In South Asia a large number of patients seek treatment for TB from private practitioners (PPs), and there is increasing international interest in involving PPs in TB control. To evaluate the feasibility, effectiveness and costs of public-private partnerships (PPPs) for TB control, a PPP was developed in Lalitpur municipality, Nepal, where it is estimated that 50% of patients with TB are managed in the private sector. From the clinical perspective the PPP was shown to be effective. The aim of this paper is to assess and report on the costs involved in the PPP scheme.The approach to costing took a comprehensive view, with inclusion of costs not only incurred by health facilities but also social costs borne by patients and their escorts. Semi-structured questionnaires and guided interviews were used to collect start-up and recurrent costs for the scheme.Overall costs for treating a TB patient under the PPP scheme averaged US$89.60. Start-up costs per patient represented 12% of the total budget. Half of recurrent costs were incurred by patients and their escorts, with institutional costs representing most of the rest. Female patients tended to spend more and patients referred from the private sector had the highest reported costs.Treating TB patients in the PPP scheme had a low additional cost, while doubling the case notification rate and maintaining a high success rate. Costs incurred by patients and their escorts were the largest contributors to the overall total. This suggests a focus for follow-up studies and for cost-minimisation strategies. Tuberculosis is a leading cause of death worldwide  and, altIn Nepal, over 14,000 new cases of smear positive (i.e. infectious) tuberculosis are notified each year , and it In Lalitpur Municipality, District Public Health Office (DPHO) is responsible for the TB control programme. The DPHO took responsibility for day-to-day management of the PPP scheme. This included providing training for laboratory staff and Directly Observed Treatment (DOT) supervisors, supervising workers involved in TB control, ensuring supply and distribution of medicines, and ensuring that five Treatment Centres (TCs) followed standard NTP recording and reporting guidelines.A semi-governmental hospital , three NGOs , Nepal Anti-TB Association (NATA) and Care & Fair) and one private nursing home  were inAn agreement was reached that microscopy services for the PPP would be provided by a laboratory at Hargan's Nursing Home, two laboratories run by NGOs, and a laboratory at Patan hospital. DOT would be provided by all five TCs. TCs had a memorandum of understanding with the DPHO; other agreements were informal.PPs were asked to refer patients to the DOTS centres rather than providing DOT themselves; patients also self-referred. Although not specifically targeted under the PPP scheme, pharmacists could also refer suspected cases to the TCs. PPs in the scheme were seen as complementary to the public DOTS services, rather than replacing them, and were encouraged to use referral facilities for TB laboratory tests, DOT and complicated cases.No financial incentives were paid to PPs, laboratories or TCs, and no other incentives were provided other than signs that confirmed participating DOTS centres were accredited by the NTP. Treatment centres paid their own staff's salaries. Patients paid the consultation fees when they visited private practitioners. As is normal in Nepal, the NTP covered costs of training, drugs for DOTS centres , laboratory supplies (but not for private labs), and materials for recording and reporting. Full details of the organisation of the Lalitpur PPP are published elsewhere .Two other groups were considered to be indirectly related to the PPP scheme: volunteers and traditional healers. The volunteers included in this study were unpaid local people, selected by Lalitpur municipality and responsible to the DOTS centre for community awareness, late patient tracing and visiting PPs and private pharmacies who referred TB cases to DOTS centres to deliver 'thank you' follow-up letters. Volunteers also provided a mechanism for the partnership to interact with PPs. They were provided training by YUHP in collaboration with the DPHO.In this context traditional healers are local people who practice ethnomedicine and other alternatives to western medical practice and who are easily accessible by almost all households in Nepal. Traditional healers in Nepal are mainly faith-based and can be distinguished by the types of services  they offer ,19.Clinical assessment demonstrated that the PPP met the international target for treatment success, and increased the number of TB patients receiving care under the DOTS strategy. The establishment of the PPP showed an overall increase of case notification of sputum positive patients in the study area from 54/100,000 before implementation in 1998 to 102/100,000 following implementation in 2001. Treatment success rates were over 90%, exceeding the international target of 85%, and less than 1% of patients defaulted. Details of the evaluation of the effectiveness of the PPP, and lessons learnt, are published elsewhere .Although the PPP had been demonstrated to be clinically effective, it was considered important to assess its costs to allow others to assess feasibility of replicating the PPP scheme elsewhere . This paThe study used a comprehensive approach to describing costs, including costs incurred by DOT centres, patients and their escorts, under the PPP scheme. This enables assessment of the long-term sustainability of the PPP ,24.Semi-structured interviews were used to collect data on costs. Recurrent costs were collected during the financial year 2001–2002. All costs were collected in Nepali Rupees (NRs), which were converted into US$ at the 2002 exchange rate of 1US$ = 78 NRs .The costs for conducting this study (research project costs) were treated as not directly related to the PPP and, although reported in this paper, were not included as part of costs for treating a TB patient.All interviewees gave verbal informed consent prior to being interviewed. Ethical approval for this study was given by the Nepal Health Research Council.institutional costs, including costs incurred by the DPHO and by each of five DOTS Centres or TCs; patient-related costs including charges, direct travel costs and opportunity costs for time lost in travel both by patients and their escorts; and costs of involving volunteers in the PPP.Data on start-up and recurrent costs were collected in three major fields: Start-up costs were estimated per patient and recurrent costs per patient per month. Costs were then calculated for treating a TB patient on average throughout the whole source of treatment. The average duration of treatment was assumed to be 8 months across all three standard treatment categories, although in practice this may vary slightly depending on the treatment category Figure . This waTable Two approaches for costing analysis were deployed, based respectively on means and medians of costs within the different fields. Although both median and mean values are reported in the paper, we emphasise the importance of medians of costs: in our view these are more representative and allow study results to be more usefully extrapolated to the rest of Nepal and to other similar contexts. However, mean values have also been reported to permit comparisons with other studies.The following three sections provide the details of the data collection methodologies to assess costs incurred by institutions, volunteers and patients respectively.The study assessed all start-up and recurrent institutional costs related to the PPP scheme, including training, supervision, staff salaries, costs of medicines, managerial set-up, logistical supplies, transportation and NTP central administrative costs.DPHO start-up costs included the managerial set-up of the scheme as well as training of PPs, TC staff and volunteers. Administrative costs of training PPs and volunteers were included in DPHO start-up costs. Recurrent costs included costs of staff, procurement and delivery of medicines, refresher training courses and regular (4-monthly) DOTS workshops involving staff from the TCs. All five TCs worked independently and supervision and monitoring was done by the DPHO.Training costs were covered primarily by the DPHO. Training costs reported under different TCs represent costs necessitated by staff cover during training.Costs of medicines for each treatment category of patients were calculated based on standard Nepal NTP treatment guidelines for each of three treatment categories of TB patients. The costs of medicines reflect market prices in 2002/2003 and were estimated using government procurement information provided by the NTP. These costs were down-inflated by 2.9% – the official inflation rate for 2002  – to obtA total of 23 volunteers were interviewed to obtain estimates of their costs.Whilst administrative costs of training volunteers and PPs were regarded as part of DPHO start-up costs, the opportunity costs for the time spent on training was included in volunteers' start-up costs. These were estimated as two days salary  for 43 people.The recurrent expenses incurred by volunteers were recorded separately. These comprise costs reported for three major types of activities, namely tracing patients, visiting DOTS centres and meetings with PPs. Volunteers' costs include actual travel expenses as well as the opportunity costs of their time spent on the above activities.Patients' costs were obtained by interviewing patients from each TC. Data were collected from patients registered in TCs in the period May 2002 to January 2003. Ten patients from each TC were randomly selected using the NTP TB register and TB treatment cards. Of the 50 patients interviewed, 27 were male and 23 were female; 26 patients were in treatment category I, 21 patients in category III and 3 in category II.Patients' costs were collected across the following categories: costs incurred with traditional healers; charges during diagnosis and treatment, which included consultation and investigation charges with the latter comprising costs of sputum smear microscopy, X-ray examination, routine blood tests, Mantoux test and Lung Function Test (LFT); and travel costs including opportunity costs for patients and their escorts. Patients' costs include income losses due to TB illness during the period from when they first sought diagnosis from any source  until the diagnosis of TB was confirmed.actual income rates for each patient, with daily rates varying from US$1.60 to US$3.08.No intervention was made to bring traditional healers into mainstream TB control; costs related to traditional healers represent those reported by patients. Patients' opportunity costs were estimated using Our estimates show that the median total cost involved in treating a TB patient in the PPP scheme is US$89.60 including start-up costs Table . The meaOur estimate of the overall cost of treating a TB patient under the PPP scheme does not include research project costs (start-up research project costs were estimated as US$15 per patient and recurrent project costs were US$34 per patient per 8-month course).The distribution of start-up and recurrent costs across institutions, volunteers and patients is given in Table Total start-up costs per patient were US$11.10, 64% of which were DPHO costs, 32% TC costs and about 5% volunteers' costs.Start-up costs for both DPHO and TCs include four main categories: training, procurement of equipment, managerial set-up and social mobilisation Table . About hRecurrent costs show a more complex picture. As shown in Table A breakdown of institutional recurrent costs is shown in Table DPHO recurrent costs were made up of costs for procurement and distribution of medicines and supplies (70%), staff (20%) and training-related costs (10%). Central administrative costs of the Nepal NTP were estimated to be $7.20/patient treated and were added to the DPHO recurrent costs.A breakdown of total reported TCs' recurrent costs shows that 38% of these costs were incurred by Patan hospital and that staff costs dominated . The relatively low cost of supplies and transportation within TCs is due to the fact that