Phylogenetic analysis of three genes of Penguinpox virus, a novel Avipoxvirus isolated from African penguins, reveals its relationship to other poxviruses. The genes corresponding to Vaccinia virus G8R (VLTF-1), A3L (P4b) and H3L were sequenced and phylogenetic trees (Neighbour-Joining and UPGMA) constructed from MUSCLE nucleotide and amino acid alignments of the equivalent sequences from several different poxviruses. Based on this analysis, PEPV was confirmed to belong to the genus Avipoxvirus, specifically, clade A, subclade A2 and to be most closely related to Turkeypox virus (TKPV), Ostrichpox virus (OSPV)and Pigeonpox virus (PGPV). Fowlpox virus (FWPV) and Canarypox virus (CNPV) has increased due to their successful use as vaccines on commercial flocks and their extensive use and testing as vaccine vectors [Leporipoxvirus genus [Chordopoxvirus genera suggesting that the species within the Avipoxvirus genus are highly divergent. A novel avipoxvirus, Penguinpox virus (PEPV) was isolated from an African penguin (Spheniscus demersus) that was brought into the Southern African Foundation for the Conservation of Coastal Birds (SANCCOB) [Turkeypox virus (TKPV) and Quailpox virus, confirmed that it was indeed a novel avipoxvirus [Vaccinia virus (VACV) derived late promoter P11 linked to the β-galactosidase reporter gene, resulting in transient gene expression.Interest in the avipoxviruses, notably  vectors -8. The g vectors ,10. Analus genus . This leSANCCOB) . Lesionspoxvirus . Infectipoxvirus . It is cfpv126 locus), was chosen for analysis in order to position PEPV in the larger chordopoxvirus group. Two additional genes, which are less highly conserved  and the virion envelope protein p35 ) were selected for analysis in order to determine the relationship of PEPV to other avipoxviruses previously analysed at these loci [One highly conserved gene, VLTF-1  as opposed to the CNPV-like group of viruses (clade B) or the Psittacine viruses (clade C) in both N-J and UPGMA trees. FWPV and CNPV orthologues showed an average of 75% nucleotide identity to each other for the various isolates at this locus. PEPV showed an average of 90% nucleotide identity to the various FWPV isolates and 74% identity to the various CNPV isolates. PEPV was found to belong to subclade A2 with 98% similarity to Falconpox virus (FLPV) and Albatrosspox virus (ABPV), and 100% homology to both the TKPV isolates (TKPV66 and TKPV98), the Ostrichpox virus (OSPV) isolate and two of the Pigeonpox virus (PGPV) isolates (PGPVTP2 and PGPVP).The P4b gene encodes a 75.2 kDa virion core protein, which is highly conserved amongst all poxviruses  and thisFigure Avipoxvirus, and go on to show that PEPV belongs to subclade A2 with the highest degree of similarity to TKPV isolates 98 and 66. Because there was no OSPV sequence available for the envelope protein p35 we can only speculate that PEPV and OSPV are highly similar based on the degree of similarity at the P4b gene. Previous work has shown that OSPV, TKPV and FWPV are genetically, antigenically and biologically related. Ostrich chicks housed in an enclosure that had previously housed pox infected turkeys became infected with a poxvirus and immunisation of susceptible chickens with this poxvirus protected them from FWPV challenge [Meleagris gallopavo) and chicken  IL-10, with 27% amino acid identity followed by the IL-10 like protein found in CNPV with 26% amino acid identity. The CNPV IL-10-like protein showed only 20% identity to the proteins from chicken and turkey. PEPV infection is evident in juvenile African penguins especially in the summer months when the penguins spend the majority of their time on land (moulting and/or nesting) and there are large numbers of mosquitoes present, which act as mechanical vectors to transmit virus . Further research is underway to determine whether outbreaks in different avian species are caused by the same virus or by different but closely related viral species.Taken together these analyses confirm that PEPV belongs to the genus hallenge . OSPV ishallenge ,24. The hallenge . AnalysiPEPV was grown on the chorioallantoic membranes of embryonated hens' eggs as described previously  to produAccession numbers of all loci used in this study Table .For further information on sequences used in this analysis please refer to .The authors declare that they have no competing interests.OC carried out the molecular genetic, sequence alignment and phylogenetic studies and drafted the manuscript. ND and ALW participated in the design of the study, analyses and interpretation of data and revision of the manuscript. All authors read and approved the final manuscript.


