Up to now, costs attributable to adverse events (AEs) and preventable AEs in the Netherlands were unknown. We assessed the total direct medical costs associated with AEs and preventable AEs in Dutch hospitals to gain insight in opportunities for cost savings.Trained nurses and physicians retrospectively reviewed 7926 patient records in 21 hospitals. Additional patient information of 7889 patients was received from the Dutch registration of hospital information. Direct medical costs attributable to AEs were assessed by measuring excess length of stay and additional medical procedures after an AE occurred. Costs were valued using Dutch standardized cost prices.The annual direct medical costs in Dutch hospitals were estimated at a total of euro 355 million for all AEs and euro 161 million for preventable AEs in 2004. The total number of hospital admissions in which a preventable AE occurred was 30,000  and more than 300,000  bed days were attributable to preventable AEs in 2004. Multilevel analysis showed that variance in direct medical costs was not determined by differences between hospitals or hospital departments.The estimates of the total preventable direct medical costs of AEs indicate that they form a substantial part (1%) of the expenses of the national health care budget and are of importance to hospital management. The cost driver of the direct medical costs is the excess length of stay (including readmissions) in a hospital. Insight in which determinants are associated with high preventable costs will offer useful information for policymakers and hospital management to determine starting points for interventions to reduce the costs of preventable AEs. Throughout the world health care workers and policymakers are trying to improve patient safety. The Dutch Patient Safety Research Program, showed Studies in various countries outside Europe have shown that AEs in hospitals are associated with high direct medical costs -9 which As health care costs are rising hospitals are forced to reduce costs. Implementation of patient safety improvement interventions in hospitals is often hampered by the investment costs. Thus insight in costs of preventable AEs may help to increase the sense of urgency and prioritise areas to improve patient safety from an economic perspective in addition to the patient and health care perspective.In this paper we present the total direct medical costs associated with AEs and preventable AEs in Dutch hospitals with regard to subgroups of university and general hospitals. A separate analysis was focussed on AEs occurring in patients who died in hospital. The costs were calculated in two ways, by using the data as determined by the reviewers and with the use of estimation of expected LOS in hospital based on administrative hospital data. In addition, we assessed determinants of AE related costs, such as type of patient admission and AE characteristics, to give insight in the opportunities for cost savings.The instruments of our study were based on the protocol of the Canadian Adverse Event Study, which was originally used by the Harvard Medical Practice Study,10. The We have performed a retrospective patient record review study in a random, stratified sample of 21 of the 101 Dutch hospitals: 4 university, 17 tertiary teaching and general hospitals. To measure the difference in incidence between hospital types, the sample of hospitals was stratified for hospital type. Proper representation of urban and rural setting in the sample was verified. Eligible hospitals had at least 200 beds, an emergency department and an intensive care unit. A large subsample of deceased hospital patients was included to determine the occurrence of potentially preventable deaths in hospitals more precisely than in previous studies. The power was estimated to detect a difference in AE rates between different hospital types. The parameters in the power calculation were based on the results of the Canadian Adverse Events Study. AssumingTo measure the difference in incidence between hospital types a selection of 800 hospital admissions per hospital type were necessary to detect a difference from 2% to 3% by an incidence between 3% and 7%. From each hospital, we randomly selected 200 admissions (> 24 hours stay) of discharged patients and 200  admissions of deceased hospital patients in 2004, excluding admissions of psychiatry, obstetrics and children < 1 year old. Ethical approval for the study was obtained from the Medical Ethical Research Committee of the VU University Medical Centre.The nursing, medical and, if available, out-patient record of the sampled admissions were reviewed by 66 trained nurses and 55 trained physicians in a three stage review process between August 2005 and October 2006. In the first stage, a nurse screened the records by using 18 screening criteria indicating potential AEs. The 18 screening criteria were:1. Unplanned admission before index admission (admission reasons are related to the index admission)2. Unplanned readmission after discharge from index admission3. Hospital-incurred patient injury (Permanent or temporary injury obtained (acquired) during index admission)4. Adverse drug reaction5. Unplanned transfer from general care to (an) intensive care (unit)6. Unplanned transfer to another acute care hospital (after unexpected deterioration of the patient)7. Unplanned return to the operating room8. Unplanned removal, injury or repair of organ during surgery9. Hospital-acquired infection or sepsis10. Other patient complication11. Development of neurological deficit not present on admission12. Unexpected death13. Cardiac or respiratory arrest14. Injury related to abortion or delivery15. Inappropriate discharge to home16. Dissatisfaction with care documented in the medical record17. Documentation or correspondence indicating litigation18. Any other undesirable outcome not covered aboveIn the second stage, two physicians independently reviewed the records with one or more positive screening criteria.For the analysis on patient record level, patients with more than one AE during admission, the AE with the most severe outcome was used for further analysis. Of the initial 8415 sampled records, 8032 were eligible for a first stage review (screening success rate was 95%). Three-hundred eighty three records were unavailable or were inadequate (for example twice sampled or admission was to short). In the second stage 106 records were excluded during screening [The definition of an AE in our study was threefold: an 1) unintended  injury which results (2) in temporary or permanent disability, death or prolonged hospital stay and (3) is caused by the health care management rather than the patients' disease unintend. A nurseAdditional administrative hospital information on diagnosis (ICD-9-DE), secondary diagnoses, acute or non-acute admission, main intervention, secondary interventions, expected LOS, and reason for admission were retrieved for each admission from the LMR maintained by Prismant.The main focus of costs calculated in this study was the health care sector. Only direct medical costs could be taken into account due to the nature and design of this study. Direct medical costs attributable to AEs were assessed by excess length of stay (LOS) during the index admission and re-admission, and by additional medical procedures during the excess LOS as a consequence of the AE.Excess LOS during the index admission attributable to AEs was calculated by taking the average of the two independent estimates of the physician reviewers and rounded upwards. The excess LOS was determined based on the clinical expertise of the physician reviewers. If they were unable to determine excess LOS, we imputed the excess LOS based on the LMR, notably the difference between expected and observed LOS. The estimates varied from no extra bed days to all bed days of the index admission. For re-admissions attributable to AEs in the index admission we imputed the national average hospital stay in 2004 (7.3 days). Additional medical procedures attributable to AEs were assessed by the physician reviewers, indicating for each AE whether additional interventions or treatment were necessary. Additional information on these interventions was added from the administrative hospital data starting from the date of the AE.The excess LOS and the excess medical procedures were multiplied by unit costs to estimate the total excess direct medical costs. Dutch guideline prices of 2003 were used to estimate the costs of one hospital day of standard or intensive care in a university and general hospital -14  were calculated, corrected for the overrepresentation of patients admitted to a university hospital and for the overrepresentation of patients who died in hospital, using 'complex samples' option in SPSS 15.0. Detailed information on the weighing of the sampling frame in order to gain representativeness for the Dutch population of hospitalised patients and patients which died during hospitalisation is described in detail previously. As the unit price of a hospital day in university hospitals is higher than in general hospitals, we analysed the costs attributable to the excess LOS and medical procedures of AEs separately in both groups. Mean total excess costs per hospital admission in which an AE occurred was estimated using weights to adjust for the complex sampling frame. As cost-data were skewed to the right, we used non-parametric bootstrapping, adjusting for the sampling frame, to calculate CIs of cost estimates. Non-parMoreover, determinants which may influence the costs attributable to AEs, such as age, diagnosis, admission department and medical or surgical responsible specialty, and type of AEs were first analysed univariate. Average costs and 95% CIs which were calculated per subgroup using bootstrapping techniques to resolve the skewed distribution. In addition, a stepwise multivariate multilevel analysis, as data were clustered within hospitals and hospital departments, was used to study the relation of these determinants with the costs of AEs and to test our univariate associations. Within all steps of the multivariate multilevel analysis, data were adjusted by a normalisation procedure for the overrepresentation of university hospitals and deceased patients. The dependent variable in the model was either the costs of AEs or the excess LOS (based on the LMR). Independent variables added to the model were successively age, sex, admission type (urgent or elective), main diagnosis groups (ICD9-DE), Charlson comorbidity index and surgical or non-surgical admission department. The amount of variation in costs of AEs caused by the hospital level or hospital department level was expressed by the intraclass coefficient (ICC).The national estimate of costs attributable to AEs in 2004 was calculated by multiplying the total amount of Dutch hospital admissions with the AE rate and by subsequently multiplying this with the average costs. The same was done for costs attributable to preventable AEs.SPSS for Windows version 15.0 was used for most statistical analyses, only the multivariate multilevel analyses were performed using MLwiN version 2.0.As it is sometimes difficult to determine the excess LOS due to the AE based on record review, we used a different approach in calculating excess LOS. For each individual admission in our sample we obtained the expected LOS from the LMR. Within the LMR, for each hospital admission, the expected LOS is calculated based on the following characteristics of the patient and the national mean length of stay that is associated with these characteristics. The cha• Age, divided in 5 classes ;• Primary diagnosis. ;• Procedures, classified by the Dutch Classification System of Procedures (procedures depend on the diagnosis of the patient. It included five procedure groups)Together these three parameters produced about 5 * 1,000 * 5 = 25,000 possibilities of expected