procurement and transportation of medicines is covered by the DPHO. Care & Fair reported the highest costs of utilities and others among the TCs. These high costs are thought to be exceptional.As shown in Table Costs reported by patients are broken down into 6 major categories reflecting the diagnosis and treatment periods. It is worth noting that medicines were provided to patients free of charge: these costs are part of the institutional recurrent costs. Costs of travel of both patients and their escorts have been differentiated from charges for diagnosis and treatment consultations.Total reported patients' recurrent costs Table  compriseMedians of costs incurred with traditional healers and other expenses were zero: that is, more than 50% of all patients did not report such costs. The largest median costs were for the diagnosis phase, and for opportunity costs of treatment-related travel.Overall diagnosis-related costs were further divided into consultation charges (20%) and investigation-related charges (80%). .The majority of patients' non-medical costs comprised the opportunity costs for time lost due to travel to and from TCs, whilst financial transportation charges were reported in about 20% of cases and represent 18% of total patient-reported recurrent costs. The median cost of escorting patients was higher for female patients (US$1.83) than for male patients (US$0.11).Treatment-related costs were a relatively small proportion of overall patient-related costs: this is to be expected as the NTP bears all the medical costs of treatment . Though the median opportunity cost for the working time (rather than treatment time) lost by patients directly due to TB was zero in this study, for individuals these costs can represent a significant share of recurrent costs. For example, two patients reported not being able to work for 2 and 3 years: the corresponding maximum cost reported was US$1,755. Median patient and escort travel expenses were zero, reflecting the proximity of TCs to patients' homes/work places.Patients' costs were also broken down by sex and source of referral Table . The higOverall costs incurred by female patients were considerably higher than those reported by male patients. However, costs reported by male patients who were self-referred and referred from semi-government facilities were much higher than female patients, although the relatively small sample sizes within each category , may mean these findings are unrepresentative.This study was designed to describe rather than compare costs. The costs of treating a TB patient within the PPP are estimated as US$89.60. The additional costs per patient to the public sector (start-up costs US$10.60 and recurrent US$35.60) associated with introduction of the scheme are small for the benefit of doubling the case notification rate. Two studies have been published that compare public sector costs and cost-effectiveness of TB control in PPP schemes ,28, but Several studies have estimated the mean costs of treating TB patients: in Pakistan estimated total costs of treating a TB patient varying from US$102 to US$180 ; in IndiWhilst mean values of total costs are from 2.6 to 3.8 higher than those estimated in Bangladesh, the median values of costs for treating one patient in our study are similar to those estimated in Bangladesh – slightly higher than in DOTS provided by BRAC (US$64) and lower than in the government DOTS programme (US$96) .Likewise, NTP central level administrative costs in our study comprised 16% of the total public sector costs. This is comparable to the situation in Bangladesh  where puA major component of overall costs was patients' or social costs Figure . Our finSeveral studies demonstrate that the DOTS strategy is more cost-effective than other approaches to TB control. As shown in Figure The dominance of recurrent costs over start-up costs mirrors the pattern generally seen when establishing a conventional DOTS programme. This pattern was also seen in Pakistan .Analysis of start-up costs across different TCs re-affirmed a trend of larger facilities having generally higher institutional overheads. A high start-up institutional cost per patient was incurred by Patan Hospital (US$48.5) Table . This isWe have also found generally high start-up costs per patient incurred by one NGO. After a follow-up with the TCs we found that this largely due to their inability to distinguish between start-up costs that were attributed to the PPP scheme and other similar activities performed by the institution.On the other hand, the relatively low start-up costs of Hargan's NH arise because no costs reported for managerial set-up and social mobilisation. This is explained by the previous focus of this private clinic on TB control, leading to a reduced need for start-up investments.As for institutional recurrent costs, the DPHO and TCs incurred roughly the same total costs, indicating a need for strong financial commitment at all levels of the district health system. Staff costs and costs for procurement and distribution of medicines and supplies dominated within total institutional costs, representing respectively 46% and 32% of overall public recurrent costs.Within TCs, the high recurrent costs per patient incurred by one of the NGOs (US$31.30) are largely explained by the relatively high cost of utilities (US$17.80 per patient), which, again, suggests an inability to draw a clear line between costs attributed to the PPP scheme and other institutional overheads.All other TCs incurred recurrent costs in the range US$8.20 to US$21.30 per patient, which is considered reasonable, given the number of patients administered in each TC which varied from 48 to 191 and the size of the institution itself.One of the messages which emerges from the above discussion is that high-cost institutions such as Patan Hospital (with high start-up costs) and Care and Fair (with high recurrent costs) may have a significant impact on the overall costs of the PPP scheme. This effect is likely to diminish if larger numbers of TCs are involved.The costs of involving volunteers in the PPP scheme appear to be relatively low. Greater involvement of volunteers in the PPP scheme might be an interesting area to explore further as a potential for cost-minimisation strategies for the PPP scheme.In this study we also assumed that volunteering had opportunity costs for patient tracing, meetings with PPs and visiting DOTS Centre, although in practice it was a voluntary donation of time in contrast to the involuntary time lost by TB patients. . However, caution may be necessary to avoid overloading volunteers and to forestall volunteers' "compassion fatigue".Patients' costs, including costs incurred by their escorts (in the form of direct charges for services and less obvious expenses such travel costs and time lost), represent the majority of estimated recurrent costs per patient and, perhaps, the most interesting area to explore in more detail. One emerging conclusion is that the significant financial burden on TB patients and their families may serve as a factor restricting patients from entering into, and completing, the full course of treatment. Further research is needed to confirm, further quantify and address this, and potential cost minimisation strategies should address, first of all, this aspect of recurrent budget costs.A breakdown of patients' costs reveals a dominance of diagnosis-related charges over treatment-related charges. During treatment, consultation and investigation costs were similar, whereas during diagnosis investigation costs dominated.Since the median month at which patients' costing data was collected was 7 months, i.e. towards the end of the average 8-months treatment course, we can be confident that most treatment-related charges were included in the study results. Clearly, most of the patients' expenditure occurred during diagnosis, whilst treatment-related charges represent a small fraction of the total patient expenses. The main conclusion emerging from this is that, from a costing perspective, attention should be given to cost-minimisation strategies during the diagnosis stage and in particular investigation-related costs as a strategy to reduce overall patient costs.Transportation costs were reported in about 20% of cases. This relatively low frequency is normal for the urban context of our study. However, these, combined with the opportunity costs of time lost due to travel, represent a significant share of total costs within the study sample. This is primarily because TB treatment involves multiple visits to health facilities. If the decision is made to explore ways of decreasing patients' costs, this is seen as a potential area on which to focus attention.An important feature of our study is the inclusion of patient opportunity costs due to time lost in travel which accounted for a substantial proportion of overall costs. We would therefore encourage other researchers to include the opportunity costs for economic days lost due to TB within social costs.Another interesting finding is the relatively high costs incurred by patients who seek assistance from traditional healers. The frequency of these is relatively low (20%), but costs incurred for traditional healers reported by patients can be as high as US$141 Table .The traditional sector plays a significant role in determining patients' health in Nepal. Two potential strategies to address this issue are to encourage a move away from expensive healers providing ineffective treatment, or to equip these healers with basic knowledge of TB and clear instructions on referring potential TB cases.Treatment of female patients tends to cost more. This is probably because of cultural values in Nepali society, which require female patients to be escorted. Understanding this gender differences in seeking TB care is another area where research is needed.Although this study was not designed to make statistical comparisons between costs for men and women, the cost differences between female and male patients represent an interesting area for a follow-up study.The high patient-related costs seen in this study are likely to be similar for other TB control initiatives, whether Government or private. This suggests the need to address patient costs, including issues of equity and gender related to patient costs, more widely in TB control.This study has demonstrated that this PPP is a financially acceptable way of doubling TB case-finding while maintaining high success rates. There is, therefore, evidence to support scaling up this initiative in Nepal and in similar contexts. However, TB Programme managers will need to consider the degree to which PPs are willing to cooperate, as well as other contextual factors. Three particular issues are highlighted for consideration. Firstly, staff costs in the assessed PPP scheme are low compared to a number of other health systems. Secondly, an important feature of the initiative is the use of unpaid volunteers. Where this is not feasible, their roles may need to be replaced by paid staff, increasing total costs. Lastly, social (patient) costs estimated in this study, as in other TB control initiatives, represent a significant share of total costs, indicating a need to address issues of equity and gender to control costs to TB patients and their families.The author(s) declare that they have no competing interests.All authors contributed equally toward the preparation of this paper and are considered the first authors. More specifically, DKK participated in the study design, led the data collection, participated in analysis and preparation of the manuscript; TNM led the data analysis and coordinated the preparation of the manuscript; ATG coordinated the study, participated in data analysis and preparation of the manuscript; JNN conceived of the study, participated in the design of the study, advised on data collection and analysis and participated in the preparation of the manuscript; SCB participated in the design of the study, coordinated the data collection and participated in the data analysis and the preparation of the manuscript.The pre-publication history for this paper can be accessed here:


