, then Eq. 10 would be simplified as The No-Constraint model, in which there is no selection pressure on amino acid replacements , In the mechanistic codon substitution model, selective constraint In the other codon models, the equal amino acid constraint The shape parameter A mutation rate at each site may vary in each branch, especially long branches, of a phylogenetic tree. If the variation of mutation rate is synchronized among sites, it will be reflected by the length of each branch. The unsynchronized portion of rate variation among sites is considered. Here, a simple approximation for the variation of mutation rate over time is provided. The mutation rate matrix This approximation for the variation of mutation rate over time is very simple and does not require any additional computational time, although the performance will be limited in comparison with a more complete approximation Substitution models are evaluated by using five datasets of codon sequences; (1) divergent and (2) closely-related chloroplast-encoded genes, (3) fast-evolving interspecific and (4) highly-polymorphic intraspecific mitochondrial genes, and (5) slowly-evolving nuclear genes.Synechocystis PCC6803, which was the outgroup sequence in their analysis, is not used in the present analysis. The codon sequences were obtained from the NCBI RefSeq database of organelle genomes. The total codon length of aligned genes is equal to Dataset cpDNA-9: Divergent codon sequences consisting of 45 protein-coding genes from 9 chloroplast genomes, whose protein sequences were used to estimate the cpREV10 by Dataset cpDNA-55: Codon sequences consisting of 52 protein-coding genes from 55 chloroplast genomes of the major angiosperm lineages, which are genome sequences available in the NCBI RefSeq database out of the 64 genomes analyzed in Dataset mammalian-mtDNA: Interspecific mammalian mitochondrial codon sequences consisting of 12 protein-coding genes from 69 mammalian species Dataset human-mtDNA: Intraspecific human mitochondrial codon sequences consisting of 12 protein-coding genes from 53 human races Dataset nDNA: Codon sequences of the 10 most slowly-evolving genes out of the 2789 nuclear genes of 10 mammals that were analyzed by Homologous codon sequences are aligned every gene by ClustalW2 Model selection must be pursued with considerable attention The naming convention of the present models is briefly described in First, each gene in a dataset is separately aligned and then all aligned sequences are concatenated. The maximum log-likelihood values of a given phylogenetic tree of concatenated genes for various codon substitution models are listed in In the case of mitochondrial genes, i.e., mammalian-mtDNA and human-mtDNA, the models based on mtREV always show the smallest The empirical codon substitution model KHG performs significantly better for chloroplast-encoded and nuclear-encoded genes than all the amino acid substitution models converted into the codon models. It has often be insisted that synonymous substitutions are saturated between distantly related genes and so substitution analyses at the codon level hardly include more information than those at the amino acid level. However, a fact that KHG performs better even for the distantly related sequence family (cpDNA-9) than the models converted from cpREV10 indicates that codon sequences include more information than amino acid sequences even in the case of distantly related sequences.Borentheria, Xenarthra, and Afrotheria.If the amino acid substitution models converted into codon models are compared with the mechanistic codon substitution models, the superiority of the codon substitution models will be clearer. For all datasets, the mechanistic codon models with the various estimates of selective constraints show significantly lower The EI model, in which the selective constraints were evaluated on the basis of average contact energies between residues in proteins In the concatenating analyses of multiple genes, it is assumed that all genes have no difference in equilibrium codon frequencies, nucleotide exchangeabilities, and the variations of mutation rate and of selective constraint. These assumptions are not always appropriate. Thus, the separating analyses of multiple genes have been carried out. The The mechanistic codon substitution model performs better for a wide range of sequences from highly-homologous to highly-diverged sequences than both nucleotide and amino acid substitution models. This is because it takes into account both mutational tendencies at the nucleotide level and selection at the amino acid level.Significance of rate variation over sites in proteins has been demonstrated in nucleotide substitution models and amino acid substitution models The discrete gamma distribution with 4 categories has been used to emulate both the variations of selective constraint and of mutation rate over sites. The models with variable selective constraints and with variable mutation rates are specified by dG4s and dG4r, respectively. However, in both the concatenating analyses and the separating analyses of genes, the Selective constraints against amino acid replacements at each site must reflect both structural and functional constraints on a residue type at each site, which are required for a protein to fold into a unique native structure and to properly function, and vary among residue sites in a protein. Here a simple analysis of site dependencies of selective constraints has been performed to ascertain the correlation between selective constraints and structural constrains at each site.Site dependencies of selective constraints are evaluated The correct estimation of branch length is critical for the estimations of phylogeny and divergence times. It is known that branch-length estimation is significantly influenced by model selection. Yang et al. In Branch lengths estimated by the models with the variation of mutation rate (dG4r) and with a uniform substitution rate are both roughly proportional to those shown on the abscissa, i.e., those estimated by the model with the variation of selective constraint (dG4s). However, as pointed out by Yang et al. When the different types of models are compared with each other, the correlation of branch lengths between the models is not always good. In In the result, except for the datasets consisting of highly-homologous sequences, the variation of selective constraint is a better model than the variation of mutation rate, and assuming the variation of mutation rate leads to the overestimation of branch length. Even for highly-homologous sequence families, the model with the variation of selective constraint may not be too bad, because the differences of AIC and BIC between the models with variable mutation rates and with variable selective constraints are not significantly large, and the branch lengths estimated by those models are almost proportional to each other.Codon substitutions requiring multiple nucleotide changes can be caused by either multiple steps of single nucleotide changes or single steps of multiple nucleotide changes. In the present mechanistic codon substitution model, codon mutations by multiple nucleotide changes in infinitesimal time are taken into account. The mechanistic codon substitution models with the various selective constraint matrices all indicate On the other hand, the parameter for multiple nucleotide changes is not significant for the datasets human-mtDNA and nDNA that consist of closely-related or highly-conserved sequences, and whose phylogenetic trees consist of short branches only. This fact indicates that multiple nucleotide changes rarely occur in short evolutionary periods, and multiple nucleotide changes detected in relatively long branches of cpDNA-9, cpDNA-55, and mammalian-mtDNA may result from compensatory substitutions that shortly succeed single nucleotide substitutions, or other mechanisms. A possibility of successive single compensatory substitutions for multiple nucleotide changes was pointed out by Bazykin et al. bThe site-specific variation of amino acid substitution rate over time was first discussed as a covarion model by Fitch and Markowitz The present model includes a parameter The mechanistic codon substitution models specified with a suffix dG4s, in which selective constraints are variable across sites, all indicate As shown in One of the advantages in mechanistic codon substitution models over amino acid substitution models is that mutational tendencies at the nucleotide level can be estimated. The estimation of mutational tendencies by mechanistic codon substitution models must be more precise than by nucleotide substitution models applied to all codon positions, because selection at the amino acid level is taken into account.Transitional substitutions have been noted to occur more frequently than transversions In the present mechanistic codon substitution model, single nucleotide mutations are modeled by the GTR model and multiple nucleotide mutations in infinitesimal time are assumed to occur independently at each position of codon, and selective constraints on amino acids are approximated by a linear function of the empirical selective constraints. It has been shown that even the Equal-Constraint model performs far better for a wide range of sequences from highly-homologous to highly-diverged sequences than both the No-Constraint model and the amino acid substitution models converted into the codon substitution models. The No-Constraint model is a nucleotide substitution model extended to allow multiple nucleotide changes in infinitesimal time. On the other hand, the codon substitution model converted from the amino acid substitution model is extended here in such a way that the special case of The present analyses have also shown that the mechanistic models with the amino acid dependent selective constraints do not only perform far better especially for phylogenetic trees consisting of relatively long branches than the Equal-Constraint model, but better even for phylogenetic trees consisting of short branches. This result indicates the superiority of the selective constraint matrices It was pointed out The present analyses show that multiple nucleotide changes in infinitesimal time are statistically significant in long branches as well as the variation of mutation rate over time. It has been also shown that the variation of amino acid substitution rate over sites results from variable selective constraints rather than variable mutation rates at least in the phylogenetic trees of cpDNA-9, cpDNA-55, and mammalian-mtDNA including long branches. Branch lengths will be overestimated for these datasets if the variation of mutation rate over sites is assumed instead of the variation of selective constraint. The capability of the mechanistic codon substitution models to extract biological knowledge from protein-coding sequences makes them superior to both nucleotide and amino acid substitution models.