LOS. Normally these calculations were only used for patients discharged alive, but for the purpose of this study the expected LOS was also calculated for the patients which died in hospital. Excess LIn total 7926 patient records have been reviewed by nurses and physicians. Additional information from the LMR could not be acquired for 37 patients due to record linkage problems. The missing patients were randomly divided over the 21 hospitals. The study population for the following results consisted of 7889 hospital admissions. Characteristics of the 7889 admissions are shown in table In the 7926 patient records the nurses found one or more screening criteria in 54% of the nursing and medical records. In the second stage the physician reviewers found 774 AEs in 663 hospital admissions. Within these admissions the 663 patients suffered from one or more AE, comprising 216 discharged and 447 patients which died during hospitalisation. The 37 hospital admissions that could not be linked contained no AEs.Excess LOS attributable to the AE could be estimated by the physicians in 96% of the cases. In the other cases we imputed the excess LOS based on the data from the LMR. In 22% of the AEs the physician reviewers indicated that the admission itself was caused by the AE, thus the AE occurred in a previous admission but was detected in or was the reason for the index admission. On average, the excess LOS due to the AEs was 10.1 days (95% CI: 6.4 – 11.9) for university hospitals and 8.9 days (95% CI: 6.9 – 9.6) for general hospitals Table . This diThe medical procedures attributable to AEs, mainly re-operations, accounted for approximately 15% of the medical costs attributable to AEs Table . Non-invAlthough the number of preventable AEs in university hospitals was lower than in general hospitals, the totThe excess LOS (5.6 days to 16.8 days) and consequently the excess costs (€2979 to €6649) of preventable AEs increased when the impairment or disability was more severe  was higher than the mean duration of a hospital stay in 2004 (7.3 days). Extrapolating these figures results in an estimate of 31,164 hospital admissions in which a preventable AE occurred or the entire admissions was caused by an AE and in 320,680 bed days attributable to preventable AEs in the Netherlands in 2004.Medical costs attributable to AEs during hospitals admissions in 2004 were more than € 355 million (95% CI: €316 million – €398 million) for all AEs and more than €161 million (95% CI: €134 million – €195 million) for preventable AEs. The national health care budget in 2004 was €14.5 billion therefore all AEs resulted in 2.4% and preventable AEs 1.1% of the national health care budget.In the sensitivity analysis the excess LOS is calculated by subtracting the observed LOS with the expected LOS based on national reference values. The analysis of observed LOS and expected LOS showed a lower, but trend wise similar, result as the analysis based on reviewers excess LOS. The mean excess LOS was 5.9 days and the total costs of AEs (including readmissions and procedures) €4,446 per AE. The mean excess LOS of preventable AEs was 4.4 days and the total costs €3,634 per preventable AE. Within admissions without AEs the observed LOS was slightly lower, patients are on average 0.5 days shorter in hospital than expected.Extrapolation of the results of this study showed that the total potentially preventable direct medical costs of AEs in Dutch hospitals are more than €161 million in Dutch hospitals in 2004; this is approximately 1% of the annual hospital budget of €14.5 billion.  The costThere are several reasons to assume that our cost estimation is conservative. Firstly, other research has shown that due to inefficiencies after things go wrong, the average costs of a hospital day becomes more expensive after an AE. We usedAlthough it is sometimes difficult to attribute the excess LOS or the additional procedures to the AE, the two approaches we used to calculate the total expenses showed similar results. Where the excess LOS based on the reviewers viewpoint may be an overestimation of the true excess LOS, the difference between the expected and observed LOS based on the LMR data may be an underestimation. The expected LOS is an estimation based on yearly data including admissions with AEs. The use of observed LOS minus the expected LOS may lead to an underestimation of the excess LOS. Nevertheless, despite these limitations, excess LOS gives a useful estimate of the direct medical costs associated with AEs and the potential cost savings of preventable AEs. Moreover because the admissions without an AE show on average no excess LOS . Patients with an AE stayed in hospital on average 4.6 more days than expected. The LMR is not an economic database, but an administrative database, so there are reasons to assume registration bias. Nevertheless, results of internal validation of our data showed that the registered information in the LMR provided by the hospitals was in good accordance with the information from the patient records. With respect to the most important data such as LOS, admission department and main diagnosis the results were good.We used price indices appropriate for cost estimates determined from a societal perspective although the perspective of our study was focussed on the hospital. Since there were no cost estimates available from a hospital perspective and the most important elements are included in the standard unit price of a hospital day, we do nA recent study has shown that yearly €85 million of health care costs were caused by potentially preventable hospital admissions related to medication related adverse events in outpatient care. As our Our study only focuses on the direct medical costs of AEs in hospital. From a societal perspective outpatient health care, loss of income and premature death are also important and the estimation of costs related to AEs would have been higher when estimated from a societal perspective.Although judgement of presence of AEs is difficult, retrospective patient record studies currently offer the best method available to assess incidence of AEs. By makiThe estimates of the total potentially preventable direct medical costs of AEs indicate that they form approximately 1% of the expenses of the national health care budget. The cost driver of the direct medical costs is the excess LOS in the hospital.Despite the limitations by retrospective analysis of AEs, the costs of AEs in the Netherlands, estimated in this study are high and show that improvement of patient safety by preventing AEs may be cost saving. If the financial system is such that the hospital or hospital department profits of this cost reduction, it may offer an economic incentive to invest in patient safety. Insight in the determinants of potentially preventable AEs offers a starting points for patient safety interventions.AE: adverse event; ICC: Intraclass coefficient; ICD-9-DE: Dutch version of the International Classification of Diseases, Ninth revision, Clinical Modification (ICD-9-CM); LMR: Dutch registration of hospital information; LOS: length of stayThe authors declare that they have no competing interests.LH wrote the manuscript, prepared the study protocol and instruments, and collected and analyzed data. MB wrote the manuscript, is the general supervisor of the retrospective record study, contributed to the design and conception of the study, and coordinated the study. CW is the general supervisor of the Dutch patient safety research program, contributed to the design and conception of the study, and contributed to the manuscript. MZ collected data and contributed to the manuscript. RW prepared the review forms within a web-based program, managed the acquisition of data, and contributed to the data analysis and to the manuscript. PS analyzed the data and contributed to the manuscript HA contributed to the manuscript and reviewed patient records. GW contributed to the design and conception of the study and has been involved in revising the article critically for important intellectual content . MT contributed to the design of the study and contributed to the manuscript. All authors gave their approval of the final version of the manuscript.The pre-publication history for this paper can be accessed here:


V is the least toxic metabolite, increasing evidence suggests that MMAIII may be the most cytotoxic and genotoxic As intermediary metabolite.Arsenic is a carcinogen to which 35 million people in Bangladesh are chronically exposed. The enzymatic transfer of methyl groups to inorganic As (iAs) generates monomethylarsonic (MMA) and dimethylarsinic acids (DMA) and facilitates urinary As (uAs) elimination. This process is dependent on one-carbon metabolism, a pathway in which folate and cobalamin have essential roles in the recruitment and transfer of methyl groups. Although DMAWe examined the associations between plasma cobalamin and uAs metabolites.We conducted a cross-sectional study of 778 Bangladeshi adults in which we over-sampled cobalamin-deficient participants. Participants provided blood samples for the measurement of plasma cobalamin and urine specimens for As measurements.p = 0.01] and positively associated with %MMA . Both of these associations were stronger among folate-sufficient participants , and the differences by folate status were statistically significant.Cobalamin was inversely associated with the proportion of total uAs excreted as iAs (%iAs) [unstandardized regression coefficient (b) = –0.10; 95% confidence interval (CI), −0.17 to −0.02; III, our findings suggest that in contrast to folate, cobalamin may not favorably influence As metabolism.In this group of Bangladeshi adults, cobalamin appeared to facilitate the first As methylation step among folate-sufficient individuals. Given the toxicity of MMA Ingestion of arsenic through contaminated drinking water is a major health concern in Bangladesh, where approximately 35 million people are chronically exposed . This exS-adenosylmethionine (SAM) serving as the methyl donor (V). Upon reduction to MMAIII, it then undergoes a second methylation to dimethylarsinic acid (DMAV). Methylation has generally been considered a detoxification pathway that generates the least toxic organic mammalian arsenical metabolite, DMAV, and facilitates urinary excretion  with arsenite in the presence of a reducing agent can produce MMA and trace amounts of DMA nonenzymatically. We observed no association between plasma cobalamin and uAs metabolites in our previous cross-sectional study of 300 adults , and vitamin B6 are required for the recruitment and transfer of methyl groups in one-carbon metabolism. Other nutrients, including protein, betaine, and choline, also contribute to the availability of methyl groups ultimately used in SAM biosynthesis. Arsenic retention in tissues is increased with dietary methyl donor deficiency , and uri0 adults . Becausein vitro work of To examine the influence of a full range of plasma cobalamin concentrations on As metabolism, we conducted a cross-sectional study in which we oversampled cobalamin-deficient participants . We also set out to replicate the n = 412 with plasma cobalamin < 185 pmol/L). For efficiency, we combined these individuals with an additional 366 nondeficient participants who already had uAs metabolite data, to incorporate a broad range of cobalamin nutritional status. This process yielded a total of 778 participants. All participants had provided blood and urine samples, and demographic information was obtained from a baseline survey. All study subjects gave informed oral consent to Bangladeshi field staff physicians before participating in the study. The research protocol was approved by the Institutional Review Board of Columbia University and the Bangladesh Medical Research Council.Using biologic samples from our original cross-sectional study of a random sample of 1,650 adults [the Nutritional