Data were retrieved from two primary health care centres in southeastern Sweden.The objective was to explore the usefulness of the morbidity risk adjustment system Adjusted Clinical GroupsA cross-sectional observational study. Data from electronic patient registers from the two centres were retrieved for 2001 and 2002, and patients were grouped into ACGs, expressing the individual combination of diagnoses and thus the comorbidity. Costs per patient were calculated for both years in both centres. Cost data from one centre were used to create ACG weights. These weights were then applied to patients at the other centre. Correlations between individual patient costs, age, gender and ACG weights were studied. Multiple linear regression analyses were performed in order to explain and estimate patient costs.The variation in individual patient costs was substantial within age groups as well as within ACG weight groups. About 37.7% of the individual patient costs could be explained by ACG weights, and age and gender added about 0.8%. The individual patient costs in 2001 estimated 22.0% of patient costs in 2002, whereas ACG weights estimated 14.3%.ACGs was an important factor in explaining and estimating individual patient costs in primary health care. Costs were explained to only a minor extent by age and gender. However, the usefulness of the ACG system appears to be sensitive to the accuracy of classification and coding of diagnoses by physicians. Accurate methods are needed for estimating the burden of morbidity in a population to enable resource allocation corresponding to the health care needs of the population. This is particularly important in relation to the funding of primary health care (PHC) centres, as there is greater variation in terms of need between localities than between regions or counties . DetermiIn countries with a national health care system, where public authorities are responsible for health care provision in regions and smaller areas, interest has focused on demographic and socio-economic determinants of health care needs. In this regard, the underprivileged area score (UPA), or the Jarman index, was developed in the UK . The indIn countries with a health care system primarily based on individual health insurance, interest has focused on measures based on individual patients' characteristics. In the USA, several instruments have been developed to compensate for differences in case-mix and to adjust for differences in risk . One of The ability of the ACG system to measure individual comorbidity and the burden of morbidity in a population is of special interest from a PHC perspective, since a GP cares for a number of individual patients who often suffer from several diseases, all of which are normally treated by the GP. A retrospective study was conducted on encounter data from a three-year period in a Swedish county council and it is published in this issue of BMC Public Health.The aim of this study is to explore the usefulness of the ACG system, in comparison with age and gender, in explaining and estimating patient costs on an individual level in a Swedish PHC setting.® in Ödeshög, and Swedestar® in Ryd. The Swedish PHC version of the International Classification of Diseases and Related Health Problems (ICD-10) was used for labelling health problems . Dat. DatPatiThe yearly cost per patient was subsequently calculated by adding all the costs for all contacts in PHC for that patient during each year.All costs were calculated in Swedish crowns (SEK) (exchange rate June 2001: 1 Euro = SEK 9.20).Only patients with at least one registered IDC-10 code during a calendar year were included in the ACG grouping, and consequently only these patients were included in the creation and application of ACG weights. Data on patients at both PHC centres with registered diagnoses during 2001 and 2002 were extracted from the EPRs. The patients were assigned to ACGs using version 6.0 of the ACG software . This isThe data on individual patient costs from Ödeshög were used to create the ACG weights. Patients with costs more than three standard deviations above the mean were excluded in order to prevent a few extremely costly patients from producing a false high mean cost for some ACGs. The calculation of weights was done by using each individual patient's costs for 2001 and for 2002 at Ödeshög, which were then applied to the categories of patients in terms of ACGs. Cost data from both years were used in order to get a sufficient number of individual yearly costs in each ACG. Relative ACG weights were then calculated and defined as the mean cost for each ACG divided by the mean cost for all ACGs.The relative weights of ACGs (from Ödeshög) were assigned to each patient at Ryd based on that patient's individual ACG assignment. Patients at Ryd with costs more than three standard deviations above the mean were excluded. The usefulness of ACG in explaining individual patient costs was explored in a concurrent model by comparing data from Ryd separately for 2001 and 2002.The relative ACG weights applied  were compared with ACG weights from Ryd, from the USA (a database at Johns Hopkins University) , and froThe usefulness of ACG in estimating patient costs for individuals was explored in a prospective model by comparing data from 2001 with costs from 2002 at Ryd.Spearman's correlation coefficient (Spearman's rho) was used for bivariate correlation for each year between the variables age, gender and ACG weights in a concurrent model. Stepwise multiple linear regression analysis was then used, for each year, to explore the variation of individual patient costs with age, gender, and ACG weights as the independent variables.In order to estimate the predictive usefulness on an individual level in a prospective model, another stepwise multiple linear regression analysis was performed with the individual patient costs in 2002 as the dependent variable and age, gender, ACG weights in 2001, and individual patient costs in 2001 as the independent variables.® version 11.5 was used.The statistical software SPSSThe mean cost per patient at Ödeshög was higher compared with Ryd both in 2001 and 2002  for ACG weights, 0.308 for age, and 0.119 for gender in 2001 in the concurrent model. In 2002 the results were similar. Age, gender and ACG weights  together explained 38.5% of the individual patient costs in 2001 , 0.203 (USA), 0.320 , and 0.337 .The costs of individual patients in 2001 turned out to be the most important factor, by 22.0%, for estimating the individual patient costs at Ryd in 2002 in the prospective model Table . The abiIn this study the ACG weights were found to be a major factor in a concurrent model, explaining patient costs during one and the same year. Age and gender explained about 11% of the individual patient costs, and when the ACG weights were added, the explanatory ability was improved and reached 38.5%. The ACGs with more complex constellations of types of morbidity were generally more resource consuming, indicating the influence of comorbidity. When estimating costs the next year in a prospective model, the ACG weights constituted the second most important factor, while the costs the preceding year were the major factor.It can be argued that costs from year one should not be used as a factor to estimate costs in year two in a prospective model, since most costs at a PHC centre are relatively stable. In this case, though, we studied not only costs on ACG group level, but also the yearly costs of individual patients. Thus it is worthwhile to include prior costs as a variable in the analysis in order to reflect the year to year variability in the individual patient's costs, when evaluating the potential of the ACGs in the prospective model.The results in this study were in accordance with findings in a Canadian study based on physician claims, where age and gender explained 9% of the variation in costs, a figure that increased to 53% when Aggregated Diagnosis Groups were added . The abiWith respect to the cost calculations, the contact-based analysis of individual patient costs can be considered sufficiently detailed to provide a reliable distribution of costs between patients. Registration of patient contacts in the EPR was a prerequisite for enabling the caregiver to record medical information, and consequently the drop-out rate was low.The original diagnoses were coded in ICD-10 and converted to ICD-9-codes. The crossover mapping was checked manually to ensure that it was as precise as possible. The fact that ACG grouping is not based on the number of encounters with a specific diagnosis, but on the mere appearance of a diagnosis , makes the ACG system less sensitive to variations in the completeness of diagnosis recording. The effects of physician preference are further reduced, since the ACG system categorises diagnoses in only 32 types of morbidity (the ADGs).In our study the ACG weighting appears to be sensitive to the accuracy with which physicians enter diagnoses into the EPRs. The coding situation in PHC in Sweden is reported to be quite accurate, but there is still a need for improved quality . Due to To explain the influencing factors in the concurrent model in more detail, it could be appropriate to analyse the case-mix of patients at Ryd a bit further. As can be seen in Table The creation of ACG weights was limited by the fact that not all patients (i.e. only those with at least one diagnosis per year) were included. However, since the costs of all patients without any diagnosis constituted no more than 2.5% of the total, this limitation probably affects our results to only a minor degree. A further limitation of the ACG weights was that the population of Ödeshög was small. This was mitigated to some extent by aggregating data from two consecutive years. Nevertheless, there were still some ACGs with only a few patients, making these weights uncertain. Constructing weights for a larger population might have resulted in greater explanatory ability. However, weights provided by the ACG software , based oWhen exploring the predictive usefulness, the individual-based approach is limited by the fact that it is based on a sub-population of individuals (i.e. only patients who are included both years), which is not fully representative for the whole population. However, our figures give an idea of the usefulness of the ACG weights in estimating individual patient costs, as well as an idea of the continuity of individual patient costs.Efforts to construct a more comprehensive database on costs per patient in PHC are ongoing, based on the same population as reported in the adjacent article (with PHC data for three years from a Swedish county) in this issue of BMC Public Health. That analysis might lead to a higher degree of certainty when using relative ACG weights. The possibility of integrating data on drug prescriptions, as is indicated in a new version of the ACG grouping software, might improve the usefulness of the system in concurrent as well as prospective models.It would have been advantageous to test the significance of a socio-economic variable, such as the Care Need Index, in explaining and estimating patient costs . HoweverIn the future, measures will be required to reduce the variation and enhance the quality of diagnostic coding in PHC. However, it is unrealistic to expect that this problem can be totally eliminated, as the criteria used for diagnostic labelling by different physicians have been shown to vary . Thus, iThe comorbidity, expressed by the individual constellation of morbidities in the ACG system, was associated with a large proportion of the variation in PHC costs. Age and gender could explain individual patient costs to a minor degree. Accordingly, the ACG system has the potential to be a useful instrument for describing and explaining concurrent resource consumption in PHC, even on the PHC centre level, as well as for estimating future costs for health care in a prospective model. As the ACG system appears to be sensitive regarding the accuracy with which physicians register diagnoses, measures will be required to reduce this variation and enhance the quality of diagnostic coding in PHC. Individual socio-economic data, as well as other health related measures, will probably be needed in future studies in order to reinforce the explanation and estimation of individual patient costs.The authors declare that they have no competing interests.SGE collected the data and carried out the first calculations of the results, and drafted the manuscript. LC handled the ACG software, rechecked and revised the calculations and drafted the manuscript. CJÖ collected the data from Ödeshög, participated in the statistical analysis and drafted the manuscript. GHN sketched the design of the study, drafted the manuscript and helped to co-ordinate the work. LAB participated in the design of the study, collected the data on ACG weights, drafted the manuscript and co-ordinated the work.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