Despite the progress in techniques for cancer prevention, detection, and treatment, as well as for increasing the public awareness in recent years, this disease is projected to become the leading cause of death worldwide. Advancements in omics, analytical procedures, and high throughput screening in the last five years have led to the realization that human diseases and especially cancer are more complex than were originally conceived. Cancer is not a static entity that can be easily monitored and manipulated. It is characterized by a dynamic and time-dependent network of constantly altered molecular and cellular interactions between players in different pathways. This network is not invariable and rigid but is constantly reshaped and altered conforming to the pliable signaling processes/responses implicated. Its complexity is apparent by the fact that the disease state is not a disruption of a single node or specific nodes in the network organism but is organism-patient dependent, thus requiring personalized perspective approaches. Numerous challenges hamper effective cancer treatment and development of effective drugs such as ineffective therapeutic drug concentration reaching the tumor site, life-threatening side-effects caused by nonspecific tissue distribution of anticancer agents, and acquired resistance of the cancer cell upon chemotherapy that triggers cross-resistance to a wide range of different drugs. targeted drug delivery should give its place to navigated drug delivery since it is not only the cytotoxic drug that targets a specific cellular location but rather a vehicle that navigates the course of the loaded drug to the appropriate site of action. Such drug loaded and navigated vehicles in order to enhance the selective uptake of the cytotoxic agent by the tumor cells and spare the normal cells, should consist of a multidimensional architecture  to the formulation of a navigated vehicle which could spatio-temporally deliver the drug in the correct location and the appropriate time. Thus, the term  Figures  and 2. T Figures , and theIn line with the challenges raised by the complexity of cancer, the aim of the present thematic issue is to provide an accumulation of innovative molecularly targeted cancer therapies and entrepreneurial methods of drug delivery in cancer . It contA constant developmental effort is being conducted for innovative nanoparticles to meet unmet needs in the transportation of imaging and therapeutic agents for cancer diagnosis and therapy. This happens since the nanoscale of these particles allows sculpting of diversity in their capabilities thus, enabling them to respond to a diverse array of functional requirements. Therefore, nanoparticles have been considered as appropriate vehicles to provide an ideal platform for personalized approaches to cancer diagnosis and therapy in cancer disease management. A. D. Miller presents a rigorous and descriptive overview on the status of lipid-based nanoparticles (LNPs) in cancer diagnosis and therapy. Special focus is given on LNPs that conform to the ABCD nanoparticle structural paradigm , D: targeting layer-receptor specific ligand) and to triggered, multimodal imaging theranostic drug-nanoparticles for cancer therapy. Clinical attrition rates are a critical issue in drug development. In oncology, fourfold higher rates of attrition have been determined in respect of other indications. This clearly pinpoints the unmet necessity to steer for novel and traditionally unexplored drug targets to be employed in the drug discovery process towards the development of novel anticancer drugs. In this light, V. Buxhofer-Ausch et al. describe the capacity of members of the organic anion transporter family (OATP) to serve as tumor biomarkers and effective cancer drug targets. The importance of these drug targets is due to their implication on the uptake of clinically important drugs and hormones, thereby affecting drug disposition and cell penetration. An OATP-targeted therapy holds promises to combat cancer efficiently and with lesser side effects due to the tissue specific expression of different OATP members and specifically their differential expression in various cancer and normal tissues. Enzymes of the sulfatase pathway could offer another appealing cancer drug target. S. Lena et al. provide a comprehensive review about the expression and function of enzymes of the sulfatase pathway, particularly of steroid sulfatase (STS), in breast, endometrial, ovarian, and colorectal cancer. Furthermore, it highlighted the applicability of STS inhibitors to function as enzyme-based cancer imaging agents applied in the biomedical imaging technique positron emission tomography for the diagnosis and therapy of estrogen-sensitive cancers.Since cancer is considered not only as a genetic but also as an epigenetic disease and tumorigenesis involves multiple genetic and epigenetic alterations that contribute to the transformation of normal cells towards a malignant phenotype, epigenetics should also be the focus of discovering novel cancer drug targets. E. Hatzimichael and T. Crook reviewed important advances in the field of cancer epigenetics and specifically provide an overview of the clinical use of epigenetics as cancer biomarkers and current progress in the utilization of epigenetic drugs in solid and blood cancers. Navigated delivery of cytotoxic agents to special sites or organs is a challenging issue that needs to be addressed in order to surpass systematic toxicity. These challenges become even more exigent for drug delivery in brain tumors where intrinsic difficulties are met due to the hurdles faced to cross the brain-tumor and blood-brain barriers. To establish a targeting vehicle for glioblastoma cells, T. Kasai et al. exploited the capability of chlorotoxin to selectively bind to matrix metalloproteinase-2 and other proteins on glioma cell surfaces once it was fused to human IgG-Fcs and displayed on the surface of bionanocapsules. This chlorotoxin loaded bionanocapsule showed specific affinity to the surface of glioma cells and internalized into the cytoplasmic space suggesting that is a promising drug delivery system for targeting glioblastoma.In order to establish a target-based agent to be specifically delivered to osteosarcomas, K. Hayashi et al. focused on a lectin (Eucheuma serra agglutinin) both as a tumor-targeting agent and as an antitumor agent. Lectins are carbohydrate binding proteins that are highly specific for sugar moieties on the surface of tumor cells. The authors formulated a drug delivery system containing PEGylated Span 80 vesicles which immobilized the specific lectin. They noted that the specific system was not only selectively targeting osteosarcoma cells but was also cytotoxic to the targeted cells emphasizing the dual role adopted by lectins both as navigating and cytotoxic agents.To achieve an effective approach for enhanced uptake of anticancer drug-loaded vehicles by tumor cells, M. W. Ndinguri et al. explored cell surface proteoglycan CD44 targeting as a way to selectively deliver therapeutic agents encapsulated inside colloidal delivery systems. CD44 has been recognized as a contributor to tumor chemoresistance and as a cancer cell and cancer stem cell biomarker due to its overexpression in cancer compared to normal cells . To target CD44, the authors used a triple-helical peptide sequence derived from type IV collagen that was incorporated in doxorubicin-loaded liposomes . The CD44-targeted liposomes were found effective in reducing tumor size, highlighting their potential to be used for selective chemotherapeutic treatment of CD44 overexpressing tumor cells.The immune system has evolved to protect the body against microorganism invasion and thereby prevent diseases. Thus, cancer vaccines could boost the immune system to enable it to combat cancer more effectively. In vaccine development, a major aim is to induce strong, specific T cell responses. This is achieved by targeting antigens to cell surface molecules on dendritic cells (DCs) that efficiently stimulate T cell responses. The paper by V. Apostolopoulos et al. focuses on the most attractive cell surface receptors expressed on DCs to be used as targets for antigen delivery for cancer immunotherapy. The DC cell surface receptors  which induce cellular responses and show promise as targets for vaccine design against cancer are highlighted. Along these lines, K.