Influences on Arsenic Toxicity study ], we anaBlood samples were collected by venipuncture from participants who had been sitting for 10–15 min. The samples were collected into EDTA-containing tubes that were immediately placed in IsoRack cool packs . Samples were transported by hand  to our field clinic in Araihazar within 4 hr of collection. The samples were then centrifuged at 4°C to separate plasma and cells. Plasma was stored at −80°C and shipped frozen on dry ice to Columbia University for analysis. Urine samples were collected in 50-mL acid-washed tubes, frozen at −20°C, and shipped on dry ice.Plasma cobalamin and folate were analyzed by radioimmunoassay  as previously described . The witWe used graphite furnace atomic absorption (GFAA), with a detection limit of 5 μg/L, to measure total As in well-water samples. Samples found to have a concentration < 5 μg/L were reanalyzed by inductively coupled plasma-mass spectrometry (ICP-MS), for which the detection limit was 0.1 μg/L .Total uAs was measured in the Columbia University Trace Metals Core Laboratory by GFAA spectrometry using the Analyst 600 graphite furnace system , according to the method of III+V), MMA (MMAIII+V), and DMA (DMAIII+V) after subtracting AsC and AsB from the total.As metabolites were speciated using HPLC separation of arsenobetaine (AsB), arsenocholine (AsC), arsenate, arsenite, MMA, and DMA, followed by detection using ICP-MS. We calculated the percentages of iAs , MMA, and DMA were purchased from Sigma . Arsenate was purchased from PerkinElmer. All other chemicals were analytical reagent grade. Water was distilled and deionized. The assay for nonenzymatic arsenite methylation was done using 0.05 M Tris-HCl buffer at various pH values containing 10 mM GSH. At each pH, we used molar ratios of CH3B12:arsenite of 1:5, 3:5, and 5:5. We performed three replicates for every combination of pH and molar ratio. The reaction mixture was incubated for 1.5, 3, 6, 9, 12, and 30 hr at 37°C. At each time point we aliquoted 50 μL of the mixture, diluted it with mobile phase (10 mM ammonium phosphate plus 10 mM ammonium nitrate), injected it onto the HPLC column, and detected concentrations of MMA, DMA, AsIII, and AsV by ICP-MS with dynamic reaction cell technology (ICP-MS-DRC). Calibration standards of a mixture of As metabolites were similarly processed. ICP-MS-DRC coupled to HPLC separates and detects 7 As metabolites chromatographically separated by anion exchange with the use of a PRP-X100 column .Because our epidemiologic data suggested a role for cobalamin in the generation of MMA, but not DMA, we sought to replicate the findings of Plasma nutrients and uAs metabolites were not normally distributed. We therefore used nonparametric tests where appropriate and natural log transformations that may either create approximately normal distributions for dependent variables in linear regression analysis, improve linearity in the relationship between an independent and dependent variable, or reduce the impact of extreme values of an independent variable in fitting linear models. We calculated descriptive statistics separately by sex for general characteristics as well as for plasma nutrient and uAs metabolite concentrations. We tested for sex differences using the Wilcoxon rank-sum test for continuous variables and the chi-square test for categorical variables. The correlations between plasma cobalamin and covariates and between cobalamin and the percentage of As metabolites in urine were examined using Spearman’s correlation coefficients. We used linear regression models to examine associations between plasma cobalamin and the outcome variables %iAs, %MMA, and %DMA in urine. Potential confounders considered for inclusion in the regression models were age, sex, urinary creatinine, body mass index (BMI), and total uAs. We used a Wald test to detect differences in the covariate-adjusted associations between cobalamin and uAs metabolites by sex, folate status, or water As status. We ran separate linear regression models to examine the covariate-adjusted associations between plasma cobalamin and each of the uAs metabolites and to examine the differences in these associations by sex, folate status, or water As status. Analyses were performed using SAS 9.1 . All statistical tests were two-sided with a significance level of 0.05. The As metabolite concentrations detected in the nonenzymatic laboratory assay experiments were expressed as a percentage of total detected metabolites.The general characteristics and plasma nutrient concentrations of the study participants by sex are shown in r = −0.26, p < 0.0001; males: r = −0.47, p < 0.0001).Because we oversampled cobalamin- deficient participants, a large proportion of the sample had cobalamin deficiency: 57.6% of women and 46.8% of men had < 185 pmol/L. We used cutoff values of < 9.0 nmol/L for folate deficiency , and ≥ 1Mean water As concentrations did not differ significantly by sex . As exper = 0.18, p < 0.0001). Both %iAs and %DMA were weakly inversely correlated with cobalamin concentration, and these associations appeared to be the same in males and females  after adjusting for age, sex, urinary creatinine, total uAs, and BMI. These associations were stronger among females than among males, but the difference was not statistically significantly (p = 0.02) and positively associated with %MMA  only among folate-sufficient subjects . We also examined associations between plasma cobalamin and uAs metabolites by water As concentrations ≤ 50 and > 50 μg/L (p = 0.002); this difference was statistically significant (p = 0.005). Plasma cobalamin was significantly positively associated with %MMA in both strata of water As concentrations. Cobalamin was not significantly associated with urinary %DMA in either strata of water As.In bivariate analysis, plasma cobalamin was inversely correlated with both water As and uAs/gCr . We note females . In line females , cobalamficantly . Cobalamficantly . Plasma  50 μg/L . The inv3B12:arsenite and was highest with a molar ratio of 5:5 at a pH of 7.4. The amount of MMA also increased as the incubation period increased but appeared to plateau at approximately 9 hr. Very small amounts of DMA  were also detected after 6–9 hr of incubation (data not shown).Incubation of arsenite with methylcobalamin in the presence of GSH resulted in the production of MMA . The amoThis study complements our previous work in Bangladesh, in which we have investigated the contributions of folate and tHcys to variations in As methylation. In our study region in Araihazar, Bangladesh, the prevalence of cobalamin deficiency (8% of men and 13% of women < 151 pmol/L) is much lower than that of folate deficiency (57% of men and 39% of women < 9 nmol/L) . The curIn addition to our previous cross-sectional study , six othV. In this study, maternal cobalamin was also inversely correlated with maternal urinary %iAs  and positively correlated with %DMA , although these associations were of borderline statistical significance (In two other studies, researchers examined cobalamin in relation to As among pregnant Bangladeshi women. ificance .73iAsIII methylated to DMA from 53% to 11%, whereas the synthesis of MMA actually increased from 10% to 13% of total 73As. Similarly, AS3MT knockout mice synethsize as much, if not more, MMA than wild-type mice (in vitro experiment.Our findings of stronger associations between plasma cobalamin and uAs metabolites among women than among men are likely explained by the higher prevalence of cobalamin deficiency among these women. The modification of the associations between cobalamin and uAs metabolites by folate status is consistent with the role of 5-methyl THF as the methyl donor for the conversion of homocysteine to methionine by methionine synthase, the reaction for which cobalamin is a required cofactor and 5-methyl THF is a cosubstrate; methionine synthase activity is reduced when 5-methyl THF is limiting. It is unclear, however, why in the presence of adequate folate, cobalamin is not associated with increased %DMA. We hypothesize that cobalamin facilitates the first methylation step and that the reaction has a tendency to stop at MMA, although the underlying mechanism cannot be determined from our data. Interestingly, ype mice . These rin vitro methylcobalamin experiments, modeled after those of 3B12:arsenite on the production of MMA, and we omitted SAM, a possible source of methyl groups, from the reaction mixture. Not surprisingly, our results indicated that increasing the molar ratio of CH3B12:arsenite resulted in increased production of MMA. We also observed that the nonenzymatic methylation of arsenite by methylcobalamin was increased at pH levels closer to the normal pH of blood and other tissues than at pH 7.8. Whether the findings from our in vitro experiment, along with those of in vivo deserves further investigation.Our V+III in urine because of the difficulty of preserving the valence state. However, MMAV is relatively nontoxic, whereas MMAIII is highly toxic (III, which is not stable and is readily oxidized to MMAV. Our observed positive association between cobalamin and total %MMA in urine, taken together with recent evidence regarding the toxicity of MMAIII, suggests that any influence of cobalamin on As-associated disease risk may not be favorable.The implications of our finding that cobalamin is associated with %MMA, but not with %DMA, are not entirely clear. However, a higher %MMA and lower %DMA in urine has been associated with increased risks of As-related skin lesions, skin cancer, bladder cancer, and peripheral vascular disease . These sly toxic . Tseng (We measured plasma total cobalamin rather than methylmalonic acid or holotranscobalamin, two measurements that may be more sensitive indicators of cobalamin deficiency . Althougin vitro experiment confirm that incubation of iAs with methylcobalamin can generate small amounts of MMA nonenzymatically. This finding is consistent with in vivo cell culture studies suggesting that alternative mechanisms for biosynthesis of MMA, independent of AS3MT, likely exist (III may be the most toxic As metabolite, our findings raise the possibility that, in contrast to folate, cobalamin supplementation may not favorably influence As metabolism.In summary, among this group of Bangladeshi adults with a high prevalence of cobalamin deficiency, cobalamin appeared to facilitate the methylation of iAs to MMA, particularly among women and among folate-sufficient individuals. The results of our ly exist , 2009. G


The purpose of this study was to use an immunization protocol with Moloney sarcoma virus (MSV-M) as active immunogen against exogenous and endogenous leukaemia. The s.c. route was chosen since it offered advantages over the i.m. route: the primary sarcomas were smaller, the regression faster, there were fewer recurrences and there was good persistent immunity. Strong protection was obtained against primary leukaemias induced by Friend leukaemia virus (FLV), Moloney leukaemia virus (MLV), Rauscher leukaemia virus (RLV), Precerutti-Law leukaemia virus (PLLV/T2), and H179A leukaemia virus. It was not possible to protect against leukaemia induced by Gross leukaemia virus (GLV). With transplantable leukaemias the results varied: partial protection was observed against H110 leukaemia  and R14 leukaemia (induced by X-irradiation) whilst no protection was obtained against P277 leukaemia (induced by Moloney leukaemia virus). As for spontaneous leukaemias, immunized BALB/c mice showed an increased incidence over the controls, while in F1 (Swiss x AKR) mice the incidence was similar but the latent period was shorter. Furthermore, in long-term observations the MSV-M-immunized mice showed an increased mortaltiy, which could be related to (1) new phenotypic mixtures between MSV-M and leukaemia viruses; (2) reactivation of MSV-M sarcoma-genesis with age, and (3) genotype susceptibility to MSV-M.