RNA viruses have high error rates, and the resulting quasispecies may aid survival of the virus population in the presence of selective pressure. Therefore, it has been theorized that RNA viruses require high error rates for survival, and that a virus with high fidelity would be less able to cope in complex environments. We previously isolated and characterized poliovirus with a mutation in the viral polymerase, 3D-G64S, which confers resistance to mutagenic nucleotide analogs via increased fidelity. The 3D-G64S virus was less pathogenic than wild-type virus in poliovirus-receptor transgenic mice, even though only slight growth defects were observed in tissue culture. To determine whether the high-fidelity phenotype of the 3D-G64S virus could decrease its fitness under a defined selective pressure, we compared growth of the 3D-G64S virus and 3D wild-type virus in the context of a revertible attenuating point mutation, 2C-F28S. Even with a 10-fold input advantage, the 3D-G64S virus was unable to compete with 3D wild-type virus in the context of the revertible attenuating mutation; however, in the context of a non-revertible version of the 2C-F28S attenuating mutation, 3D-G64S virus matched the replication of 3D wild-type virus. Therefore, the 3D-G64S high-fidelity phenotype reduced viral fitness under a defined selective pressure, making it likely that the reduced spread in murine tissue could be caused by the increased fidelity of the viral polymerase. RNA viruses have the highest error rates in nature, resulting in the likelihood that each virus differs from other viruses in the population by one or more mutations. The consequence of this “infidelity” is that the viral population as a whole, under selective pressure from the immune system or antiviral drugs, may benefit from adaptive changes in a subset of its members. Therefore, it has been theorized that RNA viruses need high error rates to survive in complex environments. We tested this hypothesis using a drug-resistant poliovirus that contains a mutation in its polymerase that reduces errors during replication. We found that this high-fidelity mutant virus has reduced growth in mice, a complex environment where mutations may be required for growth and spread within the infected animal. At least part of this attenuation is likely due to the high fidelity of this mutant virus, since it was unable to compete with the low-fidelity version of the virus in the context of a defined selective pressure. Therefore, it is likely that mutations do benefit viral populations, especially in complex environments such as an infected animal or human. RNA viruses have the highest replicative error rates in nature, with approximately one mistake made per 1,000–100,000 nucleotides copied . As a reA promising new class of antiviral drugs, RNA virus mutagens, takes advantage of the cost of error catastrophe. Ribavirin is a nucleoside analog mutagen, which is incorporated into the viral genome during replication and increases error frequency by promiscuous base-pairing . RibavirIf a viral quasispecies is important to the survival either of individual genomes or of the population during growth under standard laboratory conditions, the higher fidelity of 3D-G64S virus should result in an attenuated phenotype. Therefore, we wondered if the 3D-G64S virus would have growth defects in tissue culture, a relatively simple environment, or in mice, a complex environment with many selective pressures. Laboratory strains of mice are not susceptible to poliovirus infection; however, mice transgenic for the human poliovirus receptor, CD155, are susceptible to injected poliovirus –11. WhenIn this study, we found that 3D-G64S poliovirus is less pathogenic than wild-type virus in mice, but displays little growth defect in tissue culture. This attenuation in mice was at least partially due to the increased fidelity of the 3D-G64S virus in the context of a defined selected pressure, since it was unable to compete with 3D wild-type virus in the context of a revertible attenuating point mutation. Therefore, we argue that, in the face of the myriad selective pressures encountered by poliovirus during murine infection, the decreased variability in the population generated by the relatively high-fidelity 3D-G64S polymerase may be a distinct disadvantage for the growth and spread of the mutant virus.6 PFU (plaque-forming units), an amount slightly over the LD50 for these animals  and the other with three nucleotide substitutions . In each case, the entire PCR-generated region was confirmed by sequencing.The 3D-G64S mutation in the viral 3D-coding region was introduced by site-specific mutagenesis, which added a sitivity , was int6 HeLa cells or 2 × 105 PVR-MEF were infected at 37 °C at an MOI of 10 PFU/cell, harvested at various times post-infection, and lysed by repeated freeze-thawing. Cell-associated virus was quantified by plaque assay on HeLa cell monolayers  ATP , and was used at a 1:5 ratio with the unlabeled 3D reverse primer in PCR reactions. The PCR reactions contained 1 unit Taq  and were done according to the supplier protocol, using a buffer that contained 50 μM potassium glutamate, 20 μM potassium HEPES (pH 8.4), 3 μM MgCl2 and 0.1 mg/ml BSA. The PCR products were digested with BstBI (New England Biolabs) at 65 °C for 3–16 h, and products were ethanol-precipitated in the presence of glycogen (Invitrogen). Pellets were resuspended in denaturing gel loading dye , heated to 80 °C for 2 min, and loaded on gels that contained 7% polyacrylamide and 8M urea. Gels were dried under vacuum on DE81 paper  and exposed to PhosphorImager screens.RNA was reverse transcribed using 5–10 μg of RNA per cDNA synthesis reaction using Superscript II reverse transcriptase (Invitrogen) and reverse primer 5′ATAGGTTCCCAGAA GCCATTCTC3′ for the 3D polymerase-coding region or 5′CCATGTCGAACGCAGAGCGCCTGG3′ for the 2C-coding region. Twenty percent of the reverse transcription reaction was PCR amplified using 5′CCGCATTTGGAAGTGGATCTACTCAGCAG3′ and 5′CCATGTCGAACGCAGAGCGCCTGG3′ primers for the 2C-coding region and primers 5′GCTTCACCTGGTGAAAGCATTGTGATCG3′ and 5′ATAGGTTCCCAGAA GCCATTCTC3′ primers for the 3D-coding region. The 3D reverse primer was end-labeled using T4 polynucleotide kinase , and 125 μCi [γ-The cDNAs products from viral RNAs purified using the QiaAmp kit (Qiagen) or from cDNA-encoding plasmids were PCR-amplified for sequence analysis at the F28S locus. The PCR reactions were performed as above, except that no radiolabel was added. Products were run on 1% agarose gels and products were excised, purified using the Qiaex II kit (Qiagen), and sequenced commercially .http://www.ncbi.nlm.nih.gov/entrez) accession number for serotype 1 poliovirus is NC002058.The Genbank (


A procedure for including activity against enveloped viruses in the post-contamination treatment of hands has been recommended, but so far no European standard is available to implement it. In 2004, the German Robert Koch-Institute (RKI) and the German Association for the Control of Virus Disease (DVV) suggested that vaccinia virus and bovine viral diarrhea virus (BVDV) should be used as test viruses in a quantitative suspension test to determine the activity of a disinfectant against all enveloped viruses.We have studied the activities of three commonly-used alcohol-based hand rubs  against vaccinia virus and BVDV, and in addition against four other clinically relevant enveloped viruses: herpes simplex virus (HSV) types 1 and 2, and human and avian influenza A virus. The hand rubs were challenged with different organic loads at exposure time of 15, 30 and 60 s. According to the guidelines of both BGA/RKI and DVV, and EN 14476:2005, the reduction of infectivity of each test virus was measured on appropriate cell lines using a quantitative suspension test.10-steps within 15 s, irrespective of the type of organic load. Similar reductions of infectivity were seen against the other four enveloped viruses within 15 s in the presence of different types of organic load.All three alcohol-based hand rubs reduced the infectivity of vaccinia virus and BVDV by ≥ 4 logCommonly used alcohol-based hand rubs with a total alcohol concentration ≥ 75% can be assumed to be active against clinically relevant enveloped viruses if they effectively reduce the infectivities of vaccinia virus and BVDV in a quantitative suspension test. Candida albicans, which may serve as the clinically relevant test strain for determining activity against yeasts. To determine virucidal activity, EN 14476:2005 should be used [During the past few years, many healthcare workers have changed from antimicrobial liquid soaps to alcohol-based hand rubs for the post-contamination treatment of hands, as suggested by the CDC guideline for hand hygiene . The two be used . However be used . Such a  be used . BVDV se be used -14.To date, however, there is no evidence to show that the two chosen test viruses cover other clinically relevant enveloped viruses in respect of stability against disinfectants. That is why we have tested the activities of three different alcohol-based hand rubs against the two suggested test viruses, and in addition against four other enveloped viruses that represent some of the most relevant or recently-emerging enveloped viruses in human medicine.The following alcohol-based hand rubs were tested: Preparation A, based on isopropanol , n-propanol  and mecetronium etilsulfate ; preparation B, based on ethanol ; and preparation C, based on ethanol . All hand rubs were manufactured by Bode Chemie GmbH & Co. KG, Hamburg, Germany.10-steps to be measured were used in all experiments. Infectivity assays were performed between 2003 and 2006 according to the test method of the Bundesgesundheitsamt  and the German Association for the Control of Virus Disease (DVV) [Virus suspensions that allowed a reduction of at least 4 logse (DVV) , which u10TCID50/ml)• Vaccinia virus strain Elstree, passaged and cultured in buffalo green monkey cells (BGM cells) (range of virus titres: 7.6–9.9 log10TCID50/ml)• Bovine viral diarrhea virus (BVDV) strain NADL, ATCC VR-534, passaged and cultured in KOP-R cells (range of virus titres: 5.5–6.4 log10TCID50/ml)• Herpes simplex virus (HSV) type 1 MacIntyre, ATCC VR-539, passaged and cultured in Vero cells  (range of virus titres: 7.5–8.1 log10TCID50/ml)• Herpes simplex virus type 2, ATCC VR-540, passaged and cultured in Vero cells (range of virus titres: 7.6–8.5 log10TCID50/ml)• Human influenza A virus, Panama strain 2007/99 (H3N2), passaged and cultured in Madin-Darby canine kidney epithelial cells (range of virus titres: 5.5–5.9 log10TCID50/ml). This virus was used as surrogate for H5N1 owing to biosafety considerations.• Avian influenza A virus/duck/Ukraine/1/63 (H3N8), passaged and cultured in Madin-Darby canine kidney epithelial cells . One part by volume of virus suspension was mixed with nine parts by volume of aqua bidest. or with one part by volume of organic load and eight parts by volume of aqua bidest.One experiment was performed for each hand rub, each exposure time, each virus and each type of organic load.Different types of organic load were used :▪ No organic load (aqua bidest.)