Schizophrenia is a severe, chronic, and costly illness that adversely impacts patients' lives and health care payer budgets. Cost comparisons of treatment regimens are, therefore, important to health care payers and researchers. Pre-Post analyses ("mirror-image"), where outcomes prior to a medication switch are compared to outcomes post-switch, are commonly used in such research. However, medication changes often occur during a costly crisis event. Patients may relapse, be hospitalized, have a medication change, and then spend a period of time with intense use of costly resources (post-medication switch). While many advantages and disadvantages of Pre-Post methodology have been discussed, issues regarding the attributability of costs incurred around the time of medication switching have not been fully investigated.Medical resource use data, including medications and acute-care services  were collected for patients with schizophrenia who switched antipsychotics (n = 105) during a 1-year randomized, naturalistic, antipsychotic cost-effectiveness schizophrenia trial. Within-patient changes in total costs per day were computed during the pre- and post-medication change periods. In addition to the standard Pre-Post analysis comparing costs pre- and post-medication change, we investigated the sensitivity of results to varying assumptions regarding the attributability of acute care service costs occurring just after a medication switch that were likely due to initial medication failure.Fifty-six percent of all costs incurred during the first week on the newly initiated antipsychotic were likely due to treatment failure with the previous antipsychotic. Standard analyses suggested an average increase in cost-per-day for each patient of $2.40 after switching medications. However, sensitivity analyses removing costs incurred post-switch that were potentially due to the failure of the initial medication suggested decreases in costs in the range of $4.77 to $9.69 per day post-switch.Pre-Post cost analyses are sensitive to the approach used to handle acute-service costs occurring just after a medication change. Given the importance of quality economic research on the cost of switching treatments, thorough sensitivity analyses should be performed to identify the impact of crisis events around the time of medication change. Pre-Post (or "mirror-image") designs are studies where one compares outcomes prior to and after some event – such as a medication change. The focus here is on pre-post comparisons of economic outcomes from patients switching medication treatment for schizophrenia. In long term treatment for schizophrenia, treatment switching is common, and there is a great need for information on treatment options for patients requiring a medication switch -3. Due tHealth-care decision makers often utilize observational data to assess economic outcomes ,12. In cPre-post analyses have several important disadvantages that have been well described in the literature . These iPre-post analyses are not, in general, suited to address broader questions of interest requiring multiple comparison groups. For instance, in the treatment of schizophrenia one might be interested in evaluating the multiple treatment options at the point of medication change, including switching to one of several medications or changing the dose of the current medication. One option in such instances is to utilize randomization to several treatment options for comparison at the time point where patients were in need of a medication change – as was done in the Clinical Antipsychotic Trials of Intervention Effectiveness (CATIE) ,16,17. IAnother limitation of pre-post designs for cost comparisons that has received less attention is the issue of costs incurred while on treatment B (the medication switched to) which may have been due to the failure of treatment A . For instance, while there are many reasons for switching treatment, the most common is lack of efficacy , which iOur objective in this paper is to highlight the issue of overlapping or carry-over costs from a failed prior treatment onto the next treatment in pre-post medication switch analyses. We accomplish this by examining the sensitivity of pre-post analyses of changes in costs of care to various assumptions regarding the attributability of acute-care service costs occurring immediately after a medication switch. Data from a naturalistic schizophrenia study is utilized for the analysis.Data for this post-hoc analysis came from a cost-effectiveness study of first-line treatment with atypical and conventional antipsychotics over a 1-year period ,24. ThisPatients were defined as having switched medications if they discontinued the medication to which