J Immunother20: 89–100) recently suggested that the critical infectious event occurs during pregnancy, and identified the polyoma virus JC as a candidate agent. In the present study we investigated whether genomes from the JC virus, and closely related BK virus, could be detected in leukaemic cells. No positive results were obtained suggesting that JC virus is unlikely to play a direct role in leukaemogenesis. © 1999 Cancer Research CampaignEpidemiological evidence suggests that childhood leukaemia, and possibly common acute lymphoblastic leukaemia in particular, may have an infectious aetiology. Smith (1997


Schizaphis graminum, have been separated into biotypes based on host preference and their ability to overcome resistance genes in wheat. Recently, several biotypes were found to differ in their ability to transmit one or more of the viruses that cause barley yellow dwarf disease in grain crops, and vector competence was linked to host preference. The genetics of host preference has been studied in S. graminum, but how this may relate to the transmission of plant viruses is unknown. Sexual morphs of a vector and nonvector S. graminum genotype were induced from parthenogenetic females and reciprocal crosses made. Eighty-nine hybrids were generated and maintained by parthenogenesis. Each hybrid was evaluated for its ability to transmit Barley yellow dwarf virus-PAV and Cereal yellow dwarf virus-RPV, and for its ability to colonize two wheat genotypes each expressing a different gene that confers resistance to S. graminum. The F1 genotypes were genetically variable for their ability to transmit virus and to colonize the aphid resistant wheat, but these traits were not genetically correlated. Individual F1 genotypes ranged in transmission efficiency from 0–100% for both viruses, although the overall mean transmission efficiency was similar to the transmission competent parent, indicating directional dominance. The direction of the cross did not significantly affect the vector competency for either virus, suggesting that maternally inherited cytoplasmic factors, or bacterial endosymbionts, did not contribute significantly to the inheritance of vector competency in S. graminum. Importantly, there was no genetic correlation between the ability to transmit Barley yellow dwarf virus and Cereal yellow dwarf virus-RPV in the F1 genotypes. These results taken together indicate that multiple loci are involved in the circulative transmission, and that the successful transmission of these closely related viruses is regulated by different sets of aphid genes.Clonal populations of the aphid,  The molecular and cellular basis of virus-vector interactions that regulate transmission are not well understood, but it is clear that genetic elements within both the virus and the vector ultimately determine if a particular species or individual within a species of arthropod is able to transmit a particular virus strain . EnvironCulicoides variüennis to bluetongue virus  and Cereal yellow dwarf virus-RPV   and biotype F (Sg-F) (Two genotypes of F (Sg-F) , were obF (Sg-F) . To induF (Sg-F) .S. graminum biotypes was correlated with host preference. To determine if virus transmission phenotype in the aphid was genetically linked to the virulence phenotype in wheat, the Sg-F and Sg-C parents and their F1 progeny were evaluated for their virulence on the wheat cultivars, Amigo and Largo that express the Gb2 or Gb3 gene, respectively. Virulence is characterized by necrotic spots with chlorotic halos that appear at feeding sites of the aphids. Avirulence is characterized by small, chlorotic spots that appear at the feeding sites, but the spots do not increase in size or become necrotic. Wheat seedlings at the two-leaf stage, three per 4” pot, were infested with approximately 20 adult aphids. Aphids were introduced onto the soil and allowed to distribute themselves on the wheat plants. The plants and aphids were enclosed in one cage and maintained at 18 ± 2°C, 16:8 (L:D) regime for 12 days. Observations of necrosis were made at 3 to 4, 7 to 8 and 11 to12 days after infestation. Damage was scored as 1, 2, 3, 4 depending on if necrosis was observed at 3 to 4 days after infestation, 7 to 8 days after infestation, 11 to 12 days after infestation or no necrosis, respectively. Experiments were repeated 2 to 4 times for each F1 genotype and approximately eight genotypes were evaluated within each experiment depending on availability of aphids. The Sg-F and Sg-C genotypes were used as controls in each experiment.The Sg-F and Sg-C biotypes were originally separated using differential cultivars of wheat possessing different resistance genes . Sg-F isRhopalosiphum padi. During these experiments, virus source plants were inoculated by Sg-F. Virus source leaves were collected approximately 3–4 wk after inoculation. The base of the detached leaf was wrapped in cotton, moistened, and inserted into a 2 cm diameter plastic tube so that the cotton at the base of the leaf formed a plug at the end of the tube. Adult aphids were collected from a 3 week-old colony, dropped into the tube and allowed to crawl onto the leaf. A foam stopper was used to seal the top end of the tube to prevent aphids from escaping. The adults were allowed to feed on the leaf and produce young for 24 hrs prior to being removed. The nymphs were removed 48 hrs later; therefore, they were allowed a 48 to 72 hr acquisition access period depending on when they were born. This technique was used to minimize the handling of young aphids, which are easily damaged. At the end of the 48 to 72 hr acquisition access period, a majority of the nymphs had molted to second instars. Three aphids were transferred to each recipient non-infected plants (‘Coast Black’ oat) to determine transmission efficiency. Aphids were allowed a 3 to 5 day inoculation access period and then the plants were fumigated with DDVP  in a closed chamber, moved to a greenhouse and observed for symptom development for 3 to 5 weeks. Acquisition and inoculation access experiments were conducted in plant growth rooms maintained at 18 ± 2°C, 16:8 L:D regime. Virus transmission efficiency was calculated as the percentage of the total number of plants infested with viruliferous aphids that become infected. The transmission efficiency data were transformed as an arcsin square root prior to analysis, although the untransformed data are presented in figures. Usually each transmission experiment evaluated 8 to 10 F1 genotypes plus the two parents using 12 to 16 recipient plants for each aphid genotype. Transmission experiments were repeated 2 to 5 times for each F1 hybrid. All leaves used as virus source tissue were tested for the presence of virus using a double antibody sandwich enzyme linked immunosorbent assay ? 2) How did the mean transmission efficiency of all the F1 genotypes compare to the mean transmission efficiency of each parent ? 3) Was the transmission phenotype in F1 genotypes influenced by the direction of the cross ? The means of each level of fixed effects  were calculated in PROC MIXED as least-square means (LSMEANS statement), while the value for each level of the random effect (each clone within the F1 hybrid population) was estimated as a best linear unbiased predictor or BLUP (option SOLUTION). The variance components and broad-sense heritabilities (H2) were determined as the ratio between the total genetic variance and the total phenotypic variance, for each virus strain separately, by extracting between-genotypes (i.e. genetic) and environmental components of variance from one-way ANOVA's on F1 genotypes within each virus type using PROC MIXED . Genetic correlations between the transmission efficiency of the virus isolates were calculated in F1 genotypes as product-moment correlations of clone means  Was the transmission efficiency among the F1 genotypes genetically variable  was significantly more severe for efficient vectors of PAV and least severe for intermediate vectors (Gbs gene)  .S. graminum genotypes, Sg-F and Sg-C, behaved similarly to previous studies  and negative (Sg-C) controls in each of the F1 hybrid experiments. Due to the variation in the transmission efficiency of the parent genotypes across all experiments and a need to compare the transmission efficiencies of the F1 genotypes with each other, we calculated a corrected transmission efficiency (TE) for each F1 genotype using the following formula: TEcorr = 1-((TESgF -TEF1)/(TESgF -TESgC)). An actual mean transmission efficiency was calculated for each F1 genotype (TEF1). The mean transmission efficiency for each of the parents (TESgF and TESgC) was calculated using only the data from the experiments a particular F1 was tested in andthen the mean value for that F1 was corrected based on the parent values. If the mean transmission efficiency of the F1 genotype was lower than the mean for Sg-C, its transmission efficiency was 0%. Similarly, if the mean transmission efficiency of the F1 genotype was higher than the mean for Sg-F, its transmission efficiency was 100%. In most cases, the mean of the F1 genotype was between the mean values for the parents. The range of corrected transmission efficiencies of RPV and PAV for each F1 hybrid is shown in F1 genotypes were genetically variable for transmission of PAV  and RPV , indicating that vector competence segregated in the F1 generation. This suggests that at least one of the parents, for at least one of the loci involved, was not fixed as the F1 generation would otherwise be homogeneous. This