▪10% fetal calf serum▪ 0.2% bovine serum albumin▪ 0.03% bovine serum albumin ("clean conditions" according to EN 14476:2005)▪ 0.3% bovine serum albumin with 0.3% washed sheep erythrocytes ("dirty conditions" according to EN 14476:2005).To determine the cytotoxicity of the disinfectants, two parts by volume of aqua bidest. were mixed with eight parts by volume of the disinfectant, diluted with ice-cold cell culture medium and inoculated into permissive cells. Controls for the different organic loads consisted of one part by volume of aqua bidest, one part by volume of organic load and eight parts by volume of the disinfectant. Any microscopic changes in the cells were recorded when the tests were read for cytopathic effects (CPE). This control allowed cytotoxicity and viral CPE to be clearly differentiated.2 for the appropriate incubation time (3–10 days). Cultures were observed for the presence or absence of CPE. The infective dose (TCID50) was calculated according to the method of Spearman [Infectivity was determined in a micro-procedure by end-point dilution titration. At the end of each exposure time, the test mixture was immediately diluted with ice-cold cell culture medium, and 100 μl of each dilution were placed in 8 wells of a sterile polystyrene flat-bottomed 96-well microtitre plate  with a preformed cell culture monolayer. The microtitre plates were incubated at 37°C with 5% COSpearman  and KärbSpearman . Titre rRF = a - b10 TCID50/ml of the control titrationa = log10 TCID50/ml of the test virus titration (after hand rub exposure)b = log10-steps (inactivation ≥ 99.99%) was regarded as evidence of sufficient virucidal activity against the tested virus according German and European guidelines [A reduction of infectivity of ≥ 4 logidelines .10-steps in all cell lines. Only hand rub C was toxic to MDCK cells at 1:1000 dilution.The cytotoxicity of the hand rubs was 2 log10-steps within 15 s, irrespective of the type of organic load , whereas "efficacy" is used to describe the effect measured in experiments under practical conditions ("in vivo"). Suspension tests are important for determining the spectrum of activity. Nevertheless, better recommendations can be derived from experiments under practical conditions  such as the ASTM method E-1838-02 . HoweverAll hand rubs were tested at exposure times of 15, 30 and 60 s. The 30 s exposure time best resembles the practical use of hand rubs in hygienic hand disinfection. All the hand rubs tested have a total alcohol concentration of 75% or more. It would also have been interesting to study alcohol-based hand rubs with a lower alcohol concentration to verify the data in a more challenging experimental setting. However, hand rubs with 70% alcohol or less have been shown to have only limited bactericidal activity and should therefore not be used in hospitals ,24. ThusWe also found that all the types of organic loads tested in these experiments hardly impaired the activities of the hand rubs against the different enveloped viruses. It has previously been shown using feline calicivirus that the type of organic load may significantly influence the mean reduction of viral infectivity in a test under practical conditions . That isViral infectivity was reduced to below the limit of detection in all experiments at 15 s  irrespective of the viral species, the type and amount of organic load, and the type of hand rub. It is technically almost impossible to evaluate application times shorter than 15 s in such a test because the disinfectant activity is terminated by immediate dilution. Moreover, it would have only limited clinical relevance, since a single application of an alcohol-based hand rub usually ensures a contact time of 25–30 s before the alcohol is evaporated .Activity against both test viruses in a quantitative suspension test does of course not include the inactivation of non-enveloped viruses in vitro. Entero-, rota-, adeno- and noroviruses e.g. are in general more resistant than BVDV and vaccinia virus. A virucidal activity  of a hand disinfectant is difficult to achieve requiring normally a high concentration of ethanol  or some 10-steps. It will be important to confirm our results with selected enveloped viruses including surrogate viruses applying test methods using the finger pads or the whole hand of volunteers.Overall, commonly-used alcohol-based hand rubs with 75% alcohol or more can be assumed to be active against clinically relevant enveloped viruses if they reduce the infectivities of vaccinia virus and BVDV in the presence of different standard organic loads by ≥ 4 logThe first author is paid employee of Bode Chemie GmbH & Co. KG, Hamburg, Germany, which funded the study.GK, JS and HR participated in the study design and coordination. JS carried out the experiments. GK drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Respiratory viruses are a major cause of respiratory tract infections. The prevalence of a virus-positive respiratory sample and its significance in patients requiring mechanical ventilation remain unknown.We conducted a cohort study in all consecutive adults ventilated for more than 48 hours admitted to a 22-bed medical intensive care unit during a 12-month period. Respiratory samples at the time of intubation were assessed by culture, by indirect immunofluorescence assay or by molecular methods in systematic tracheobronchial aspirates. Patients with a virus-negative respiratory sample at the time of intubation were considered unexposed and served as the control group.P = 0.12), with chronic obstructive pulmonary disease/asthma patients  and with admission between 21 November and 21 March  were independently associated with a virus-positive sample. Among the 122 patients admitted with respiratory disorder, a tracheobronchial aspirate positive for respiratory viruses at the time of intubation  was independently associated with better survival, controlling for the Simplified Acute Physiology Score II and admission for cardiogenic shock or cardiac arrest. Among the remaining 65 patients, a virus-positive sample on intubation did not predict survival.Forty-five viruses were isolated in 41/187 (22%) patients. Rhinovirus was the most commonly isolated virus (42%), followed byherpes simplex virus type 1 (22%) and virus influenza A (16%). In multivariate analysis controlling for the Acute Pathophysiology and Chronic Health Evaluation II score, patients with respiratory disorder at admission (adjusted odds ratio, 2.1; 95% confidence interval, 0.8–5.1; We confirmed the pathogenic role of respiratory viruses in the intensive care unit, particularly rhinovirus. We suggest, however, that the prognostic value of virus-associated respiratory disorder is better than that of other causes of respiratory disorder. Respiratory viruses represent an important role in the etiology of community-acquired pneumonia in adults -3. RespiIn a previous work we investigated the incidence of nosocomial viral ventilator-associated pneumonia . The aimAll consecutively intubated adults admitted to the intensive care unit (ICU) in the University Hospital of Caen between September 2003 and September 2004 were screened, as previously reported .Patient characteristics recorded at the time of intubation included age, sex, main reason for ICU admission, scoring of disease severity within the first day in the ICU – assessed by the admission Simplified Acute Physiology Score type II , the AcuThe main reasons for ICU admission  included cardiac arrest, septic shock, cardiac shock, mixed shock, hemorrhagic shock, respiratory distress alone (without other associated organ failure), acute renal failure, coma, intoxication, surgery and other. In addition, clinical outcomes assessed by the occurrence of ventilator-associated pneumonia and death were recorded.According to French legislation at the time of the study and given the observational nature of our study, no ethical committee approval was requested and thus no informed consent was obtained from the patients.Details of the virologic methods for virus detection are published elsewhere . BrieflyChlamydia pneumoniae and Mycoplasma pneumoniae were also detected by PCR assay.The following viruses were tested: parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3 and parainfluenza virus 4, influenza virus A, influenza virus B and influenza virus C, respiratory syncytial virus, metapneumovirus, rhinovirus, coronavirus 229E and coronavirus OC43, adenovirus, cytomegalovirus and herpes simplex virus. Respiratory specimens were processed for PCR or RT-PCR at the end of the study period. One positive sample and several negative control samples were included for each infectious agent, which were treated identically to the virus samples throughout. Results of conventional methods for viral isolation, routinely performed (in case of respiratory disorder), were transmitted weekly to the clinicians. Antiviral drugs could be used during the study period for proven herpes simplex virus or cytomegalovirus infection in immunocompromised patients.Pneumonia was defined as any acute septic episode with respiratory symptoms  and a radiographic infiltrate that was neither preexisting or of other known cause . Pneumont test or the Mann-Whitney nonparametric test, when appropriate. The confidence intervals of percentages were based on normal approximation.Quantitative data and qualitative data were expressed as the mean ± standard deviation or as the median (range) and percentage , respectively. Categorical variables were compared using the chi-square test or Fischer's exact test, when appropriate. Quantitative variables were compared using the Student We modeled the probability of a positive virus respiratory sample using a logistic regression model. Because we hypothesised that the pathogenic role of respiratory viruses in the respiratory tract may differ when associated with respiratory symptoms or not, we examined outcome according to the presence (group 1) or the absence (group 2) of respiratory disorder at admission. To assess the impact of the virus respiratory sample on time to death and time to ventilated acquired pneumonia, we constructed Kaplan-Meier curves and Cox models.P < 0.1 in the univariate analysis was chosen for multivariate modeling in both the logistic and the proportional hazards models. Multivariable modeling is a tradeoff between model complexity and parcimony. Because of our relatively small sample size, we selected a level of alpha risk <0.25 to remain in the multivariable model [P < 0.05, and all tests were two-sided.A stepwise selection of variables associated with outcome at le model  to avoidWe used EPI-INFO version 6.04dfr software  for data collection, and used EPI-INFO and SAS version 9.1 software  for data analysis.Among 653 patients admitted to our ICU during the study period, a tracheobronchial aspirate was taken for viral studies in 187 patients, as shown in Figure Forty-five viruses were isolated from the respiratory specimens of 41 patients Table . RhinoviP = 0.0012), admission between 21 November and 21 March (P = 0.003) and admission with respiratory disorder (P = 0.03) were significantly more prevalent in patients with a virus-positive sample. In addition, patients with a virus-positive sample had a nonsignificant lower Acute Physiology and Chronic Health Evaluation II score compared with patients with a virus-negative sample . In multivariate analysis controlling for Acute Physiology and Chronic Health Evaluation II score and respiratory disorder, the COPD/asthma patients and admission between 21 November and 21 March remained significantly associated with a virus-positive sample, as shown in Table In univariate analysis Table , COPD/as P = 0.00 and admiP < 0.006) but not in group 2 (P = 0.94), where only a higher Simplified Acute Physiology Score II (P < 0.002) and admission for cardiac arrest or cardiogenic shock (P = 0.07) predicted time to death, as shown in Table The Kaplan-Meier curves of survival according to the presence of respiratory viruses in group 1 and in group 2 are shown in Figure 2 P = 0.9, where oThe present study reports that respiratory viruses, as systematically screened with sensitive methods at the time of intubation, are common (22%) among adults ventilated for more than 48 hours, regardless of the reason for admission to the ICU. Rhinovirus was the most commonly isolated virus. We have identified, for the first time in this setting, three risks factors associated with a virus-positive sample – namely, admission with respiratory disorder, COPD/asthma and admission