they were initially randomized and then initiated therapy with a different antipsychotic within 14 days. Acute-care services were defined as hospitalizations, partial hospitalizations, and visits to emergency departments (EDs). Data on each patient's use of such services were systematically abstracted from medical records, patient self-reports, and the study sites' administrative databases. Cost estimates applied to each unit of resource use have been previously described in detail . In brieTo examine the impact of carry-over (overlapping) costs, we analyzed switching data using 4 different approaches – each making different assumptions regarding the attributability of costs and timing of incurred costs potentially due to the initial (pre) treatment. To standardize the 2 evaluation periods to a common duration, we chose to evaluate the last 60 days on the Pre-treatment and the 1st 60 days on the Post-treatment. The goal was to use a short enough duration to avoid deletion of patients from the analysis (or imputation of missing data) who only had short durations in the study pre- and/or post-switch and long enough to provide a more stable assessment of costs while on the medication. As secondary analyses, we utilized a 30-day window. Costs in the post-period were computed regardless of additional subsequent switches in medications. Costs are presented on an average daily basis to allow for comparisons across methods on a standard time scale. The 4 analysis approaches were:Costs incurred during the 60 days leading up to the last day on the initial medication are defined as 'Pre' period costs. Costs incurred starting from the day after the initial medication was discontinued for the next 60 days are defined as 'Post' period costs.Costs of acute care services that began while on the initial medication, but continued into the 'Post' medication period, were excluded from the analysis. Otherwise, calculations were the same as for the Standard Pre-Post Switch Analysis.Calculations follow the Standard Pre-Post Switch analysis except that the first 14 days of the Post period were excluded from the calculations. The 2-week cutoff was selected as most hospitalization durations were less than 14 days, so the impact of hospitalizations beginning in the 'pre' period would be addressed and since research suggests that impact of new medication could be observable by 2 weeks -28.Calculations follow the Standard Pre-Post Switch analysis except that costs occurring in the first 14 days in the post period are assigned to the Pre-treatment period.Paired differences in costs (Pre-Post) were compared statistically (with a null hypothesis of no change in mean costs) using both a paired t-test and, due to the skewness of the cost data, a nonparametric bootstrapping approach. The bootstrapping approach was utilized as we desired a nonparametric approach due to the anticipated skewed cost data and a test for differences in means as opposed to medians. Means are the appropriate parameter of interest as health care payers are responsible for the cost of the entire population, and N-times-the-median may not be representative of the total costs given skewed distributions ,30.A total of 664 patients were randomized, though 13 never started on their assigned medication . Of the Table Figure Figure None of the changes in costs were statistically significant from zero, and confidence intervals on the differences in pre-post cost changes for the 4 approaches are large and overlapping. For example, the 95% two-sided confidence interval for the standard approach was  and for assigning the first 14 days of post costs to the pre period was . Due to the high variability in total costs of care, such studies must be fairly large in order to have enough power for relevant cost comparisons. However, the focus here was not on establishing whether a change in costs occurred, but to compare the methodologies.The current analyses have demonstrated that assumptions about costs potentially attributable to failure of the first medication can have a directional impact on results when assessing the economic impact of changing medications using a pre-post analysis. The difference in estimated changes in daily costs in this study varied by over $12 per day across the methods utilized (between -$9.69 and +$2.40). Acute care service costs, which is the largest cost component in the treatment of schizophrenia, is often associated with relapse and with changes in medications, and thus must be considered carefully. Given the variation in results depending upon the attribution of crisis care costs around the time of switch, sensitivity analyses are critical to proper understanding of such data.Caution regarding the analysis