-C. Sheng et al. investigated the effect of IFN-gamma on DC functional maturation and DC meditated helper T cell activation, in the presence and absence of Toll-like receptor (TLR) ligation. The authors demonstrated an adjuvant effect of IFN-gamma on DC maturation and T cell stimulation and proposed a novel use of IFN-gamma together with Toll-like receptor agonists to enhance antigen-specific T cell responses, for applications in the development of enhanced vaccines and drug targets, against diseases, including cancer.An alternative to the systemic drug delivery is the convection-enhanced delivery (CED) on the basis of which agents are delivered directly into the tumor and the surrounding infiltrative edges with continuous, positive-pressure infusion, thus allowing direct access to the tumor bed, achieving high local concentrations of the drug with minimal systemic absorption. The paper by J. Yun et al. focuses on the authors preclinical and clinical experience with CED in glioblastoma and highlight the challenges and potency of this methodology. Special emphasis is given onto the translational goals of this work.M. Thanou and W. Gedroyc provided a comprehensive overview on the utilization of MRI-guided focused ultrasound (MRgFUS) as a new method of drug delivery. This methodology combines a high intensity focused ultrasound beam that can be used as an external stimulus to activate drug delivery in tissues and Magnetic Resonance Imaging system (MRI) which visualizes patient anatomy and controls the treatment by continuously monitoring the tissue effect. The advantages of being noninvasive as well as controlled and focus could establish this methodology as valuable tool in clinic to increase drug targeting and tissue specific drug delivery.The transport of anticancer drugs and their consequence on tumor cells implicates an array of physical and biochemical processes. Since multiple steps are involved in the drug-transport, drug-release, and drug-uptake pipelines, mathematical models have become an indispensible tool to analyze the transport processes and predict the outcome of anticancer treatment. Mathematical modeling of cancer provides a tool to assist our realization on the interaction and drug-mediated perturbation of such complex processes, thus, fruitfully contributing to the optimization and refinement of drug delivery. W. Zhan and X. Y. Xu present the development of an improved mathematical model that was applied to an idealized geometry consisting of tumor and normal tissues. They predicted the efficacies of direct intravenous administration and thermosensitive liposome-mediated delivery and illustrated that thermo-sensitive liposome-mediated delivery provides a lower drug concentration in normal tissues than direct infusion of non-encapsulated drug as also a significantly higher peak intracellular concentration. These computational results furnish a projection on the effectiveness of two different treatments, within a mathematical framework, and set the basis to develop and corroborate optimized treatments with reduced risk of associated side effects as also effective tumor cell killing in a short time period of treatment.Due to the complexity of the disease, efficient cancer therapy can emerge only upon interscience collaboration. This special issue aims to accumulate current knowledge on molecularly targeted cancer therapies and innovative methods of drug delivery in cancer. We believe that this accumulative knowledge will assist to accelerate progress developing more precise navigated drug delivery in cancer based on innovative tools.Andreas G. TzakosAndreas G. TzakosEvangelos BriasoulisEvangelos BriasoulisTheresia ThalhammerTheresia ThalhammerWalter JägerWalter JägerVasso ApostolopoulosVasso Apostolopoulos


The National Rural Health Mission (NRHM) has initiated community-based institutions at various levels of the health system e.g. Arogya Raksha Samithi (ARS) and Planning and Monitoring Committee (PMC) at Primary Health Center (PHC) level while the Village Health and Sanitation Committee (VHSC) at the revenue village level. These institutions are intended to create awareness about health services, and to empower communities to demand their entitlements. They are also responsible for planning and monitoring of public health and sanitation programs based on local needs.However, some barriers including the asymmetry of information and power among health service personnel and community representatives, inefficient decentralization mechanisms, and excessive bureaucracy hinder the effectiveness of these committees. Thus, the Karnataka experience towards communitization has unfortunately been ambivalent.In this context, an innovative community-based monitoring initiative was devised with objectives of (1) making community-based monitoring operational; (2) developing a sense of ownership among community representatives about their PHCs; (3) bridging information gaps among community representatives to help them articulate local problems and begin to look for their solutions; (4) enhancing demand for health services through community engagement; and (5) building a low-cost technology based platform for rapid analysis and dissemination of information generated through community-based monitoring of health services.This innovative community-based monitoring of health services initiative has been implemented in all rural PHCs of the Mysore district. A questionnaire with close-ended questions (yes/no type) was devised through a community consultation attended by medical doctors, academicians, and community representatives. This questionnaire containing 79 questions on various aspects of PHC functioning was then installed on a toll-free Interactive Voice Response System (IVRS). Selected VHSC and ARS members were oriented to answer the IVRS-based questionnaire. Any selected community member could answer the questionnaire for their respective PHC.Responses generated through IVRS-based questionnaire were validated through physical verification and statistical assessment of error. Using this base-line community-based monitoring database, a district-wide ranking of rural PHCs was carried out. The ranks were disseminated among the community as well as to the Department of Health and Family Welfare.Community representatives responded to the IVRS-based monitoring questionnaire for three consecutive months. Although the medical officers are apprehensive about the community-based monitoring of their PHCs, they concur with the ranks and positions of individual PHCs.Community representatives are interested in monitoring of health services and in working with the PHC staff, provided that the suggested changes are visible to them. Preliminary analysis of the community’s responses suggests that presence of good physical infrastructure does not necessarily translate into better service delivery. While the T Narasipura block has the highest density of PHCs is the Mysore district, it ranks lowest among all the blocks in the Mysore district in terms of overall utility of the PHCs.In two of the PHCs, changes brought due to community monitoring have been documented. The district health officer is interested in using the community-based monitoring information as a tool for reviewing PHC progress under NRHM.This project validates the view that community-based monitoring of health services can be an authentic tool for measuring performance of PHCs and opens up the possibilities to replicate such experiments for other grassroots government institutions as well. The project demonstrates the use of low-cost technology in minimizing data entry/integration issues that adversely affect usability of existing community-based monitoring mechanisms.The selection of members of VHSCs and ARS for the project was made with the intention to encourage them to bring improvements wherever they can in their role as members of such committees. While positive indications of progress exist, it is too early to conclude that this form of monitoring can also bring sustainable improvements in service delivery at PHCs.The community-based monitoring and ranking system reports the situation, as it is, without blaming anyone for it. This allowed the monitoring process to evolve into a joint assessment, rather than a community led inspection of the PHC.Based on these encouraging outcomes, the project aims to target the planning and implementation of activities to be carried out at the PHC level. For this reason, the PMC members shall participate as respondents to the community-based monitoring questionnaire. The advocacy activities of the project will be more focused on need based planning and guaranteeing service delivery in the public health system.None declared.None declared.