during the winter endemic viral season. These factors highlight that the diagnosis of respiratory viral infection should be focused for patients with a respiratory disease, and support the hypothesis of the clinical impact and pathogenic role of viral infection. In addition, we suggest that the ICU mortality might be lower in viral-associated respiratory disorder than in nonviral-associated respiratory disorder. A virus-positive sample had no impact on the time to ventilator-associated pneumonia, as previously reported in a smaller sample of this cohort .Our finding differs from previous studies assessing the microbiologic pattern of severe pneumonia ,21,22 orThe molecular method used in this study for viral detection is recognised as the most sensitive technique ,28. NoneAccording to previous studies focusing on patients at high risk for viral disease ,25,28,33In the present study, the proportion of rhinovirus (42%) was higher than reported in ICU patients ,9. WhileIn contrast, influenza virus is recognised to play a major pathogenic role during flu outbreaks in the winter-spring season. A causal relationship between influenza virus infection and hospitalisation for respiratory or cardiac failure has been shown in vaccine effectiveness studies ,38.Staphylococcus aureus to internalise into pneumocytes with a mechanism that involves the virus-induced release of IL-6 and IL-8 and the overexpression of ICAM-1 [We failed to demonstrate that patient exposure to respiratory viruses significantly increased the risk of ventilator-associated pneumonia. It is commonly reported that respiratory viruses could facilitate bacterial infection of the airways, by damaging the respiratory epithelium . Some exf ICAM-1 . Finallyf ICAM-1 ,44 or ref ICAM-1 .In the subgroup of patients with respiratory disorder, those with virus-positive samples surprisingly had a better survival. This result should be interpreted cautiously because it relies on the control group . This finding does not question the severity of virus-associated respiratory disorder, but simply suggests that the prognostic may well differ from other causes of respiratory disorder. It has been reported that the clinical severity and inflammatory responses in COPD exacerbations could be modulated by the nature of the infecting organism ,25.We are aware of limitations. The monocenter design of the study, the relatively small number of included patients, patients' underlying disease heterogeneity as well as the fact that 18 patients (8.7%) were eligible but not screened may limit the interpretation and relevance of our data. Because the systematic search for bacteria was not obtained at the time of intubation, 'virus-associated respiratory disorder' does not necessarily mean virus-induced respiratory disorder. In addition, the PCR might be too sensitive and we cannot exclude an asymptomatic carriage of respiratory viruses in the airways in some cases. In the future, a quantifying viral load might be another approach to improve the diagnostic accuracy.The results reported here may have important implications for the design and power analysis of a randomised controlled trial using antiviral drugs. With a 12% mortality rate in the control group , the room for improvement in patients with viral pneumonia would be lower than that for respiratory disorder overall (34%). An appropriate sample size would consequently be necessary to demonstrate the clinical impact, if any, of antiviral drugs.We have reported and described the prevalence of virus-positive respiratory samples taken at the time of intubation in ventilated adults, contributing to improving epidemiological knowledge in the critical care setting. Using the most sensitive methods for viral detection, we were able to identify that 22% of our patients had viruses in their airways. The detection of respiratory viruses in the respiratory tract, however, was not always associated with respiratory symptoms, as demonstrated by the 12% asymptomatic carriage in group 2 Figure , group 2• Respiratory viruses screened at the time of intubation are common among adults ventilated for more than 48 hours, especially in patients admitted for respiratory disorder.• Rhinovirus is the most commonly isolated virus.• We suggest that virus-associated respiratory disorder may be associated with a lower clinical severity and better prognosis, as compared with other causes of respiratory disorder.• Further studies are necessary to confirm the importance of viral infections as a cause of acute respiratory failure in patients admitted to the ICU, and to address the role of antiviral therapy in this patient population.COPD = chronic obstructive pulmonary disease; ICU = intensive care unit; IL = interleukin; PCR = polymerase chain reaction; RT = reverse transcriptase.The authors declare that they have no competing interests.CD and SV wrote the experimental protocol and initiated the study. AV and FF performed the virologic assessments. J-JP and CD computed the statistical analysis and were involved in the interpretation of the results. CD drafted the manuscript, which was critically revised by J-JP, SV, FF, AV, DdC, MR and PC. All authors read and approved the final manuscript.


Objective: To continue evaluation of the use of acyclovir suppression in late pregnancy after first episode genitalherpes simplex virus (HSV) infection, using an open-label study design.Methods: Ninety-six women diagnosed with genital herpes for the first time in the index pregnancy were prescribedsuppressive acyclovir 400 mg orally three times daily from 36 weeks until delivery in an open-label fashion.Herpes cultures were obtained when patients presented for delivery. Vaginal delivery was permitted if no clinicalrecurrence was present; otherwise a Cesarean delivery was performed. NeonatalHSV cultures were obtained andinfants were followed clinically. Rates of clinical and asymptomatic genital herpes recurrences and Cesareandelivery for genital herpes were measured, and 95% confidence intervals were calculated.Results: In 82 patients (85%) compliant with therapy, only 1% had clinical HSV recurrences at delivery. In an intentto treat analysis of the entire cohort, 4% had clinical recurrences .Asymptomatic shedding occurred in 1% of women without lesions at delivery. Two of the four clinical recurrenceswere HSV-culture positive. No significant maternal or fetal side-effects were observed.Conclusions: In clinical practice the majority of patients are compliant with acyclovir suppression at term. Thetherapy appears to be effective at reducing clinical recurrences after a first episode of genital herpes complicating apregnancy.


The virus causes infections in humans, pigs, and several other domestic animals. It has also been isolated from fruit bats. The pathogenesis of Nipah virus infection is still not well described. In the present study, Nipah virus replication kinetics were estimated from infection of African green monkey kidney cells (Vero) using the one-step SYBRThe qRT-PCR had a dynamic range of at least seven orders of magnitude and can detect Nipah virus from as low as one PFU/μL. Following initiation of infection, it was estimated that Nipah virus RNA doubles at every ~40 minutes and attained peak intracellular virus RNA level of ~8.4 log PFU/μL at about 32 hours post-infection (PI). Significant extracellular Nipah virus RNA release occurred only after 8 hours PI and the level peaked at ~7.9 log PFU/μL at 64 hours PI. The estimated rate of Nipah virus RNA released into the cell culture medium was ~0.07 log PFU/μL per hour and less than 10% of the released Nipah virus RNA was infectious.® Green I-based qRT-PCR assay enabled quantitative assessment of Nipah virus RNA synthesis in Vero cells. A low rate of Nipah virus extracellular RNA release and low infectious virus yield together with extensive syncytial formation during the infection support a cell-to-cell spread mechanism for Nipah virus infection.The SYBR Henipavirus of the Paramyxoviridae family [® Green I dye-based quantitative real-time RT-PCR (qRT-PCR) amplification assay was established and the assay was used to examine the kinetics of Nipah virus replication in cultured African green monkey kidney (Vero) cells.Nipah virus, an enveloped, non-segmented, negative-stranded RNA virus is a recently discovered zoonotic virus belonging to the genus e family ,2. The ve family -5. No Nie family  and in Be family -10. In te family ,9 and Nie family . The orie family ,13 and se family . These se family . During e family ,19 and te family  and culle family . There we family ,14,18 ane family ,22. The e family . Applicae family  and fluoe family  for the Nipah virus infected Vero cells showed significant cellular morphological changes beginning at eight hours post-infection (PI). Cell fusion and syncytial formation were noted and the frequency of these giant multinucleated cells increased as the infection progressed Figure . At 48 h7 PFU/mL. Nipah virus RNA was detected from as low as one PFU/μL using the similarly diluted RNA in the qRT-PCR assay  value of 76°C and DNA sequencing of the fragment verified its identity (data not shown). The standard curve plot determined using the tenfold serial dilutions of the Nipah virus RNA had a coefficient of correlation of r2 = 0.996  were in the range of 1.26 to 3.55%. These suggested a very high reproducibility of the SYBR® Green I-based qRT-PCR amplification assay. Using the commercially available Nipah virus Armored RNA® , it was determined that the SYBR® Green I-based qRT-PCR had a sensitivity of five to 5 × 105 RNA copies/μL in RNA copy number. A linear correlation between the RNA extracted from Nipah virus inoculum (with a virus titer of ~1.0 × 107 PFU/mL) and the Nipah virus Armored RNA® (RNA copy number) was established  was expected, as the primers could not differentiate between infectious and noninfectious RNA-containing virus particles such as the defective interfering particle. No amplification, however, was obtained when the genomic RNA of human parainfluenza virus type-3, dengue virus type-2, human enterovirus 71 and Japanese encephalitis virus were used in the SYBR® Green I-based qRT-PCR amplification. On the other hand, fluorescence signals indicating amplification was obtained when Hendra virus genomic RNA template was used. A DNA fragment with a Tm value of 80.8°C was consistently obtained using the NIP-NF3 and NIP-NR1 primer pairs .The NIP-NF3 and NIP-NR1 primer set designed for the study amplified the Nipah virus nucleocapsid (N) gene sequence to give a fragment of ~178 bp. The detection limit of this one-tube RT-PCR system was at ~100 PFU/μL and has a dynamic range of five logs Figure . This way Figure . The ampL Figure . Beyond 6 Figure . In addid Figure . Using ts Figure . The Tm ® Green I-based qRT-PCR amplification. The standard plots for this assay was established using RNA extracted from Nipah virus inoculum with an estimated titer of ~1.0 × 107 PFU/mL. The amount of viral RNA from the amplification assay was expressed as equivalent log PFU/μL as we were interested only in the proportion of virus RNA that corresponded to the estimated number of infectious virus particles. A significant increase in the intracellular Nipah virus RNA level was noted beginning at eight hours PI  and the calculated infectious virus particles in PFU, it was determined that on average, at least 10% (± 0.1%) of the released virus in the cell culture supernatant was infectious.The kinetics of Nipah virus RNA synthesis in infected Vero cells was examined quantitatively by determining the amount of intra- and extracellular Nipah virus RNA using the SYBRI Figure . The increported ,27. The ® Green I-based qRT-PCR amplification assay. The method was sensitive, highly reproducible and specific enough to differentiate against most other RNA viruses except Hendra virus, a closely related zoonotic virus. The characteristic shift in the Tm value that differentiates Nipah virus from Hendra virus was obtained perhaps due to the differences in the G+C content of the amplified DNA fragment. The identity of the amplified fragment was confirmed by DNA sequencing. The ability to amplify and differentiate Hendra virus from Nipah virus using the melt curve analysis is potentially useful in the surveillance of Nipah virus infection in animals, particularly in wild animals as both viruses are known to have a common reservoir host, fruit bats [Findings from the present study reaffirmed earlier reports that Nipah virus replicated productively in Vero cells. In this study, however, the kinetics of the virus replication was estimated using the SYBRuit bats -16,28. Fuit bats  had onlyuit bats . In thatuit bats .