is not unusual when parental genotypes used for the crosses are drawn from a natural population. The broad-sense heritability (H2) was equal to 0.60 and 0.42 for PAV and RPV transmission, respectively, indicating that a significant proportion (60% and 42%) of the variation in the ability of F1 genotypes to transmit PAV and RPV was due to genetic differences between F1 genotypes. There was directional dominance found for transmission of both viruses, as F1 genotypes displayed an ability to transmit PAV  and RPV  with similar efficiencies as Sg-F. The mean transmission efficiencies for the F1 population were significantly higher than Sg-C for both PAV  and RPV  . There wAlthough the direction of the cross did not have a significant effect on the transmission efficiency of both PAV  or RPV  , the F1 The ability to generate sexuals and then maintain parthenogenetically reproducing hybrid aphid genotypes is a powerful tool for genetic analysis. We were able to maintain genetically stable genotypes and repeatedly assay each parent and hybrid genotype for multiple quantitative phenotypes. In purely sexual species, it is necessary to generate ”recombinant inbred lines” in order to obtain replication. However, inbreeding depression and residual genetic variation is common in recombinant inbred aphids , biasingS. graminum is common. The role of S. graminum in the epidemiology of BYDV/CYDV in wheat is not well characterized in the central and western United States, although vector populations do exist in both regions . All of this leads us to believe that symbionin plays a minor, if any, role in genetic regulation of BYDV/CYDV transmission by S. graminum.Furthermore, we have no data to indicate that symbionin plays a significant role in the lack of BYDV-PAV or CYDV-RPV transmission by Sg-SC. The direction of the cross was not a major factor in transmission efficiency , suggests et al.  found thS. graminum that differ in where the barrier to transmission  is located. These aphid genotypes will be invaluable in studies aimed at identifying genes and gene products that are involved in regulating the movement of virus across specific tissues in the aphid. Coupled with other technologies to study the genomics and proteomics of virus transmission, and information being developed on virus binding proteins in aphids (The ability to generate and maintain stable genotypes of vector and nonvector aphids within the same species will allow continued study on the mechanisms of circulative virus transmission. n aphids , genetic


The virus is probably maintained on the population level by amplification and transmission in maternity colonies. Myotis dasycneme, M. daubentonii, Pipistrellus nathusii,P. pygmaeus). The lineages formed a monophyletic clade of bat coronaviruses found in northern Germany. The clade of bat coronaviruses have a sister relationship with a clade of Chinese type I coronaviruses that were also associated with the Myotis genus (M. ricketti). Young age and ongoing lactation, but not sex or existing gravidity, correlated significantly with coronavirus detection. The virus is probably maintained on the population level by amplification and transmission in maternity colonies, rather than being maintained in individual bats.We tested 315 bats from 7 different bat species in northern Germany for coronaviruses by reverse transcription–PCR. The overall prevalence was 9.8%. There were 4 lineages of group I coronaviruses in association with 4 different species of verspertilionid bats ( Coronaviruses are enveloped viruses with plus-stranded RNA genomes of 26–32 kb, the largest contiguous RNA genomes in nature  were marked with aluminum bands for tracking during an ongoing survey and protection program established by the Schleswig-Holstein Federal Ministry of Environment. In 1 case, a radio transmitter  was used for bat tracing. While being kept in bags, bats produced fecal pellets that were collected with clean tweezers and spiked into RNAlater RNA stabilization solution  for sample processing. Duplicate sampling of bats was prevented by marking the toes of captured bats with nail polish upon first catching. Procedures were consistent with national guidelines for the capture, handling, and care of bats.Field work was conducted at 8 sites in a 7,834-kmwww.megasoftware.net), and analyzed by bootstrapped phylogenetic analysis by using the neighbor-joining algorithm. A nucleic acid distance matrix was also calculated by Mega 4. All analyses on the 334-bp fragment were repeated with the complete 440-bp fragment for validation. All phylogeny and homology results were equivalent, with the exception of a smaller number of complete sequences that were available for the analyses. Results are therefore not shown. All statistical procedures were done with the Statgraphics V 5.1 software package . Sequences from northern German bat coronaviruses can be retrieved from GenBank under accession nos. EU375853–EU375875. Isolation of virus was attempted from diluted RNAlater suspensions, as well as from some fecal pellets suspended separately in phosphate-buffered saline. Vero and CaCo2 cells, as well as primary cell cultures from Carollia bat lung and kidney, were used. No virus growth could be confirmed by RT-PCR (data not shown).Fecal pellets suspended in RNAlater were homogenized by vortexing. Of the suspensions, 50 μL were introduced into 500 μL of Buffer AVL from the QIAGEN viral RNA minikit and processed further according to the instructions of the manufacturer. Elution volume was 50 μL. Nested reverse transcription–PCR (RT-PCR) was performed exactly as described previously, with primers that provided equally high sensitivity for all coronavirus groups  yielded coronavirus, with detection rates of 5.2% to 25.4% per species. Detection rates varied significantly between bat species, with M. dasycneme showing significantly higher rates than any other species . Among the M. dasycneme bats, detection rates were not equally distributed but correlated significantly with the location in which bats were caught . Similar observations were made for 3 other virus-positive species, where detection was achieved in 2 of 6 (M. daubentonii), 1 of 3 (P. nathusii), and 1 of 4 (P. pygmaeus) sampling locations.Of the 7 species studied, 5 , the highest rate of all locations sampled in the study period.Spillover of bats between colonies was not seen, with one interesting exception. In location 2, one of 7 examined t test, p = 0.026) or swarming sites . Differences between foraging and swarming sites were not significant (p = 0.609).Factors correlating with coronavirus infection were determined. Analysis was conducted on major physical properties. Approximately half of the bats (56%) were females, 30% of them lactating. Twenty-seven percent were juvenile, 12% subadult, and 61% adult. As shown in M. ricketti, which belongs to the same subgenus (Leuconoe Boie) as M. dasycneme and uses a similar ecologic niche , along with Pipistrellus bats that carried clearly distinct virus of lineages 2 and 3. Viruses therefore seemed to be more closely associated with bat species than with sampling locations. On the contrary, virus from the only virus-positive M. bechsteinii bat, a very rare and almost extinct species, clustered with lineage 1. Virus detection was successfully repeated from the same sample. These findings suggest that spillover of virus from M. dasycneme into M. bechsteinii might have occurred. In addition, nonrecent host transition of a common ancestor of Myotis-associated CoV into Pipistrellus is suggested by virus phylogeny for lineage 2 and 3 viruses  were 10% distant.To appreciate the diversity of northern German bat-CoV, a nucleotide distance matrix of 30 major taxa of coronaviruses was set up, including established coronavirus species and novel bat-CoV taxa, as recently defined . LineageSimilar to our previous studies on anti-coronavirus antibodies in African bats and recent findings of bat CoV in North America  virus; location 5 harbored all 4 virus-carrying species in 1 place. Three of 4 Myotis species yielded viruses  as M. dasycneme and uses a similar ecologic niche  Pipistrellus species would likely have resulted from a host switch of Myotis virus; Pipistrellus is not closely related to Myotis spp.  that separates both Pipistrellus virus lineages. This in turn could be a correlate of independent adaptations to P. nathusii and P. pygmaeus, respectively, after host transition.Finally, the virus observed in 2 different Should these initial observations be confirmed in future studies, implications on infection control and prevention of zoonotic outbreaks would be considerable. Targeted eradication of bats is technically impossible and ecologically detrimental. Systematic intervention in the ability of bats to carry coronaviruses might be a realistic, but remote scenario. Further research into the association of coronaviruses with natural hosts is necessary to understand their maintenance patterns and zoonotic potential.Nucleic acid distances in a 334-bp fragment of ORF1b retrievable from most coronaviruses. Pairwise nucleic acid distances in the 334-bp core fragment of ORF1 that has been completely entered in GenBank for most coronaviruses from a 440-bp amplification product. Distances between prototype bat coronaviruses or type strains of established mammalian coronavirus species of groups I and II are shown. Names of type strains or bat coronavirus prototype strains are shown in the left column. Distance values ≤0.1 are underlined.