of costs occurring around the time of switch should not be limited to pre-post designs. For instance, in clinical trials patients are typically discontinued at the point at which they have a medication change. This potentially ignores a substantial amount of the treatment costs which would then occur outside of the purview of the trial. In practical clinical trials, where patients are allowed to change medications and remain in the study, it is common to conduct an analysis including only the period of time in which the patient was on the medication of interest . Once agThe availability of data from a control group may address some of the issues associated with pre-post analyses. For instance, pre- and post-switch data for patients treated with a comparison medication would allow for additional methodological approaches to be used. Fu and colleagues  compare This study has several limitations. First, the number of patients switching was relatively small for comparisons of costs. Replication with a larger sample is needed due to the high variability in cost data. Next, we were unable to fully collect the reason for switching, which may indeed be associated with costs incurred around the time of switch. Lastly, about one-third of the nonswitchers discontinued early from the study. Some proportion of these patients likely switched medications outside of the purview of the trial, but we were unable to collect post-switch economic data for these patients. At baseline, these early discontinuers were more likely to be male; had more severe symptoms (as measured by the BPRS); and had a higher rate of prior hospitalization, substance abuse, and comorbid anxiety as compared to the switchers in this study. Thus, it is not clear if the results observed here are generalizable to such patients.Pre-post cost analyses ("mirror-image") are complicated by costs incurred post-switch that are likely due to the failure of the initial treatment. Such costs are not ignorable and can have substantial impact on the analyses. Sensitivity analyses should be performed to identify the impact of such costs.This work was sponsored by Eli Lilly and Company. The authors are full-time employees and minor shareholders of Eli Lilly and Company.DEF participated in the conception and design of the study, acquisition of data, analysis and interpretation of data, and drafting of the manuscript. AWN participated in the acquisition of data, interpretation of data, and critical revisions of the manuscript. HA-S participated in the conception and design of the study, interpretation of data, and drafting of the manuscript. All authors gave approval of the final version of the manuscript.


To understand the biological basis of these observations, we used mathematical models to explain observations made in patients with drug-resistant HIV treated with enfuvirtide (ENF/T-20), an HIV-1 fusion inhibitor. Due to resistance emergence, ENF was removed from the drug regimen, drug-sensitive virus regrown, and ENF was re-administered. We used our model to study the dynamics of plasma-viral RNA and CD4+ T cell levels, and the competition between drug-sensitive and resistant viruses during therapy interruption and re-administration. Focusing on resistant viruses carrying the V38A mutation in gp41, we found ENF-resistant virus to be 17±3% less fit than ENF-sensitive virus in the absence of the drug, and that the loss of resistant virus during therapy interruption was primarily due to this fitness cost. Using viral dynamic parameters estimated from these patients, we show that although re-administration of ENF cannot suppress viral load, it can, in the presence of resistant virus, increase CD4+ T cell counts, which should yield clinical benefits. This study provides a framework to investigate HIV and T cell dynamics in patients who develop drug resistance to other antiretroviral agents and may help to develop more effective strategies for treatment.For most HIV-infected patients, antiretroviral therapy controls viral replication. However, in some patients drug resistance can cause therapy to fail. Nonetheless, continued therapy with a failing regimen can preserve or even lead to increases in CD4 + T cells, and clinical outcomes is complex. We used mathematical models to evaluate the benefits of HIV drug therapy in the presence of drug-resistant virus. As an example, we considered resistance to enfuvirtide, the first FDA-approved fusion inhibitor. If viral load increases on drug therapy due to drug resistance, therapy with this drug may be stopped. We found that the drug resistant virus is less fit than the drug-sensitive virus in the absence of drug, and this fitness disadvantage causes the loss of drug-resistant virus