® Green I-based qRT-PCR suggest that there was a latent phase of at least eight hours following initiation of infection, a period during which no significant increase in Nipah virus RNA synthesis could be detected in the infected Vero cells. A rapid rise in the intracellular virus RNA level occurred within the next eight hours PI and this exponential rise is comparable to that of other virulent paramyxoviruses [Results from the quantification of the intra- and extracellular Nipah virus RNA synthesis using the SYBRoviruses ,31. The oviruses . There woviruses -34 and soviruses , results® Green I-based qRT-PCR assay. A short viral RNA doubling time was observed but the rate of extracellular virus RNA and infectious virus release from the infected cells were low. These suggest that Nipah virus replicates well in susceptible cells but the infection is insidious as the virus is spread slowly through the cell-to-cell spread mechanism.Nipah virus replication kinetics in Vero cells was established using the SYBR2 and infected with the pig Nipah virus strain NV/MY/99/VRI-2794. The infected cells were examined for cytopathic effects (CPE). Following manifestation of ~90% CPE, the supernatant was harvested and sedimented at 1000 × g to remove all residual cells. The supernatant was then stored at -80°C. The supernatant was later titrated for virus infectivity using virus plaque assay. Briefly, a tenfold serial dilution of the virus stock was prepared and 250 μL of each virus dilution was added in duplicates into 24-well plate containing 1 × 106 cells/well. The virus-cells mixture was incubated at 37°C for one hour. Following that, cells were washed twice with EMEM. Then, 500 μL of 0.8% agarose in EMEM supplemented with 2% FCS was overlaid on top of the cell monolayer and the plate was incubated at 37°C. On day two PI, the virus-cells mixture were fixed with 4% paraformaldehye and stained with naphthalene black. Virus infectivity titer was estimated by determining the virus dilution and the number of plaques formed.Vero cells used for Nipah virus isolation were cultured in Eagle's minimum essential medium  supplemented with 2% fetal calf serum . Cells were incubated at 37°C in 5% CO® LS  according to the manufacturer's protocol. The RNA pellet was dissolved in nuclease-free water and a tenfold serial dilutions of the RNA was made to reflect the calculated PFU of 0.1 to 1 × 106 for establishing the qRT-PCR assay standard plot. In addition, Nipah virus Armored RNA®  containing ~5 × 105 Nipah virus RNA copies per μL (Lot #10233) was used for the estimation of Nipah virus RNA copy number. The RNA was prepared according to the manufacturer's instructions to generate 0.5 to 5 × 105 copies of the Nipah virus RNA.Nipah virus RNA was extracted from Nipah virus inoculum following determination of the virus infectivity titer. RNA was extracted using the TRI Reagent4, 0.6 pmol/μL of each primer, and 1 μL template RNA. Amplification was performed in a 25 μL reaction mix using the following program: 42°C of cDNA synthesis for 1 hour, 95°C for 15 min, 30 cycles of 1 min at 95°C, 1 min at 55°C, and 1 min at 72°C and followed by final extension of 72°C for 5 min.Initial amplification of Nipah virus sequences was accomplished using the conventional RT-PCR performed in a PTC-200 thermal cycler . Primer pairs NIP-NF3 (5' GGC TAG AGA GGC AAA ATT TGC TGC 3') and NIP-NR1 (5' ACC GGA TGT GCT CAC AGA ACT G 3'), designed from the conserved region within the N gene were used. The reaction mixture consisted of 1× Access RT-PCR buffer , 0.5 μL MMV, 0.5 μL AMV, 1 mM of dNTPs, 1.5 mM of MgSO® Green I-based qRT-PCR was performed using the same set of oligonucleotide primers as above, in a 20 μL mix at 50°C for 30 min, 95°C for 15 min, and 45 cycles of 15 s at 95°C and 1 min at 60°C. The reaction mixture consisted of 1× QuantiTect SYBR® Green RT-PCR Master Mix , 0.5 μL QuantiTect RT Mix, 0.6 pmol/μL of each primer, and 1 μL template RNA. The amplification was performed in DNA Engine Opticon® System . Fluorescent measurements were recorded after each cycling step and at the end of the amplification cycle data were analyzed using the OpticonMONITOR™ 2 analysis tool. A threshold cycle (Ct) value for every sample was determined and compared to that of the standard. The standard plot was established in parallel for each experiment using known amount of Nipah virus Armored RNA®. Standard curves for the RT-PCR was accepted when the coefficients of correlation, r2 were > 0.90. All amplification standards, controls, and samples were performed in duplicates and repeated at least twice. In addition, melting curve analysis was performed routinely at the end of each amplification assays to verify the amplicon by its specific Tm. The melting curve analysis consisted of 35 cycles of incubation during which the temperature was increased from 60°C to 95°C at a rate of 0.2°C/30 s/cycle with continuous reading of fluorescence.The SYBR5 cells/well) cultured in 24-well plate were infected with Nipah virus to give an estimated MOI of 0.2 per cell. Cells were incubated with the virus for one hour at 37°C, following which the virus suspension was removed and the cells were rinsed twice with EMEM. Subsequently, EMEM supplemented with 2% FCS was added and the cells were incubated at 37°C. At selected intervals PI  the cell culture supernatant consisting of the extracellular virus was removed, centrifuged at 1000 × g and RNA was extracted using TRI Reagent® LS . The remaining cell monolayer was rinsed twice with serum free EMEM medium and total intracellular RNA was extracted using the TRI Reagent® . The efficiency of RNA extraction was consistent between all extractions at ~72.3% (± 1.5%). All the extracted RNA was stored at -70°C until needed.Adherent Vero cells (2.5 × 10The author(s) declare that they have no competing interests.The corresponding author, Sazaly AbuBakar is the principal investigator of the study, was involved in the design, supervision, data analyses and writing of the report. Li-Yen Chang performed all the laboratory experiments, analyses of data and writing of the report. A.R. Mohd Ali contributed in the virological investigations. Sharifah Syed Hassan was involved in the virological investigations and supervision for the usage of the BSL 3 facility.


In Thailand, rabies remains a neglected disease with authorities continuing to rely on human death statistics while ignoring the financial burden resulting from an enormous increase in post-exposure prophylaxis. Past attempts to conduct a mass dog vaccination and sterilization program have been limited to Bangkok city and have not been successful. We have used molecular epidemiology to define geographic localization of rabies virus phylogroups and their pattern of spread in Thailand.We analyzed 239 nucleoprotein gene sequences from animal and human brain samples collected from all over Thailand between 1998 and 2002. We then reconstructed a phylogenetic tree correlating these data with geographical information.All sequences formed a monophyletic tree of 2 distinct phylogroups, TH1 and TH2. Three subgroups were identified in the TH1 subgroup and were distributed in the middle region of the country. Eight subgroups of TH2 viruses were identified widely distributed throughout the country overlapping the TH1 territory. There was a correlation between human-dependent transportation routes and the distribution of virus.Inter-regional migration paths of the viruses might be correlated with translocation of dogs associated with humans. Interconnecting factors between human socioeconomic and population density might determine the transmission dynamics of virus in a rural-to-urban polarity. The presence of 2 or more rabies virus groups in a location might be indicative of a gene flow, reflecting a translocation of dogs within such region and adjacent areas. Different approaches may be required for rabies control based on the homo- or heterogeneity of the virus. Areas containing homogeneous virus populations should be targeted first. Control of dog movement associated with humans is essential. Rabies is not high on the list of the World Heath Organization's list of important infectious diseases, and is also often overlooked by regional, national, and local public-health professionals. The dog is the primary reservoir and vector of rabies transmission in Thailand and developing countries .To date, the evaluation of the importance of rabies has been determined solely by estimating the number of human deaths and statistics on dog rabies infectivity, which may not be a reliable indicator in developing countries . For exaIn Thailand, the substantial decline in human rabies deaths from almost 200 a decade ago to less than 20 in 2003, has occurred due to the huge and continuously escalating financial obligation in the annual budget required to supply rabies biologicals for human PEP. More than 400,000 patients received PEP in 2003, as compared to approximately 90,000 in 1991 [Ministry of Public Health (MOPH) annual report]. Moreover, annual human rabies deaths in Bangkok, where diagnostic facilities and neurologists are readily available, rose from less than 5 in 1990–1994 to 5–10 in 1995–2001 .There are no reliable statistical analyses of dog populations that could be evaluated to determine the effectiveness of the current human rabies prevention methodologies used in Thailand. One quoted figure of 6 million dogs in Thailand is undoubtedly an underestimate of the actual population present within the country. The Division of Disease Control and Ministry of Agriculture reported that between 60 to 78% of the dog population was vaccinated  in Thailand between 1995 and 2000. Experience in Latin America has shown that vaccination of a critical percentage of dogs, on the order of 40–70%, at least in major urban areas, was sufficient to interrupt canine rabies transmission and resulted in diminished human rabies deaths . HoweverA survey in 1999 by the Department of Livestock and the Bangkok Metropolitan Administration revealed that stray dog populations in Bangkok  have tripled in size, . Additionally, a 2002 survey suggested that dog populations were increasing, both in Bangkok and the countrywide, implying that the specific carrying capacity of canine habitats has not yet been saturated. Moreover, a substantial number of dogs, especially stray and community dogs, are not vaccinated.Due to budget limitation, an intensive dog vaccination and sterilization program has been in place only in Bangkok City since June 2002. Seventy-two million baht  were spent during the first 2 phases of the program (June 2002-September 2003), with the third phase (October 2003-September 2004) costing an additional 31 million baht . Although there were no human rabies deaths in Bangkok in 2002, 3 deaths were reported in 2003. Preliminary assessment revealed that less than 20 percent of the estimated dog population was sterilized and vaccinated.Without reliable data on dog ecology and surveillance of rabies infection in dogs and humans, it is not possible to develop a strategic plan for rabies prevention and control and to assess program success. Therefore, our objective was to use molecular biological techniques to characterize the presence and movement of rabies virus according to geographical locations in Thailand and use this information as baseline data to design and implement rabies prevention programs in the country. Areas with evidence of continuous gene flow, and presence of viruses of more than one genetic group or subclade, were characterized. The potential translocation of rabies virus from one area to another was evaluated in relation to natural barriers, transportation routes, human activity and socioeconomic factors.Two hundred and thirty nine brain samples  from 56 