Nearly 1 in 10 in the population experience fatigue of more than six months at any one time. Chronic fatigue is a common reason for consulting a general practitioner, and some patients report their symptoms are not taken seriously enough. A gap in perceptions may occur because doctors underestimate the impact of fatigue on patients' lives. The main aim of the study is to explore the economic impact of chronic fatigue in patients seeking help from general practitioners and to identify characteristics that explain variations in costs.The design of study was a survey of patients presenting to general practitioners with unexplained chronic fatigue. The setting were 29 general practice surgeries located in the London and South Thames regions of the English National Health Service. Use of services over a six month period was measured and lost employment recorded. Regression models were used to identify factors that explained variations in these costs.The mean total cost of services and lost employment across the sample of 222 patients was £3878 for the six-month period. Formal services accounted for 13% of this figure, while lost employment accounted for 61% and informal care for 26%. The variation in the total costs was significantly related to factors linked to the severity of the condition and social functioning.The economic costs generated by chronic fatigue are high and mostly borne by patients and their families. Enquiry about the functional consequences of fatigue on the social and occupational lives of patients may help doctors understand the impact of fatigue, and make patients feel better understood. A quarter of the population report tiredness as being a recent health problem , and forFatigue has been shown to result in high economic costs to society, especially because of its impact on employment and the need for families and friends to spend time caring for the individual . The aimParticipants for the study were recruited from 29 general practice surgeries in the London and South Thames National Health Service region. The practices covered a population of approximately 236,000 people. Between November 2003 and October 2007 patients presenting at these general practice surgeries complaining of chronic fatigue were referred by doctors if they fulfilled the following criteria. Patients had to: (i) have had fatigue for more than 3 months, (ii) have a score greater than 4 on a 11-item self-reported Chalder fatigue scale which has been shown to be valid and reliable , (iii) bThe clinical condition of the patients was measured using the Chalder fatigue scale . The HosThe use of services by patients in the six months before interview was measured using an adapted version of the Client Service Receipt Inventory . PatientThese service use data were combined with appropriate unit costs for 2006/2007 obtained from national sources -16. The Patients were also asked about their salaries (if in work) and whether they had had to stop or reduce work due to their ill health and if so for how many days or hours per week during the previous six months. This information was used to calculate the productivity losses generated by the condition, assuming that individual levels of earnings reflect productivity, based on the human capital approach . The resCosts were reported in the following three categories: (i) formal service costs, (ii) all service costs , and (iii) total costs (i.e. including production costs). Service costs are also disaggregated into service categories .Multiple regression models were constructed to identify factors that explained variation in costs chronic fatigue. The dependent variables were the three measures of costs described above . Demographic factors included in the models were gender, age, whether the patient cared for any dependants, and whether the patient lived alone. A variable reflecting the patients' perceptions of the cause of their fatigue  was included. Also included were the duration of the fatigue and functional impairment measured through the Work and Social Adjustment Scale (WASA),. Clinical factors included: total symptom score as measured by the Chalder Fatigue Scale, and depression and anxiety scores measured using the Hospital Anxiety and Depression Rating Scale. All variables were entered into the model in a single block. Cost data are often positively skewed, therefore generalised linear models with a log link and gamma errors were used .As there is uncertainty around a number of unit costs, sensitivity analyses were performed to examine how costs changed when the unit costs were varied. In the case of informal care, rather than the cost of a homecare worker used in the base-case analysis, it was assumed a cost per hour equivalent to the national minimum wage (£5.35). In the valuation of production costs it was assumed first that the fall in production was greater than the level of the wage, using a wage-production ratio of 1.2. Second, it was assumed that this ratio was 0.4, implying that 50% of employees would be able to make up for their lost time on their return to work.Inclusion of the WASA in the regression models may mask the impact that other variables have on cost. Therefore, in sensitivity analyses we repeated the model but excluded the WASA scores.The research protocol for this study was reviewed and approved by the Multi Centre Research Ethics Committee (MREC) West Midlands (MREC/02/7/71) and was approved by Lambeth, Southwark and Lewisham Primary Care Trusts (RDLSLG 142).The number of patients referred by GPs was 324. Of these, 100 did not meet the eligibility criteria and were excluded and 2 removed their consent. Therefore, data were collected for 222 patients. The characteristics of the sample are shown in Table All except one patient had contacts with their GP during the six-month period prior to interview Table  - an expTable Informal care was required by almost a third of patients, and for these patients this amounted to an average of eight hours per week. Informal care accounted for just over a quarter (26.4%) of the total costs. More than half of the patients stated that their health had affected the number of hours that they were able to work or study and this resulted in a mean lost employment cost of £2350 during the six-month period. This accounts for just over 60% of the total economic cost.The distribution of total costs is shown in Figure If the national minimum wage is used to cost informal care rather than the cost of a homecare worker there is a 17.5% reduction in costs. There would be a 30% increase in costs if it was assumed that the fall in production was greater than the level indicated by wage rates and a 30% decrease if it was assumed that 50% of employees would be able to make up for their lost time on their return to work. The regression analysis without WASA scores (not shown) did not result in substantial differences in the health care cost model. In the analysis of service and total costs, the removal of the WASA variable from the model did modify the findings. The Chalder fatigue score became statistically significant for service costs and the variable indicating the level of depression was significant associated with total costs. The level of variation explained by the models was reduced significantly, with values of 6%, 12% and 15% respectively for health care, service and total costs.syndrome, estimated at £3.5 billion for the UK [The total economic cost associated with chronic fatigue is high, with informal care and lost productivity as a result of reduced work being the main contributors. The sensitivity analyses show that the total costs are extremely sensitive to the unit costs used for informal care and the method to value the lost production. Other studies have also shown the majority of costs due to chronic fatigue are 'hidden costs' such as informal care ,24,25. Mr the UK  and betwr the UK . As far Nearly a fifth of patients had used complementary and alternative therapies professionals, which is very similar to reports in the United States ,29, AustOver one-quarter of the variation in total service costs and costs including lost employment could be explained. This percentage is similar to the one found in a previous chronic fatigue study . In the There are limitations to the study. First, there is a lack of a clear consensus as to how to calculate production costs. We used the most common method, the human capital approach, but different values for lost production were explored in sensitivity analyses. Questions in the CSRI were used to measure lost work time. The CSRI is adapted for each study but these questions have been used in numerous other studies. However, we did not measure the effect of reduced efficiency at work ('presenteeism') and so the production costs are likely to be an underestimate. Second, patient recall was used for measuring service utilisation and lost employment. This may not always be accurate, however data from patient recall can correlate well with data from other sources ,32. ThirThe cost of formal services is only a small proportion of the overall costs for patients with chronic fatigue. People with chronic fatigue frequently receive care from their families and experience lost employment. These two features account for the greatest proportion of costs. These findings reinforce the point that healthcare professionals need to be aware of the real economic strain imposed on a person with chronic fatigue and their family, and that this can have serious implications such as the breakdown of relationships . DocumenThe authors declare that they have no competing interests.RSF did the data analysis, participated in the interpretation of the data and prepared the manuscript. PM contributed to the research proposal, reviewed the analysis, participated in the interpretation of the data and participated in the preparation of the manuscript. MH, MK and AND participated in the research proposal and the conduct of the study, and reviewed the manuscript. LR originated the idea for this study, developed the research proposal with MH and MK, led on the conduct of the study, participated in the interpretation of the data and participated in the preparation of the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/10/56/prepub


The global spread of the highly pathogenic avian influenza H5N1 virus has stimulated interest in a better understanding of the mechanisms of H5N1 dispersal, including the potential role of migratory birds as carriers. Although wild birds have been found dead during H5N1 outbreaks, evidence suggests that others have survived natural infections, and recent studies have shown several species of ducks capable of surviving experimental inoculations of H5N1 and shedding virus. To investigate the possibility of migratory birds as a means of H5N1 dispersal into North America, we monitored for the virus in a surveillance program based on the risk that wild birds may carry the virus from Asia.Of 16,797 birds sampled in Alaska between May 2006 and March 2007, low pathogenic avian influenza viruses were detected in 1.7% by rRT-PCR but no highly pathogenic viruses were found. Our data suggest that prevalence varied among sampling locations, species , ages (juveniles higher than adults), sexes , date (highest in autumn), and analytical technique .The prevalence of low pathogenic avian influenza viruses isolated from wild birds depends on biological, temporal, and geographical factors, as well as testing methods. Future studies should control for, or sample across, these sources of variation to allow direct comparison of prevalence rates. Wild aquatic birds are considered the reservoir for all subtypes of influenza A viruses, with most infections thought to be inapparent . Bird-toSignificant numbers of birds migrate between Asia, where the HPAI H5N1 virus is endemic, and high latitude areas of North America. It is estimated that 1.5–2.3 million birds migrate from Asia to Alaska each year . While tIn 2006, an interagency strategic plan was developed by the US Department of the Interior (DOI), US Department of Agriculture (USDA), and state, tribal, and other organizations to conduct monitoring for early detection of the introduction of HPAI H5N1 into North America by migratory birds . The US Samples from 16,797 birds were collected between May 2006 and March 2007 , mallard [A. platyrhynchos] and northern pintail [A. acuta]) had sufficient samples of age, sex, and collection dates to allow assessment of trends in exposure from spring through autumn . When we added covariates  to the best model , there was strong support for inclusion of date of sampling as well as species, but little evidence of an interaction between date and species. Thus, while species have different overall rates of virus prevalence, all show similar patterns or trends across sampling dates , Aleutian tern [Onychoprion aleuticus], common murre [Uria aalge], and thick-billed murre [U. lomvia]) tested rRT-PCR positive for AI viruses, samples from only 2 species of shorebirds  were positive, and AI viruses were not detected in passerines.We found considerable differences in AI prevalence among taxa but, overall, our rates are somewhat lower than found in surveillance studies conducted in Europe ,24. In AThe prevalence of AI in waterfowl is typically low on wintering areas and declines further in spring, with prevalence rates in ducks of 0.4% in late winter and 0.25% as they return to nesting areas in Canada ,27. In ca priori expectations based on sampling, but similar to previous studies, we