during drug interruption. After the drug-sensitive virus replaces resistant virus, enfuvirtide therapy was re-administered. Analyzing the resulting viral kinetics, we demonstrate that despite the inability of the re-administered drug to suppress viral load because of the continued presence of drug resistant virus, therapy still provides benefit to the patient by preserving or increasing peripheral blood CD4+ T cell levels.The impact of antiretroviral drug-resistance on viral load, CD4 Observing increasing CD4+ T cell counts without viral suppression is intriguing and suggests that issues of viral fitness may play a role. Fitness costs have been associated with drug resistance not only to enfuvirtide but also to other drug classes + T cell counts and reduction in the risk of morbidity and mortality + T cell increase and to determine a general principle that may be useful in developing treatment strategies in the face of drug resistance, we performed a detailed viral kinetic analysis of a set of patients treated with enfuvirtide in which longitudinal measurements of drug sensitive and drug resistant viral levels, as well as CD4 counts, were available.Antiretroviral therapy has been used to successfully treat HIV-1 infection. However, a subset of patients develops drug resistance followed by an observable increase in plasma HIV viral load. This “virological failure” usually triggers a change in the drug regimen. Here we examine a situation in which patients had developed resistance to most common drugs and a novel agent, enfuvirtide, was added to their failing drug regimen. When resistance to enfuvirtide developed the use of this agent was discontinued in the hope that drug-sensitive virus would outcompete the resistant virus and enfuvirtide could be given again. Despite the fact that resistance developed when enfuvirtide was re-administered and viral loads were unable to be suppressed, CD4Enfuvirtide (ENF), formerly called T-20, is a 36 amino acid synthetic peptide that binds to the HR-1 region of the HIV-1 gp41 molecule, thereby preventing fusion of the viral membrane with the target cell membrane + T cell benefit associated with certain ENF-associated mutations As with other antiviral drugs, in patients treated with ENF, the high replication rate of HIV and the low fidelity of HIV reverse transcriptase can lead to the development of drug resistance Interruption of ENF in individuals with ENF-resistance is associated with a rapid decay in the resistant variant Despite marked differences in fitness of drug-sensitive and drug-resistant viruses and evidence of ongoing viral evolution, plasma HIV-1 RNA levels remain almost constant during ENF interruption + T cell data from Department of Medicine, University of California-San Francisco, CA, USA, San Francisco General Hospital, San Francisco, CA, USA and Section of Retroviral Therapeutics, Brigham and Women's Hospital and Division of AIDS, Harvard Medical School, Boston, MA, USA. Viral load and CD4+ data were obtained for three HIV-1 infected subjects  during ENF interruption who continued to receive the other drugs in their antiretroviral regimen. Before ENF interruption, subjects P1, P2 and P3 were treated with ENF for 27, 33 and 39 weeks, respectively, and each of them had the V38A mutation as the predominant virus population (more than 85% frequency). Viral load and CD4+ data were also obtained during subsequent 4-week re-administration of ENF after interruption for 76, 68 and 38 weeks, respectively. For subject P3, the data were also collected during a second interruption of ENF. Therefore, there were two data sets during ENF interruption for subject P3.We obtained wild-type and V38A mutant viral load and CD4T, cells infected by ENF-sensitive virus, sI, cells infected by ENF-resistant virus, rI, ENF-sensitive virus, sV, and ENF-resistant virus, rV. The model assumes that target cells are produced at a constant rate, λ, and die at rate dT. ENF-sensitive virus infects target cells to produce infected cells, sI, at rate sTVsβ, among which a fraction sβsTVsμ, become ENF-resistant during the process of reverse transcription of viral RNA to DNA due to mutation at rate sμ. Similarly, the infection by ENF-resistant virus produces infected cells, rI, at rate rTVrβ, with a fraction μrβrrTV undergoing backward mutation to the drug sensitive strain at rate μr. Cells infected by ENF-sensitive and ENF-resistant virus produce new virions at rates sIsp and rIrp, and die at rates sδI and rδI, respectively. Both viruses are cleared at the same rate c per virion. Whether the V38A mutation in gp41 affects viral production remains unclear. For simplicity, we assume sp = rp, and describe the resistance-associated fitness loss only by a