provinces were obtained from 25 diagnostic laboratories all over Thailand between 1998 and 2002. Samples selected for analysis were chosen to be representative of the geographical location in each province down to the scale of small districts (subdivisions of a province). Samples were not available from 20 provinces. All samples were prescreened for evidence of rabies virus using the direct fluorescence antibody test and kept frozen at -80 degree C until genetic analysis was conducted.Genetic typing was based on nucleotide sequence differences in cDNA obtained by direct one step RT-PCR amplification of the nucleocapsid (N) gene fragment from the samples. The amplified products of 414 bp (nt 1101 – 1506) were characterized by sequencing. RT-PCR and sequencing procedures were conducted as previously described [Twelve additional N sequences were retrieved from Genbank database to be outgroups for this study: a non-rabies lyssavirus Mokola Virus S59448), 3 Australian Bat Lyssavirus (ABLV) isolates , a rabies strain Pasteur Virus (PV) (M13215), 6 rabies viruses from other Asian countries  and 1 rabies isolate from Thailand (U22653). The sequences of all isolates were aligned together using program ClustalX [9448, 3 APhylogenetic analyses of the 239 N rabies sequences collected from all over Thailand clearly demonstrated that all of the isolates formed a monophyletic group with 100% boostrap supporting values, separate from Mokola Virus, Australian Bat Lyssavirus (ABLV), Pasteur Virus (PV) and other Asian rabies viruses  were very similar to those obtained from rabid dogs. No specific grouping of sequences from rabies virus isolated from non-canine species was identified. Instead, these rabies virus sequences were scattered across the tree. This finding was in accord to our expectation that the dog is a prime reservoir and transmitting vector for rabies and causes spillover to human and domestic animals and wildlife. Nevertheless, we are also aware that there may be other vectors, such as bats, and other lyssaviruses, besides genotype 1, circulating in Thailand. In fact, our recent survey in Thai bats indicated that as many as 7.5% of the bat population had evidence of lyssavirus infection by an as yet unidentified genotype(s) .The 2 major groups found in our Thai rabies phylogeny were judged to be significant with high bootstrap supporting values. Notably, these 2 major lineages resembled the putative groups A and B found in our previous study  in whichAlthough there are some overlapping areas shared between the TH1 and TH2 phylogroups, the viral transmission dynamics and evolutionary background the sub-lineages may not be similar which could explain why both have different success levels in disease dispersals. It has been proposed that the degree of differences in compartmentalization mechanisms may influence the duration that each individual canine-associated rabies variant resides in certain geographical regions . RelatioTo estimate the epidemiological characteristics of the TH1 and TH2 Thai rabies groups, a phylogeographical concept was introduced to infer their transmission dynamics ,20. CompSecondly, we suggest that transmission of rabies virus may be related to human activity, particularly human migration. Considering the phylogeographic areas of the 3 genetic subgroups of TH1 rabies virus Fig. , geneticThe theory that the spread of canine rabies virus was instigated by pet-owner translocation via transportation routes was supported in this study by the results of the distribution pattern of each subgroup of TH2 Figs. , 4, 5. MThe most convincing support for the human-facilitated rabies distribution hypothesis we propose herein is the geographical distribution of the TH2B subgroup in which all, except a few samples from the northeast, were from the southern region of the country. This phylogeographic subgroup with a 1300–1600 km spreading range, had a very strong bootstrap supporting-value (89%) on the genetic tree. We propose that this inter-regional migration path of the TH2 subgroup is explained by a rural-to-urban viral transmission polarity. ,16 The mSelection of suitable areas using molecular epidemiological techniques should be considered as a powerful tool when planning disease control strategies. For decades, Thailand has invested vast sums of money and manpower on the effort to control and vaccinate the dog population in randomly selected districts and, recently, Bangkok capital city without success. Results of this research demonstrated that Bangkok and other metropolitan cities  contain various groups and subgroups of viruses, actively circulating to and from other surrounding provinces Fig. . TherefoOn a national scale, we propose that rabies control can be successful if it is initiated in southern Thailand. This region contains only the TH2B rabies subgroup. Furthermore, it is an "island-like" area surrounding by Andaman Sea, Gulf of Thailand, and the Malaysian border. Influx from the TH1C subgroup has been restricted to an area around Ranong province, plausibly from high mountain ridges. Moreover, the majority of the population in southern Thailand are Muslims who do not keep pet dogs or feed stray dogs. Implementation of a rabies control in this region should therefore be effective in terms of a dog population reduction and vaccination campaign, and the enforcement of strict regulations regarding dog transfer.In order to test this concept of "targeting a genetically defined area", a mass rabies control compaign should be conducted in a suitable-size province with a homogeneous virus population. The province of Kanchanaburi, 19,483 km sq and about 130 km westwards from Bangkok, appears to be a good choice since, according to our study, it contains only the TH2D rabies subgroups Fig.  which arIn conclusion, we have presented a novel approach to the development of a rabies control and prevention program through the utilization of genetic epidemiology. We believe that the implementation of such a disease control program utilizing existing information on the genetics of circulating rabies viruses in a country like Thailand could be successful if the campaign target areas have been carefully selected and limited to one circulating phylogroup of virus and the movement of dogs along human transportation routes into the area is strictly enforced.The authors declare that they have no financial or personal relationships with other people or organizations that could inappropriately influence this research. All authors have access to all data in the study and held final responsibility for the decision to submit for publication.JD participated in data analysis and interpretation, phylogenetic tree construction, and writing the paper. SW participated in data collection and analysis, PCR primer design, sequencing rabies genes, and writing the paper. BL participated in specimen collection, data analysis, and writing the paper. NR participated in data analysis, figure preparation, and writing the paper. WH participated in coordinating the study with the veterinarians throughout the country, data analysis, and writing the paper. AP participated in coordinating the study with the physicians throughout the country, data analysis, and writing the paper. TH participated in study design, data analysis and interpretation, and writing the paper. The first 3 authors  contributed equally to this work. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Table 1 Taxon list of N-gene sequences of Thai rabies virus used in this studyClick here for file


Arenaviruses such as Lassa fever virus cause significant mortality in endemic areas and represent potential bioterrorist weapons. The occurrence of arenaviral hemorrhagic fevers is largely confined to Third World countries with a limited medical infrastructure, and therefore live-attenuated vaccines have long been sought as a method of choice for prevention. Yet their rational design and engineering have been thwarted by technical limitations. In addition, viral genes had not been identified that are needed to cause disease but can be deleted or substituted to generate live-attenuated vaccine strains. Lymphocytic choriomeningitis virus, the prototype arenavirus, induces cell-mediated immunity against Lassa fever virus, but its safety for humans is unclear and untested. Using this virus model, we have developed the necessary methodology to efficiently modify arenavirus genomes and have exploited these techniques to identify an arenaviral Achilles' heel suitable for targeting in vaccine design. Reverse genetic exchange of the viral glycoprotein for foreign glycoproteins created attenuated vaccine strains that remained viable although unable to cause disease in infected mice. This phenotype remained stable even after extensive propagation in immunodeficient hosts. Nevertheless, the engineered viruses induced T cell–mediated immunity protecting against overwhelming systemic infection and severe liver disease upon wild-type virus challenge. Protection was established within 3 to 7 d after immunization and lasted for approximately 300 d. The identification of an arenaviral Achilles' heel demonstrates that the reverse genetic engineering of live-attenuated arenavirus vaccines is feasible. Moreover, our findings offer lymphocytic choriomeningitis virus or other arenaviruses expressing foreign glycoproteins as promising live-attenuated arenavirus vaccine candidates. Arenaviruses such as Lassa fever virus (LFV) account for substantial mortality in endemic Third World countries and represent potential bioterrorist weapons. Live-attenuated vaccine strains would likely represent an optimal strategy for prevention, but their rational design and engineering have been thwarted by technical limitations. As an additional difficulty, arenaviruses have only four genes, all of which are needed for the infectious cycle. Unlike for other virus families, attempts at deleting a “virulence gene” would therefore interfere with the virus' viability and thereby with its immunogenicity. Using the prototype arenavirus as a model, the authors have developed the necessary tools to investigate an alternative strategy for tailoring of live-attenuated strains: Recombinants were engineered to express a foreign envelope gene instead of the natural one. Thereby, the virus was crippled but still viable. When testing such “envelope-exchange” viruses in a mouse model, they failed to cause disease. Nevertheless, they elicited rapid and long-lived immunity against overwhelming infection and lethal disease upon wild-type virus challenge. This delineates a novel general strategy for the reverse genetic engineering of live-attenuated arenavirus vaccines to be used in endemic areas or in case of a bioterrorist attack. Lymphocytic choriomeningitis virus (LCMV) represents the prototype member of the arenavirus family. The genome of these enveloped negative-strand RNA viruses  consistsMastomys, this indicated that the virus cannot efficiently sustain itself by human-to-human transmission . Numbers in the upper right quadrants indicate the percentage of epitope-specific CD8+ T cells within the total CD8+ T cell population .(A) C57BL/6 mice were infected i.v. with 10(B and C) GP33-specific (B) and NP396-specific (C) cytolytic activity of the same splenocytes as tested in (A). Symbols represent the mean ± SD of three mice per group (except for the single naïve control). (B and C) One representative of two independent experiments.(408 KB EPS)Click here for additional data file.Figure S83 PFU rLCMV/INDG intracerebrally, either in adulthood (“adult”) or within 24 h after birth . Two weeks later, all animals were killed and brain RNA was processed for RT-PCR analysis. An NP-specific amplification product was obtained using the primers NP2223f and NP2743r, as described in C57BL/6 mice were inoculated with 3 × 10(400 KB EPS)Click here for additional data file.Protocol S1Explanatory text to (80 KB DOC)Click here for additional data file.Table S1(36 KB DOC)Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank) accession numbers for the complete sequences of the cDNA-derived recombinant S segments in rLCMV/INDG, rLCMV/NJG, and rLCMV-ARM* are DQ408670, DQ408671, and DQ458914, respectively.The GenBank (