A novel characterization of Clinically Isolated Syndrome (CIS) patients according to lesion patterns is proposed. More specifically, patients are classified according to the nature of inflammatory lesions patterns. It is expected that this characterization can infer new prospective figures from the earliest imaging signs of Multiple Sclerosis (MS), since it can provide a classification of different types of lesions across patients.The method is based on a two-tiered classification. Initially, the spatio-temporal lesion patterns are classified. The discovered lesion patterns are then used to characterize groups of patients. The patient groups are validated using statistical measures and by correlations at 24-month follow-up with hypointense lesion loads.The methodology identified 3 statistically significantly different clusters of lesion patterns showing p-values smaller than 0.01. Moreover, these patterns defined at baseline correlated with chronic hypointense lesion volumes by follow-up with an The proposed methodology is capable of identifying three major different lesion patterns that are heterogeneously present in patients, allowing a patient classification using only two MRI scans. This finding may lead to more accurate prognosis and thus to more suitable treatments at early stage of MS. Multiple Sclerosis (MS) is an acquired inflammatory, demyelinating disease of the central nervous system. This disease is a major cause of disability among young adults, and is very common in the Northern Hemisphere. Moreover, the high evolution heterogeneity among the patients renders it complex to encompass and to predict at the individual level. Epidemiological and imaging data are showing that MS is a two-phase neurodegenerative inflammatory disease. The early stage is dominated by focal inflammation of the white matter (WM), and the latter stage is dominated by diffuse lesions of WM, gray-matter (GM) and spinal-cord lesions The MS onset can be studied in Clinically Isolated Syndrome (CIS) patients. These patients experienced an initial attack suggestive of demyelination, but do not yet meet the criteria for MS even though they are highly likely to develop it The use in MS of contrast agents specific of active macrophages offers an opportunity to better understand the occurrence of irreversible disability and could help to predict it. To our knowledge, no previous study has been carried out using USPIO on CIS patients. Moreover the current classification of patients is based either on their total lesion load or disability index without analyzing the specific lesions evolution, the proposed framework aims at providing more specific figures. The overall challenge is to propose a new clinical and data processing paradigm in order to study MS at the disease onset, to provide a better understanding of the early pathogenic mechanisms and to predict the disease evolution at the patient level. In this context, we propose a new framework, based on a two-tiered classification , to find spatio-temporal patterns in CIS patients, using MRI volumes enhanced by USPIO and by Gd and focusing on the longitudinal evolution of lesions. These clusters of patterns are validated by means of statistical analysis, and a correlation with future chronic hypointense lesion load after 21 months follow-up. T2 Total Lesion Load (TLL) by follow-up is also reported for the sake of completeness. This validation is relevant, as these figures correlate with long-term disabilities Twenty-five CIS patients (17 women and 8 men), aged MR imaging was performed within 3 months after the onset of the disease in different hospitals using either a 3T Verio  or a 3T Achieva  scanner according to a multi-center protocol. The protocol contained axial 2D T1 SE sequences, pre, 5 minutes post Gd injection and 24 hours post USPIO injection, with TR/TE 500/8.4 ms, T2 TSE imaging with TR/TE 6530/84 ms, axial 2D FLAIR with TR/TI/TE 10000/2600/80 ms and 3D FLAIR with TR/TI/TE 5000/1800/273 ms. A Three neuroimage specialists  reviewed all images independently and blindly from each other. Afterwards, the lesion detections were compared reaching an agreement among the specialists. Subsequently, a fourth physician [AM] performed the manual annotations verifying again the lesions. The chronic hypointense lesions The local institutional review board  approved the protocol; and all participants gave their written informed consent.A specific feature space has been defined for each classification tier: first for the lesion pattern classification, and then for the patient classification. The overall work-flow described in the following sections is depicted in Once the enhanced lesions are manually delineated for all ria  see ) appeareThe patient feature vector is given by all the cardinalities of the lesion pattern clusters The feature representations described in the previous subsections are used in a two-tier classification. Initially lesion patterns are identified, then the discovered patterns are used to identify specific patients which could potentially undergo a more severe course. These two steps are performed by an unsupervised clustering algorithm and a regression using the output of the first tier as the input of the second, i.e. the detected lesion patterns K-means, hierarchical clustering and Gaussian Mixture Models (GMM) are well-known unsupervised clustering algorithms Yet the choice of cluster number needs to be predefined arbitrarily. Since we are investigating the existence of possible patterns, we have no prior knowledge of the number of clusters and therefore we rely on some validity indices. Many validity indices have been proposed, the reader can refer to Once the clustering of the lesions patterns is performed, the detected The statistical significance of the lesion clusters is assessed by computing the cluster separation The goodness of fit of a regression model can be assessed through the In this section the results of the two-tiered classification are reported. First the clinical observations are reported, and then the results for the lesion pattern clusters and the patient groups.The mean EDSS score of all twenty-five patients at baseline was Comparing the manual annotations of the enhancement for both contrast agents for the same lesions, the USPIO-enhanced lesions show a different behavior than the Gd-lesions and are often visible as a mild ringing around the Gd-lesions as depicted in The expert radiologist manually delineated 103 Gd-lesions and 24 USPIO-lesions. Only one lesion was enhanced by USPIO and not Gd, such a lesion pattern was grouped in the cluster C1 described in the following section. By examining which of the active lesions converted to hypointense lesions by The clinical value of USPIO is assessed comparing the results obtained using the The pipeline results highlight whether the same patient has multiple lesions belonging to different lesion clusters This shows that the clusters differentiate the co-presence of both contrast agents, and not only the size of the lesions.The statistical significance assessed by the cluster separation The experiment was repeated using 1) the Gd features only still using the tensor-like measurements; 2) for both Gd and USPIO features and only Gd using the simpler representation given by the volume of lesions for each time point; 3) using K-means in place of the Spectral clustering algorithm. All these last variations of the experiment produced only two clusters with one cluster comprising relatively large and medium lesions by The volume of chronic hypointense lesions - reported in The proposed framework is capable of classifying lesions and MS patients from the very early stages onwards . This classification correlates closely with the volume of hypointense lesion voxels and to the T2 TLL by The Moreover, from these observations, it may be hypothesized that not all the lesions - even if enhanced by Gd - contribute in the same way to the worsening of the disease. Disability may not be directly related to a blood brain barrier breakdown, but rather to how the brain overall copes with the damages generated by MS Contrary to Different cohorts are used. In previous studies, the patients were at a more advanced stage of the disease (RRMS or progressive MS). Moreover, Vellinga et al. Most of the RRMS patients may be under treatment, which reduces the number of Gd-enhanced lesions.The studies have different time interval between MRI scans.The observation that generally half of USPIO-enhanced ringing lesions converted to hypointense lesions at The tensor-like representation for lesions is motivated by the rotationally invariant eigenvalues and the analogy to PCA. The detected clusters proved to be statistically significant in our dataset. However, both the tensor-like and volumetric representation can oversimplify the complexity of the lesion enhancement even if still informative. To complement this representation a clear indication of the hollowness of the lesion has been added in such a way to discriminate focal from ringing-like lesions. Future works can comprise to use features obtained using the Laplace-Beltrami operator A novel paradigm for spatio-temporal analysis of MS lesions at disease onset has been proposed. It has been applied using a novel contrast agent (USPIO) yielding complementary information compared to Gd. We devised a two-layer classification, first identifying lesion patterns then classifying patients in terms of predictive risk of disease progression according to the lesion patterns. Such an approach is very interesting as it allows to relate parameters extracted from patient images to invisible disease parameters such as future course of the disease.This patient classification performed at the very early clinical stages of MS correlates closely with hypointense lesions and TLLs by