found higher prevalence from rRT-PCR compared to virus isolation. For example, in one study, molecular screening yielded 14.8% AI positives compared with 8.4% by virus isolation, and in another report, viruses were isolated from 60% of the molecular positives [Contrary to our ositives ,22. Thisositives ,28. FurtMunster et al.  proposedThe sources of variation noted above complicate exposure rate comparisons among studies. For example, our study found that AI prevalence, as indicated by positive matrix gene rRT-PCR test, was detected in 1.7% of the birds tested which contrasts with the 0.06% prevalence reported by Winker et al. , who tesOur large-scale surveillance study has provided a comprehensive analysis of the status of HPAI H5N1 in Alaska, where the virus had a high likelihood of first appearing in North America if introduced by migratory birds. The detailed ornithological and virological data gathered broadens our understanding of the ecology of AI in wild birds. Our data indicate that AI prevalence varied among locations, species, ages, sexes and through time. Given the patterns detected, we caution against simple comparisons among virus prevalence rates that do not control for these sources of variation. Finally, our data indicate that rRT-PCR results and virus isolation tend to differ regarding the prevalence of avian influenza viruses detected; thus, further study will be required to determine the overall best protocol for surveillance sampling as well as more detailed study of AI viruses in general. Continued surveillance under this strategy over multiple years will allow for a better understanding of the role wild birds play in the intercontinental transmission of AI, including HPAI H5N1. We encourage others to pursue risk-based sampling for H5N1 surveillance, based on current known distributions of the virus in combination with movements of migratory birds.All migratory bird species linked to both Alaska and Asia were identified. Each species was ranked based on 5 factors related to the likelihood of contracting the virus in Asia and bringing it to Alaska :1) Proportion of the Alaskan population occurring in Asia. This factor is based on numerical probability such that the more individuals within a species that winter in, or migrate through, Asia, the higher the likelihood that one or more will encounter the virus.2) Potential for contact with a known H5N1-affected area. This factor considers if these species are using areas where HPAI H5N1 is currently known to occur. The scores for this factor are anticipated to change in future years as HPAI H5N1 continues to spread and new areas of exposure continue to be identified.Clangula hyemalis) winter in areas where HPAI H5N1 is known to occur, but these birds primarily utilize near shore marine habitats reducing their probability of exposure. Conversely, northern pintails winter in areas known to have HPAI H5N1 and utilize freshwater and agricultural habitats significantly increasing their likelihood of coming into contact with domestic fowl or domestic fowl wastes.3) Habitats used in Asia in the context of exposure potential. This factor considers the habitats used by each species in the context of the probability of being exposed to avian influenza. For example, long-tailed ducks (4) Population size in Alaska. This factor considers the total population of a species that occurs in Alaska in the summer and, similar to the first factor, is based on numerical probability and de-emphasizes rare and accidental species.5) Probability of obtaining a representative sample of sufficient size. This factor considers the likelihood of obtaining a representative sample. Alaska is very large with numerous logistical and economic constraints on access. Locations used by large numbers of some species are ephemeral and impossible to predict. Thus, this factor represents the likelihood of successful sampling.From these factors, we developed sampling plans for the highest-ranked species. Because virus exposure in wild birds would likely not be uniformly distributed among all populations within species, geographically isolated populations for each species were identified for sampling. Chen et al.  isolated® swab around the interior mucosa of the cloaca. Fecal samples were collected using a sterile Dacron® swab or by transfer of fecal material, but only when species identity was known for individuals flushed from nest or roost sites, observed while feeding, or captured in mist nets and briefly held in a clean container prior to banding. Cloacal swabs and/or swabs of fresh fecal material were immediately placed into a vial containing 1.5 ml of viral transport media [Birds were live-captured using a variety of techniques, sexed and aged using plumage, biometric or cloacal characteristics  protocol ,28,32. TAll matrix rRT-PCR test positive specimens, as well 73.6% of negative specimens, were further tested by virus isolation in embryonating eggs . Negativa priori groups and across covariates. A series of a priori models was developed and Akaike's Information Criterion (AIC)-based selection methods were used to identify the models most supported by the data [Confidence intervals for proportions were estimated following Newcombe . Logistithe data . HierarcWhen comparing the results of the rRT-PCR and the virus isolation tests, we expected virus isolation to yield higher AI prevalence than rRT-PCR. This assumption was based on the fact that we tested all rRT-PCR positive samples, but only a proportion (73.6%) of the rRT-PCR negatives, by virus isolation. Accordingly, we employed 1-tailed tests under a null hypothesis that rRT-PCR exposure rates would be less than virus isolation exposure rates.None of the authors has any financial interest or conflict of interest with this article. Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the US government.HSI led the laboratory analysis. HSI, PLF, JCF and DAR wrote the manuscript. PLF, RJD, DVD, REG, CRE, JMP, RBL, SMM, DBI, JBF, RMO, MRP, TFF, TCR developed the sampling design and directed the collection of samples. DVD and DAR coordinated field collection programs and JCP provided confirmatory virus analysis. All authors approved the final version of the manuscript.Results of avian influenza surveillance in Alaska between May 2006 and March 2007. The total number of samples collected, the number tested by matrix rRT-PCR and virus isolation in embryonating eggs, and the number of samples positive for avian influenza by both methods is presented for each of 126 species. The rRT-PCR results were used in the statistical analyses and summaries in the text.Click here for fileSelection results for logistic regression models used to describe variation in rRT-PCR prevalence among age and sex classifications. Structure and associated Akaike's Information Criterion (AIC) values for models used to describe variation in rRT-PCR virus prevalence among adults and juveniles, males and females.Click here for fileSelection results for logistic regression models used to describe temporal and species variation in rRT-PCR prevalence among age and sex classifications. Structure and associated Akaike's Information Criterion (AIC) values for models used to describe variation in rRT-PCR virus prevalence across dates of sampling among adults and juveniles, males and females.Click here for file


In the face of the dual TB/HIV epidemic, the ProTEST Initiative was one of the first to demonstrate the feasibility of providing collaborative TB/HIV care for people living with HIV (PLWH) in poor settings. The ProTEST Initiative facilitated collaboration between service providers. Voluntary counselling and testing (VCT) acted as the entry point for services including TB screening and preventive therapy, clinical treatment for HIV-related disease, and home-based care (HBC), and a hospice. This paper estimates the costs of the ProTEST Initiative in two sites in urban Zambia, prior to the introduction of anti-retroviral therapy.Annual financial and economic providers costs and output measures were collected in 2000–2001. Estimates are made of total costs for each component and average costs per: person reached by ProTEST; VCT pre-test counselled, tested and completed; isoniazid preventive therapy started and completed; clinic visit; HBC patient; and hospice admission and bednight.Annual core ProTEST costs were (in 2007 US dollars) $84,213 in Chawama and $31,053 in Matero. The cost of coordination was 4%–5% of total site costs ($1–$6 per person reached). The largest cost component in Chawama was voluntary counselling and testing (56%) and the clinic in Matero (50%), where VCT clients had higher HIV-prevalences and more advanced HIV. Average costs were lower for all components in the larger site. The cost per HBC patient was $149, and per hospice bednight was $24.This study shows that coordinating an integrated and comprehensive package of services for PLWH is relatively inexpensive. The lessons learnt in this study are still applicable today in the era of ART, as these services must still be provided as part of the continuum of care for people living with HIV. Recently there have been major improvements in access to treatment for people living with HIV (PLWH) in developing countries . ProvisiThe appropriateness of these individual ProTEST components has previously been established. Co-ordination of services is expected to improve access to prevention and care ,6. VCT hAlthough some evidence exists on the costs of these individual services  -37, therThe Zambian ProTEST pilot project began in 1999 as a collaboration including the Zambian AIDS-related TB project (ZAMBART), Kara Counselling and Training Trust (KCTT), local primary health clinics, youth friendly services (YFS), district health authorities, and the Archdiocese of Lusaka. Together they provided: VCT with IPT for TB prevention, community outreach, a clinic for management of HIV-related illness, hospice, and HBC. Although these services operated independently, through collaboration and improved referral systems they formed an integrated package. The initial impetus for the intervention was the dual HIV/TB epidemic being seen in Lusaka with its high HIV prevalence (22%)  and highProTEST Zambia was implemented in two high-density low income urban compounds in Lusaka: Chawama and Matero. Prior to ProTEST, the Chawama health clinic already had a VCT centre providing IPT as part of a study. The ProTEST Initiative in Chawama built a new building for this growing service, introduced monthly co-ordination meetings and strengthened the referral system, supported YFS to provide outreach, and established the ProTEST clinic. The costing took place in the second year of ProTEST operation. Matero was a completely new site, opening in November 2000. ProTEST established a VCT and other ProTEST services, and included chest x-rays as part of their process for TB screening. The two sites differed slightly in their spatial integration: Chawama's VCT and clinic were in stand-alone buildings within the PHC grounds, while in Matero they were situated within the main hospital building.The core components of ProTEST were considered to be co-ordination, VCT, IPT, ProTEST clinic, and outreach. Hospice and HBC were considered affiliated services. The heart of the ProTEST Initiative was coordination of the different services. This was done by the ProTEST team at ZAMBART. The team consisted of a medical doctor/manager, a clinical officer, an outreach worker/drama coordinator, a driver, and had input from a secretary (50%) and an expatriate director (20%). The team also participated directly in many of the services provided. They set up and monitored the IPT, ran the weekly ProTEST clinics, and organised the outreach programme. The clinicians provided weekly medical services to the hospice.The aim of the coordination component was to strengthen collaboration and channels of communication between traditionally vertical TB and HIV services that were serving much of the same population. The main tangible activity was meetings among the different service providers to coordinate their services and strengthen their mutual referral systems Figure . Lunch aIn Chawama, meetings ranged from 12 to 40 participants, averaging 33 participants, and in Matero they ranged from 15 to 25 participants, averaging 19 participants. Although the meetings were planned as monthly events, during the period of the costing, there were 6 meetings in Chawama. In Matero there were 5 meetings during the 3-month start-up period and 3 in the year of the costing.VCT served as the entry point for the ProTEST Initiative. Services were provided by nurses with counselling training. During pre-test counselling clients were syndromically screened for STIs, and if need be referred for treatment. Rapid HIV tests were used, making it possible to provide test results to clients on the same day. During post-test counselling HIV-infected people were screened for active TB. People with suspected active TB were referred to the ProTEST clinic for further evaluation of TB and if confirmed they were sent to the national TB services. All other HIV-positive clients with no TB symptoms were offered IPT. IPT consisted of a six-month self-administered course of isoniazid (300 mg daily), collected monthly by clients from the VCT counsellors. Additionally, VCT clients were provided information on services for PLWH.The Chawama VCT centre was located in a stand alone building in the hospital compound. It had a large reception with smaller counselling rooms around