Structure-activity relationship (SAR) is one of the foundational principles of biomolecular activity and states that similar molecules have similar biological activity. This work is focused on the utilization of enzymatic reactions in order to augment ligands with small apolar substituents. These modified molecules will then be employed for establishing the SAR of a ligand series.For this purpose, prenyltransferases (PT) seem like an attractive avenue. Their enzymatic attachment of the apolar prenyl moiety  Figure  is desirdimethylallytrypthophan synthases) and correspondingly of prenylated indole derivatives. The reactions can further be subdivided into classes of i) reverse and regular prenylation, ii) endo- and exocyclicity of the prenylation site, iii) aromaticity of the prenylation site and iv) element name of the nucleophilic atom.In the initial phase of this project, PTs and their reactions were catalogued and digitalized: A MySQL-database was established where reactions – extracted from primary literature – are stored as SMARTS strings. These reaction patterns will be used for generating a library of molecules accessible to prenylation. In order to automatically generate SMARTS, the atoms involved in bond cleavage and formation have to be enumerated. Fragmentation of the substrate followed by atom mapping onto the product molecule makes it possible to identify the nucleophilic atom in an automated manner Figure . From th


The adherence to policies of National TB Control Programme (NTP) to manage a case of tuberculosis (TB) is a fundamental step to have a successful programme in any country. Childhood TB services faces an unmet challenge of case management due to difficulty with diagnosis and relatively new policies. For control of childhood TB in Pakistan, NTP developed and piloted its guidelines in 2006-2007. The objective of this study was to compare the documented case management practices of pediatricians and its impact on the outcome before and after introducing NTP policy guidelines.An audit of case management practices of a historical cohort study was done in children below 15 years who were put on anti-tuberculosis treatment at all nine public hospitals in three districts in province of Punjab. The study period was two years pre-intervention (2004-05) and two years post-intervention (2006-07) after implementation of new NTP policy guidelines for childhood TB. There were 920 childhood TB cases registered during four years, 189 in pre-intervention period and 731 in post-intervention period. The practices changed significantly in post-intervention period for use of tuberculin skin test , and for the use of chest x-ray . Diagnostic scores were recorded for only a minority of cases (18%). The proportion of correct drugs pre- and post-intervention remained same. There were unknown treatment outcomes in 38 out of 141 cases (27%) in pre-intervention and in 483 out of 551 cases (87%) post-intervention, all among the 692 cases without documented treatment supporter.The study has shown that pediatricians have started following parts of the national policy guidelines for management of childhood TB. The documented use of diagnostic tools is increased but record keeping of case management practices remained inadequate. This seems to increase case finding substantially but the treatment outcomes were poor mainly due to unknown outcomes. Development and implementation of standardized operational tools and regular monitoring system may improve the services. The adherence to policies of the National TB Control Programme (NTP) to manage a case of tuberculosis (TB) is a fundamental step to a successful programme in any country. Since the initiation of the campaign to control tuberculosis, a positive change has been observed in managing TB cases in the Eastern Mediterranean region  with e.gThe diagnosis of pediatric tuberculosis is usually made on the basis of clinical evidence and is difficult particularly in infants and young children in whom the symptoms are often non-specific . SeveralFor the control of childhood TB in Pakistan the NTP has developed and piloted its guidelines in 2006-2007 in the public sector hospitals in ten selected districts. The policy guidelines were developed in collaboration with the Pakistan Pediatric Association and consist of protocols with a purpose to improve care of children with TB . The guiAn audit of the case management practices in a historical cohort was done by reviewing patient records. We included all children aged less than 15 years who were registered as cases of TB at the nine public hospitals (district and sub-district) in three districts of the province of Punjab. Children detected with TB at health facilities other than these hospitals such as maternal, child and primary health facilities, private clinics and para-statal hospitals were not included in the study. The study received ethical clearance from the National Bioethics Committee of Pakistan.The diagnosis was based on the combination of history and examination suggestive of TB, history of close contact with an adult case of TB, bacteriological examination, chest X-ray, TST and histopathology of tissue samples. In addition there was a scoring chart meant to help in making diagnostic decisions. A diagnostic algorithm has been presented by NTP in the guidelines to facilitate the case management process Figure . These cThe district level public sector hospitals in Pakistan usually have 'pediatrician', i.e. child specialist or doctors and health personnel taking care of children. They were informed by NTP on how to manage childhood TB including suspect screening, use of scoring chart, diagnosis and managing records. Facilities that are available in these hospitals include sputum smear microscopy and CXR to support TB diagnosis. The administration of TST is delegated to a nurse or vaccinator. The recording and reporting (R&R) of routine TB cases is the responsibility of a DOTS facilitator, usually a paramedic from the existing set up and trained on NTP routine adult TB R&R system.missing in terms of diagnosis includes missing information on the use of TST, chest X-ray or score charts. In the case of prescribing anti-TB treatment inadequacy was used to denote prescriptions that were wrong according to category and dose of any single drug.The term 'practice' in this study is the pediatrician's documented record of case management which should be based on NTP childhood TB policy guidelines. The absence of such records was termed as 'missing information'. The The NTP selected ten districts in all four provinces of Pakistan to pilot their childhood TB policy guidelines. The selection of districts was based primarily on evidence of functioning adult TB care in a district, geographic distribution and access, and willingness of districts to participate in a pilot. For our retrospective study, we selected all three districts from the province of Punjab for the study because of continued programme support including drugs and materials, which facilitated implementation under programme circumstances. All nine hospitals at district and sub-district level were included. The district level hospitals have more specialist persons and inpatient facilities available as compared to sub-district level hospitals. Otherwise they are almost similar in the context of routine TB care services including childhood TB case management. These three study districts represent southern, central and northern parts of the province. All childhood TB cases registered at these hospitals during the period under review were included in the study . The pedThe documented case management practices in two cohorts of childhood TB cases were compared. The first cohort included patients registered during the two complete years 2004 and 2005, when NTP had no particular emphasis on childhood TB ('pre-intervention') and had recently expanded the DOTS strategy for adults. The second cohort consisted of patients registered during 2006 and 2007, when new NTP childhood TB policy guidelines were being implemented ('post-intervention'). The prime sources of data were the TB registers, TB treatment cards and quarterly reports. These were mainly available as outpatient records and were part of the standard NTP recording and reporting system. A specially designed form was provided in year 2006 to the pediatricians to document the scores of individual children using the scoring chart. The TB patient registration and quarterly reporting in the public sector had been decentralized in Pakistan to the level of diagnostic centers including all the public hospitals and the rural health centers in the district. These diagnostic centers (average 12-15 in public sector in a district) register the TB cases including children and send their quarterly case finding and treatment outcome reports to the district TB coordinator office. The district TB coordinator office then consolidates these quarterly reports and transmits a quarterly consolidated district case finding and treatment outcome report to the provincial TB control office. There are regular quarterly intra- and inter-district meetings to consolidate reports to avoid errors and to facilitate the progress. A researcher visited each hospital to review the patient records and extract relevant data, using a specially designed manual tool. The quality of data extraction was ensured by another researcher by cross-checking the extracted data for missing