the edge. During the year of the costing counsellors increased from three to six. In addition to the outreach activities described below, the Chawama VCT centre used community mobilisers to draw people in for VCT, who were provided a bonus for each person presenting for pre-test counselling. In Matero, the VCT was a room within the main hospital building. There was a single counsellor. The waiting room was a bench in the hospital corridor.Outreach was provided through YFS. With support of the ZAMBART drama coordinator, YFS volunteers wrote and performed dramas in the communities and recorded them on video to provide information on HIV prevention, the ProTEST Initiative, and the benefits of VCT. Each group received a nominal allowance. A 4-month outreach project targeted at sex-workers and their clients was implemented by the Chawama YFS. YFS in Chawama was allocated two rooms in the new VCT building. In Matero the health clinic provided them with a room.The ProTEST clinics served HIV-infected individuals who had passed through VCT. These weekly clinics specialised in treating opportunistic infections and other HIV-related diseases and they were staffed by the ZAMBART doctor and medical officer. The clinicians also referred clients to community services such as hospice and HBC. Drugs were provided free of charge to patients. Treatment did not include the provision of ART during the time of this study, although ProTEST clients have more recently been able to access ART. The ProTEST clinic was held in 2 rooms of the newly built ProTEST building in Chawama, and in a consulting room in the hospital in Matero.The Archdiocese of Lusaka ran a community HBC programme throughout Zambia, including Chawama and Matero. Volunteer caregivers provided care and delivered food supplements to ill patients in their homes. With their strong links in the communities, they were in a position to refer people for VCT and into the ProTEST system.In addition to VCT, KCTT also ran a hospice which served terminally ill patients referred in from Chawama, Matero, and other compounds. The hospice had a total of 20 beds for adults. In addition to core staff, ZAMBART provided the services of a medical doctor and a medical officer to the hospice on a weekly basis. Community organisations provided about a third of the caregiver time.Costing Guidelines for HIV Prevention Strategies [Cost data collection was based on the rategies . In bothFull costs were collected for all ProTEST components, except IPT, where incremental costs of adding IPT on to routine VCT services were estimated. Full costs include the value of space, vehicles, and all operating expenses. In Matero, this also includes a depreciated value of start-up costs.All costs were adjusted to 2007 US dollars ($), including costs quoted from other studies, using a GDP deflator , the 200Financial and economic costs were estimated from a provider's perspective. Financial costs represent actual expenditure on goods and services purchased. These were based on financial accounts and financial reports. Economic costs include the estimated value of all resources used in the intervention, such as the opportunity cost of service providers attending coordination meetings, YFS and HBC volunteers, and donated goods such as HIV test kits and space. An inventory of donated time and volunteer staff input was made either from logs of donations or interviews during site visits. Unit costs were assessed for volunteer time by either the salary of the individual donating time or minimum wage, for unemployed individuals. The value of donated goods were assessed from market prices (for food donations), pharmacies  and from the internet (for HIV test kits not available in country). The annual financial cost of capital goods was calculated using straight-line depreciation, in which the total cost of the good was divided by the length of its working life. The annual economic cost of capital goods was calculated using a 3% discount rate, as recommended by Weinstein et al. .Cost data were collected from administrative records from the service providers , observation at study sites, and discussions with project staff. As ProTEST started in the Chawama site prior to the costing, it was not possible to collect full start-up costs there. Start-up costs related to integrating services in Matero were collected, and are presented separately.Costs were collected retrospectively using a combination of an ingredients-based costing methodology, whereby quantities of resources were multiplied by their respective prices to obtain total costs, and a step-down methodology, where joint costs were allocated to activities using allocation factors . Each seComponent total costs were divided by the component outcomes to obtain component average costs (unit costs). As the first point of contact with the ProTEST Initiative was through the VCT service, the number of people pre-test counselled was taken as the number of people reached by ProTEST. The impact of VCT was measured using the number of people: pre-test counselled, tested, tested HIV-positive, and completing the VCT process (receiving their results). IPT compliance is known to be a problem , therefoA univariate sensitivity analysis was performed to understand how assumptions made in this analysis affected the unit costs of ProTEST. We varied the discount rate (0% and 6%), we estimated the impact of full VCT compliance, a twice as rapid loss to follow-up for IPT, and variance in the number of ProTEST clinic visits (up and down by 25%), the allocation factor of ZAMBART joint costs . In addition, the impact of having a international director was tested by replacing the international salary with a local salary, taken as that of the local project coordinator.Component costs are presented by input in Tables Total annual economic costs of VCT were $47,598 in Chawama and $8,532 in Matero, of which 27% and 39% were for supplies. Financial costs for supplies were 30% and 18% of their economic costs, in Chawama and Matero, respectively, primarily due to large donations of HIV test kits. The total annual incremental cost of adding IPT on to VCT was $1,373 in Chawama and $1,300 in Matero, where x-rays were a routine part of the screening process.Outreach by YFS was $9,884 and $5,078 in Chawama and Matero per year, respectively. In Chawama, the higher outreach costs were largely due to a 4-month outreach intervention targeted at sex-workers and their clients. Much of the outreach by YFS was run by volunteers, leading to the economic costs being more than twice the financial costs in the new site. The annual cost of the clinic was $21,132 and $15,991 in Chawama and Matero, respectively, which contributed to 25% and 49% of total costs in each, respectively.There was a large difference between the financial and economic costs in the affiliated services, HBC and hospice, with their economic costs being 4.4 and 2.6 times their financial costs. HBC paid for only 39% of labour and 13% of supplies as their care-givers were primarily volunteers and they received large food donations from international organisations such as the World Food Programme. The hospice received 85% of their supplies in donations of which 57% was medicines supplied by the Archdiocese of Lusaka and 43% was other supplies coming from community donations. A further breakdown of personnel and supply inputs and their contribution to personnel and supplies, respectively, can be found in Table Client flows through the various ProTEST components are presented in Table The annual cost of ProTEST co-ordination was $1 and $5 per person reached in Chawama and Matero, respectively  but also measures the cost of co-ordinating these services to provide a core package of care for PLWH. Annual costs of the core package of services ranged from $32,347 in the smaller site to $84,213 in the larger site. The introduction of ProTEST has led to enhanced collaboration and improved referral systems for as little as $1 per person reached.There were a number of striking differences between the two sites. The Matero site operated on a much smaller scale than the Chawama site. The client numbers were mostly constrained on the supply-side. Group counselling in Chawama made it possible to reach many people with pre-test counselling. The Matero VCT service had no space for group counselling, there was only a single counsellor, and the health centre (including the VCT service) was closed for a couple of months. On the demand side, it takes time for people to hear about the service and start coming. When people do start presenting, new VCTs tend to attract the people with more advanced symptoms of AIDS ,50.The HIV prevalence in Matero was very high (64%), while the HIV prevalence among VCT clients in Chawama (29%) was more representative of the prevalence in the general population (22%). This difference can also be recognised in the behaviour of clients. While in Matero a very high proportion of clients completed the full process (92%), the Chawama VCT experienced high rates of people not proceeding to testing after pre-test counselling. The high drop-out rate may have a few reasons. Group counselling made it possible to pre-test counsel large groups. Many group counselling sessions were for school children who needed parental permission to test. These youths were educated about HIV at Chawama, but unable to test. Additionally, mobilisers received a bonus for each person presenting for pre-test counselling with their referral slips. The number of ProTEST clinic visits per client in Matero was 62% higher than Chawama again suggesting a more advanced stage of HIV in the clients in Matero.A number of caveats must be considered in this analysis. The allocation of indirect costs had a large influence on component costs, although not on costs of ProTEST as a whole. As start-up costs were not collected for Chawama, both site costs are presented excluding start-up costs. A number of variables were estimated retrospectively, such as staff time allocations. The line item of HIV test kits for VCT was modelled as KCTT had complemented their purchased test kits with donated kits of unknown number. Quantities were estimated by the number of HIV test performed plus an estimated 10% wastage. International prices for these kits were then applied. The HBC costs are the average costs over all sites, and are therefore not site specific. Cost-effectiveness in terms of HIV infections averted would need to be estimated in order to compare ProTEST with other HIV interventions. This requires behavioural surveys and modelling or a randomised intervention design. Although the direct costs of research were removed, time input by international staff into the daily running and co-ordination of the intervention were included at full costs. Therefore if replicating ProTEST outside of the pilot intervention setting cost, personnel cost particularly, are expected about a quarter lower Table .A more general limitation of this study is that it was conducted before the widespread availability of ART. The integration of services is however as relevant in the ART era as at the time of this study, but costs due to the provision of ART would need to be factored in.The cost of VCT was around $35 per client receiving results, which is consistent with other studies of the cost of VCT in Africa. Sweat et al. estimated the cost of VCT in Kenya and Tanzania and found a cost per VCT client of $34 and $37 (adjusted to 2007 dollars) . A key dThe larger number of annual clinic visits per patient to ProTEST clinic in Matero (4.1) than in Chawama (2.6) is consistent with the findings by Kinghorn et al. who found the annual number of outpatient visits in South Africa increased strongly from stage 1 HIV  to stage 4 HIV  . The cosIn many locations a range of preventive and clinical services for HIV exist. This study shows that it is feasible to integrate these services to provide a package of care for PLWH. Without such a continuum of care ART provision makes no sense. VCT must be expanded to allow access to ART. Some clients may not yet need ART but still need screening for other diseases, such as TB, and provision of preventive therapy. Patients accessing ART also need screening for TB and may similarly still require preventive therapies, although the additional benefit of preventive therapy in this setting is currently unclear. The provision of ART still requires a broader range of services for PLWH including symptom-; symptom management, palliative care and HBC will still be required by some patients. Without integration of services and collaboration between service providers, HIV care will not be successful.ART Anti-retroviral therapyHBC Home based careIPT Isoniazid preventive therapyPLWH People living with HIVTB TuberculosisVCT Voluntary counselling and HIV testingYFS Youth friendly servicesThe author(s) declare that they have no competing interests.All authors read and approved the manuscript. FTP conducted the cost analysis and drafted the paper. LK designed and supervised the cost analysis and participated in writing the paper. RG ran the project, assisted in collecting cost data, participated in the interpretation of the results and helped to draft the paper. HA supervised the project and assisted in writing the paper. IK collected the costs for the VCT and assisted in the cost analysis of the VCT. MH collected the costs of HBC and assisted in the interpretation of the results. PGF assisted in the overall interpretation of the study results and writing the paper.