information and inconsistency.Data were entered and analyzed using the SPSS version 15 software package. The comparisons of the two cohorts relate to diagnostic practices, to prescription of anti-TB drugs, allocation of treatment support and to treatment outcomes. For comparing group differences of categorical variables, Pearson Chi-square test was used and Student t-test was used for continuous variables. The level of significance was set at p < 0.05.There were 920 childhood TB cases registered during the four years in the three districts studied, 189 in the pre-intervention period and 731 in the post-intervention period  had a recorded patient category and these were correct in 96%. Prescription of correct drug regimen increased in absolute numbers from 46 pre-intervention to 167 post-intervention after the implementation of guidelines. However the proportion of correct drugs regimen pre- and post-intervention remained the same, 24% and 23%, respectively.During the study the treatment support allocation was recorded among 228 (25%) of all the registered cases  who were supposed to be involved in the R&R activity. This resulted in weak implementation of R&R with incompleteness of records. The use of tuberculin skin test after introduction of the NTP guidelines increased. The TST was not initially available from the programme and after the implementation of the new policy its use by the pediatricians in the programme conditions was encouraging. The TST as a tool to support diagnosis of TB among children is well established and still considered to be the best available method to detect TB infection . It can The documented use of chest radiographs to assist in diagnosis increased among both pulmonary and extra-pulmonary TB cases. The use of CXR in making the diagnosis of children with TB has been supported internationally and a majority of cases with children with pulmonary TB have CXR changes suggestive of TB . The CXRIn our study some children had sputum smear done, although this did not increase with introduction of the new policy guidelines. Very few of the cases below 5 years of age got sputum smear done which shows the challenge faced by the health care providers in getting smear done in very young children within routine programme conditions. The NTP childhood TB case management policy and involving pediatricians in TB control activities were recent activities. We observed an increase in case notification during post-intervention phase but the linkage between various investigation facilities to operationalize diagnostic algorithms within the hospital was limited. The role of laboratory services in childhood TB diagnosis including sputum smear microscopy and specimen collection by gastric lavage, was not well defined. The operational linkages between the pediatricians and laboratory services need to be strengthened in order to enhance sputum microcopy among children suspected with TB. Similar operational issues are relatively common in the start of a new initiative and gradually improve with programme implementation. More training is required for introducing techniques such as gastric lavage and induced sputum within routine programme situation in order to increase sputum smear microscopy among children. The use of the score chart was introduced by the new guidelines. The score chart helps suspect screening  and the The study has shown that the proportion of cases with correct regimen has not changed from pre-intervention to post-intervention. There is wide variation in the practices of pediatricians regarding prescribing anti-TB drugs to the diagnosed children and none of the doses prescribed were found to be recorded correctly. To have good treatment outcomes the application of standardized treatment regimen needs to be according to the relevant diagnostic category . This suPatient treatment cards are recommended for documenting treatment adherence  and chilA strength of this retrospective study is that it is based on the review of available patient records under routine programme conditions at nine selected hospitals in public sector in three districts with previous adult TB care experience and good collaboration with NTP. One major limitation is that many patient records were incomplete and it was not possible to get more information on each of the patients from the hospitals.The study has shown that pediatricians have started following parts of the national policy guidelines for management of childhood TB. In the diagnosis of childhood TB the documented use of TST, CXR and score chart increased but the same was not observed for sputum smear which actually declined. Record keeping of case management practices including diagnosis and treatment support remains inadequate. The impact of these practices on treatment outcomes was scanty with too many unknown outcomes. Perhaps the absence of operational tools to guide the process of care and monitoring to improve the case management practices and outcomes confines the achievement of desired results. Development and implementation of standardized operational tools and regular monitoring system is required to improve the performance and quality of services.The authors declare that they have no competing interests.NS has participated in study design, data entry and analysis, data interpretation, draft writing, editing and submission. SGH has participated in study design, data analysis, and data interpretation and editing whereas NAB, DAE, MAK and OM have participated in study design and data interpretation and editing. All authors read and approved the final manuscript.


Researchers from the public and private sectors convened at BBC11 to discuss advances and challenges in a wide spectrum of application areas. A key theme of the conference was the contribution of bioinformatics to enable and accelerate translational and clinical research. The BBC11 stressed the need for stronger collaborating efforts across disciplines and institutions. The demonstration of the clinical relevance of systems approaches and of next-generation sequencing-based measurement technologies are among the existing opportunities for increasing impact in translational research. Translational bioinformatics will benefit from research models that strike a balance between the importance of protecting intellectual property and the need to openly access scientific and technological advances. The full conference proceedings are freely available at http://www.bbc11.lu.The 6 In a time full of possibilities and expectations for personalised medicine, bioinformatics represents more than a technological convergence point at the intersection of various research fields.Bioinformatics has become a bridge of integration. A continuous integration of information, insights and teams all over the planet. But it also catalyzes the translation of advances from the computing into the life sciences, and from there to the clinic, and back. Bioinformatics is gradually expanding its potential to improve the life of patients everywhere, through the transformation of fundamental discoveries into advanced approaches to preventing, detecting and treating disease ,2.th BeNeLux Bioinformatics Conference  to review advances, reflect on challenges and celebrate the contributions of bioinformatics in a "post-genome" era. The conference attracted around 200 participants, including internationally-renowned guest speakers and more than 100 peer-reviewed abstract submissions from 3 continents.We met at The 6Our programme of guest and selected oral and poster presentations comprised a diverse range of biologically and clinically relevant questions, computing approaches and applications . These sPresentations and discussions addressed applications of significant relevance to the detection and treatment of cancers, infectious, neurodegenerative and cardiovascular diseases. The meeting also showcased progress in bioinformatic infrastructures and enabling technologies. Examples of the latter included next-generation sequencing tools and applications, complex information integration and annotation over the Web, and several disease- and organism-specific research resources.Among such a diversity of approaches and applications, a crucial pattern can be discerned. Bioinformatics research is putting a great emphasis on answering "when", "what if" and "why" questions using different types of biological data. When are complex events at the molecular and systems levels taking place ? When are specific disease states likely to occur? Thomas Lengauer's talk on translational bioinformatics in treating HIV infections represented an instance in this direction. What if specific perturbations are induced ? Why are observed associations or predictions biologically meaningful ? Ioannis Xenarios's talk on gene and cellular regulatory network models was an example within this research question category.Different presentations also highlighted opportunities for integrating multiples vistas of biological phenomena as dynamic webs of interactions. Examples of this included: "Visualizing genotype-phenotype relationships across cell cycle and evolutionary time scales" (Maria Secrier and Reinhard Schneider), "Prediction of a phosphorylation network in Arabidopsis thaliana"  and "Interfering with the interaction network of adenyl cyclase virulence factors" . This and future contributions would not be possible without advanced capabilities in large-scale data analysis and interpretation, for example from next-generation sequencing experiments  and from other types of "omic" data .Harnessing the huge amounts of data routinely produced by high-throughput techniques remains a challenge, as demonstrated by some of the posters presented during the conference. While microarrays still represent a fair amount of the contributions in genomics, next-generation sequencing has begun its journey towards clinical applications. This is happening thanks to the development of affordable, robust and well-standardized pipelines available for service, as introduced in "Union makes strength: building baseline Tracks from 69 open access full human genomes" (Stephane Plaisance and Mark Veugelers). Having all this information available, efforts are being put in its "organisation": building pathways, modelling networks, analysing interactions, and prioritising candidates for further downstream validation. "Analyzing gene and protein expression variance in cellular pathways using high-throughput experimental data" (Enrico Glaab and Reinhard Schneider) and "Network analysis of differential expression for drug target prioritization"  are examples of work in this direction. Given all the methods proposed, it is also worthwhile to compare how they perform, as demonstrated in "Critical assessment of candidate gene prioritization methods" .Another remarkable point is how bioinformatics is expanding beyond research environments to reach the clinicians directly: "Breaching the surface with HOPE" (Jules Kerssemakers) aims at abstracting the complexity behind resources dealing with protein interfaces, surfaces and assemblies, thus helping physicians to understand the structural effects of mutations. Furthermore, one of the three awarded posters, "Clinical data miner - an electronic data capture software framework that improves interrater agreement" , was specifically aimed at the routine clinical environment. The proposed method enhances expert robustness and facilitates interpretation in situations where "case report forms" are used.Presentations and a panel-driven debate on "Technological and Cooperation Challenges in Bioinformatics" offered crucial insights into the state of our field, including critical research directions and advances to watch in the short and longer terms.The need for cross-disciplinary cooperation in bioinformatics and in its different application domains is unassailable. If we want to develop new tools to improve human health in fundamental and clinical routine settings, new ways to share data and software will be essential. Many of such collaborations will ultimately be benefitted from different levels of data integration and access to high-performance computing. The selection of specific tools and approaches will continue being driven by domain-specific biological questions and computing requirements. For example, the use of cloud computing technologies cannot be seen as an "one-fits-all" solution in fundamental or translational biomedical research. The size and complexity of many projects requiring, for example, large-scale software development and integrative data mining demand a deeper involvement of area experts in key steps and decisions of the translational research cycle.in silico, in vitro and in vivo models. Although the estimation of the clinical relevance and utility of the majority of findings reported to date will require significant efforts, areas such as disease biomarker discovery and drug target prediction offer fertile ground for progress in the near future. The latter will be greatly facilitated by better access to carefully annotated sample cohorts (biobanking) and by the availability of user-friendly bioinformatic infrastructures.Researchers are incrementally breaking new ground to demonstrate the relevance of systems biology approaches to translational research with stronger couplings between The potential impact of bioinformatics in translational and clinical research will also depend on our capacity to harmonise the needs for freely publishing research outputs and for protecting intellectual property. This is particularly critical taking into account the advantages and opportunities offered by open-source development and open-access publishing.Bioinformatics will be expected to contribute new ways to streamline the discovery pipeline needed for the development of diagnostics and drugs in the face of high costs and stringent regulatory standards. This will be possible through advances in the interrogation and simulation of complex dynamic systems, and based on the capacity of such models and resulting analyses to be reproduced and independently validated.th BeNeLux Bioinformatics Conference discussed advances and challenges in bioinformatics across a diverse range of applications. A key theme of the conference was the mission and roles of bioinformatics in enabling translational and clinical research. Although we did not intend to provide an in-depth discussion of opportunities and challenges in translational bioinformatics, a number of important perspectives were derived from the conference and deserved to be highlighted in this article. Major opportunities and challenges include the extraction of biological meaning from next-generation sequencing and other high-throughput "omic" technologies, the development of user-friendly bioinformatic infrastructure and the demonstration of the clinical relevance of systems-based approaches.The 6The authors declare that they have no competing interests.FA drafted the manuscript with inputs or revisions from other authors. All the authors read and approved the manuscript.


A diversity of hypotheses have been proposed based on both morphological and molecular data to reveal phylogenetic relationships within the order Cetacea , and great progress has been made in the past two decades. However, there is still some controversy concerning relationships among certain cetacean taxa such as river dolphins and delphinoid species, which needs to be further addressed with more markers in an effort to address unresolved portions of the phylogeny.Tursiops and Stenella were found to be paraphyletic. Estimates of divergence times revealed rapid divergences of basal Odontocete lineages in the Oligocene and Early Miocene, and a recent rapid diversification of Delphinidae in the Middle-Late Miocene and Pliocene within a narrow time frame.An analysis of additional SINE insertions and SINE-flanking sequences supported the monophyly of the order Cetacea as well as Odontocete, Delphinoidea (Delphinidae + Phocoenidae + Mondontidae), and Delphinidae. A sister relationship between Delphinidae and Phocoenidae + Mondontidae was supported, and members of classical river dolphins and the genera Several novel SINEs were found to differentiate Delphinidae from the other two families (Monodontidae and Phocoenidae), whereas the sister grouping of the latter two families with exclusion of Delphinidae was further revealed using the SINE-flanking sequences. Interestingly, some anomalous PCR amplification patterns of SINE insertions were detected, which can be explained as the result of potential ancestral SINE polymorphisms and incomplete lineage sorting. Although a few loci were potentially anomalous, this study demonstrated that the SINE-based approach is a powerful tool in phylogenetic studies. Identifying additional SINE elements that resolve the relationships in the superfamily Delphinoidea and family Delphinidae will be important steps forward in completely resolving cetacean phylogenetic relationships in the future. Platanista gangetica) and the now nearly extinct Yangtze river dolphin or Baiji (Lipotes vexillifer), as well as those between the three delphinoid families: Monodontidae , Phocoenidae (porpoises) and Delphinidae (dolphins)  remain controversial, despite that a variety of studies have been conducted using a diverse array of systematic markers  . The BEAST analysis was executed for 20, 000, 000 generations with a random starting tree, birth-death default priors sampled every 1000 generations. Results were examined using Tracer 1.4 [Although SINE insertions allow one to construct tree topologies, they cannot be used for reliable calculation of relative branch lengths without the potential to model amplification rates of SINE markers over time. SINE-flanking sequences, however, may potentially be used for dating historical retropositional events that diagnose common ancestry, because of the probable neutral nature of evolution in nonfunctional regions of the genome . Here, eakicetus ,106 withakicetus   and the two different SINE insertions (B). Dots indicate nucleotides identical to the consensus sequence at the top. The name of the SINE family as well as the two different SINEs are indicated in a bold box. The line above the sequences represents the tRNA-related region of the SINE. Box A and Box B promoters for RNA Polymerase III are boxed and highlighted. Putative flanking direct repeats are underlined.Click here for fileAlignments of sequences for loci Turt164 (A) (including Band A and Band B) and the four SINE insertions amplified in the four species in this study (B). Dots indicate nucleotides identical to the consensus sequence at the top. The name of the SINE family as well as its subfamily is indicated in a bold box. The line above the sequences represents the tRNA-related region of the SINE. Box A and Box B promoters for RNA Polymerase III are boxed and highlighted. Putative flanking direct repeats are underlined.Click here for filePrimers used in this study.Click here for file


Lack of breast feeding is associated with higher morbidity and case-fatality from both bacterial and viral etiologic diarrheas. However, there is very limited data on the characteristics of non–breastfed infants attending hospital with diarrheal illnesses caused by common bacterial and viral pathogens. Our objective was to assess the impact of lack of breast feeding on diarrheal illnesses in infants living in urban Bangladesh.We extracted data of infants (0–11 months) for analyses from the data archive of Diarrheal Disease Surveillance System (DDSS) of the Dhaka Hospital of icddr,b for the period 2008–2011.The prevalence of breastfeeding in infants attending the hospital with diarrhea reduced from 31% in 2008 to 17% in 2011, with corresponding increase in the prevalence of non-breastfed (chi square for trend <0.001). Among breastfed infants, the incidence of rotavirus infections was higher (43%) among the 0–5 months age group than infants aged 9–11 months (18%). On the other hand, among non-breastfed infants, the incidence of rotavirus infections was much higher (82%) among 9–11 months old infants compared to those in 0–5 months age group (57%) (chi square for trend <0.001). Very similar trends were also observed in the incidence of cholera and ETEC diarrheas among different age groups of breastfed and non-breastfed infants (chi square for trend 0.020 and 0.001 respectively). However, for shigellosis, the statistical difference remained unchanged among both the groups (chi square for trend 0.240).We observed protective role of breastfeeding in infantile diarrhea caused by the major viral and common bacterial agents. These findings underscore the importance of promotion and expansion of breastfeeding campaigns in Bangladesh and elsewhere. Escherichia coli, Shigella spp., Campylobacter spp., and Vibrio are the common bacterial pathogens causing diarrhea in young children in developing countries, including Bangladesh In developing countries, diarrheal diseases, as a group, remains the leading cause of illness and the second leading cause of death among young children, particularly during the first two years of their lives. Diarrheal diseases accounted for 11% of the estimated 7.6 million under-five deaths, globally, in 2010 The human-milk glycans, which include oligosaccharides in their free and conjugated forms, constitute a major and an innate immunologic mechanism by which human milk protects breast-fed infants against infections The Diarrhoeal Disease Surveillance System of icddr,b is a routine activity of the Dhaka Hospital, which has been approved by the Research Review Committee (RRC) and Ethical Review Committee (ERC) of icddr,b and this study has been approved by the RRC and ERC of icddr,b. Verbal consent was taken from the caregivers, or guardians on the behalf of the patients, for their information to be stored in the hospital database and used for research. Although the Diarrhoeal Disease Surveillance System of icddr,b is a routine activity for hospital patients, and used to be done only with verbal consent from the parents or guardians of the patients following the hospital policy, parents or guardians were assured about the non-disclosure of information collected from them, and were also informed about the use of data for analysis and using the results for improving patient care activities as well as publication without disclosing the name or identity of their children. ERC was quite satisfied with voluntary participation, the maintenance of the rights of the participants and confidential handling of personal information by the hospital physicians and approved this consent procedure.V. cholerae, and Shigella; acute respiratory infections; and malnutrition; and provides training to health care providers on case management and research methodology. Infants (0–11 months old), constitute about 28% of all attending patients. The majority of the patients come from poor socio-economic backgrounds, who live in urban and peri urban Dhaka.The study was conducted at the Dhaka Hospital of icddr,b. Located in the Dhaka, the capital city of Bangladesh, the hospital provides care and treatment to around 140,000 patients each year, vast majority of whom are under five children. Diarrhea is the common feature among the patients, whether or not they are associated with diarrhea-related complications or other health problems, while malnutrition is a common health problem in under-five children. The hospital also conducts research on enteric infections caused by common pathogens including rotavirus, ETEC, For this descriptive study, we stratified infants by their etiologic agents then compared them by their breastfeeding status. Infants receiving any amount of breast milk at their current episode of diarrhea were defined as breast-fed and those not receiving any amount of breast milk constituted non-breast-fed infants in our analyses. Age stratification was done taking into consideration that exclusive breastfeeding should be continued till the end of age as 0–5 months (recommended age of exclusive breastfeeding), 6–8 months are the time when most of the infants receive their complementary feeding (beginning of the weaning food), and 9–11 months .th patient, regardless of age, sex and diarrhea severity) and records their socio-demographic and environmental history; nutritional and clinical characteristics; immunization status and feeding practices (this data is collected for only those younger than 3 years); hygienic practices in the family; and determines the etiology of diarrhea along with antimicrobial susceptibility of common bacterial pathogens. Trained research assistants interview patients  using a standardized questionnaire, performs anthropometry using standard techniques, and records findings in pre-tested case report forms (CRFs). A physician obtains medical history, performs thorough physical examination, and records findings in the CRFs.icddr,b maintains a Diarrheal Disease Surveillance System (DDSS) at its Dhaka Hospital since 1979 Vibrio choleraeShigella spp. A fresh stool specimen is routinely collected from the surveillance patients for screening of common enteric pathogens such as rotavirus We extracted relevant information from the electronic database of the Diarrheal Disease Surveillance System for our analyses for the period 2008–2011. In total, 2,869 infants (aged 0–11 months) of both sex, attended the hospital during this study period.V. cholerae, and Shigella) causing diarrhea with a view to describe their hospital visit patterns due to diarrheal illnesses in a large urban diarrheal disease hospital in Bangladesh. We have also calculated age stratified proportion of diarrhea pathogens among the breastfed and non-breastfed infants and yearly distribution of different diarrhea pathogens among the breastfed infants. We used Chi-square for trend to test statistical significance of the changing trend by year and age We used Statistical Package for Social Sciences (SPSS) Windows  for data entry and analysis. We calculated the proportion of both breastfed and non-breastfed infants infected with enteropathogens  . RotavirVibrio cholerae, ETEC, Campylobacter, and ShigellaShigella failed to gain statistical significance might be due to small sample size. Our observation of higher proportion of non-breastfed infants with rotavirus is also consistent with earlier observation Shigella is the most virulent organism among the all organisms that have been studied here and consequently acquiring immunity against Shigella even during continuation of breast milk sometimes may take longer period and our observation of statistical insignificant difference of proportion of Shigella isolates in infants between 6–8 months of age among the breast-fed and non-breast-fed groups might also be due to the same reason We noted lesser proportion of hospital visits by breastfed infants infected with all the enteric pathogens we analyzed than non-breastfed infants. Breast milk contains non-inflammatogenic secretory immunoglobulin A (sIgA) antibodies that protects intestinal mucosa against the attack from The overall results from the study tell us that the proportion of breast-fed infants with diarrhea is reducing, while the national data gathered from the general population without diarrhoea suggests an increase in prevalence of exclusive breastfeeding among infants. Thus the data indicates lack of protection from breast milk against diarrhoeal disease among the non-breastfed infants. However, the population who reported to the facility was the special population with disease condition resulting from the lack of adherence to the breast feeding which had a potential role for this observation.The main limitation of the study is that study observations are based on infants who attended the hospital and thus our data may not represent the general population because infants with less severe disease less often seek care from hospital facilities. However, systematic sampling of patients including infants and large data set are among strengths of our analyses. Moreover, despite variations, some differences were not statistically significant possibly due to lack of power with small sample size.In summary, the present study documented the proportion of breastfed infants significantly decreased over the period.The study finding transparently produces evidence of the protective role of breastfeeding against the most common enteropathogens that cause diarrhea compared to those who were non-breastfed. So, there is a large scale need to give more emphasis on breastfeeding promotion campaigning as well as strengthen the current resources and infrastructures for breastfeeding counseling programs. At the same time, a cohort study in different facilities may be conducted to examine if the results of our study are similar in cases of children presenting with other non-diarrheal but infectious diseases.