reduced infectivity rate, i.e., rβ = (1−α) sβ, where the fitness cost of the mutant virus, α, satisfies 0≤α≤1. ENF is a fusion inhibitor and reduces infection of target cells by free virus. We assume sε and rε are the efficacies of ENF against ENF-sensitive and ENF-resistant virus, respectively, with 0≤sε, rε≤1.A schematic diagram of the model is shown in 10 HIV RNA copies/ml). Thus, stochastic effects would not be significant and we formulate the model as a deterministic model – a standard two-strain viral dynamic model – similar to the ones in In the patient data we analyze the populations of both drug-resistant and drug-sensitive virus always remain high /a, where a denotes the fraction of CD4+ T cells that are activated. In principle, a could be time-varying or in particular depend on the CD4+ T cell count a is constant.As measured by Ki-67 antigen expression, only a small percentage of CD4sβ, rβ and the virus production rates sp, rp that we estimate include the effects of the other drugs in the background regimen. However, since the background regimen was failing to suppress HIV replication, these effects may be minimal. Moreover, the data have taken only V38A mutants into account with other mutants being included in the “wild-type”. Therefore ENF efficacy against wild-type, sε, in our model also incorporates the possible reduction in efficacy due to other mutants included in the wild-type. Further, loss of V38A mutation at rate rμ, can lead to any of a variety of viral variants that we include in the drug sensitive population. Lastly, virus variants carrying the V38A mutation may also carry other mutations, such as compensatory mutations or other drug resistance mutations, which may affect the fitness of the drug-resistant population as well as its level of drug resistance.We note that in the study we analyze s)βsTVs(1−ε and −r)βrTVr(1−ε to Eqs. (4) and (5), respectively. For the measured range of T in the subjects considered here, and the estimates of sβ and rβ determined below, sTβ and rTβ are <0.05 d−1 which is ∼500 times lower than the viral clearance rate c (23 d−1), indicating that virion loss due to infection will have negligible effect on the viral dynamics compared to the term −cV. We confirmed this by fitting the model with the terms −s)βsTVs(1−ε and −r)βrTVr(1−ε in Eqs. (4) and (5), respectively, in which we found almost no change in parameter estimates. Therefore, we neglected virion loss due to infection and left only the viral clearance term (−cV) in the V equations.We note that there is loss of some free virus due to the infection of target cells as virus must enter a cell in order to infect. To incorporate this effect, one can add the terms −sV = s/c)Is(p and r = (pr/c)IrV. This simplifies the model leaving only equations for T, sI and rI. Further, we set sI(0) = (c/sp)sV(0) and rI(0) = (c/rp)rV(0) for data fitting as well as all simulations, where s(0)V and r(0)V are determined by direct measurement at the start of interruption or the start of ENF re-administration.The dynamics of free virus is typically fast in comparison with that of infected cells + T cell death rate d = 0.01 day−1. Recent estimates show that the virion clearance rate constant, c, varies between 9.1 day−1 and 36 day−1, with an average of 23 day−1c = 23 day−1.As measured by Mohri et al. λ (target cell recruitment rate), sβ (drug sensitive virus infection rate), sμ (forward mutation rate), rμ (backward mutation rate), α (fitness cost of ENF-resistance), sp (production rate of drug sensitive virus), δ (infected cell death rate), 0T  and a (fraction of CD4+ T cells that are activated) by fitting the model to the ENF-sensitive viral load, the ENF-resistant viral load and the CD4 count data simultaneously for each patient. Since fewer data points are available during re-administration of ENF, we fix some parameters at the values obtained by estimation during ENF interruption; and only estimate sε (ENF efficacy against the sensitive strain), rε (ENF efficacy against the resistant strain), λ, 0T and a. We also fitted the data during ENF interruption and ENF re-administration allowing the initial concentrations of drug sensitive and drug resistant viruses to be free parameters, but the fit could not be improved.During ENF interruption, we estimate the parameters 10 values of the ENF-sensitive and ENF-resistant viral loads and the CD4 count for each patient were fit to the corresponding log-transformed data. Of note, to avoid the difficulty of assigning different weights to the viral loads and CD4 counts in the objective function being minimized, and give equal importance to all the widely varied values in the data set, we fitted the data in the log-scale rather than linear-scale for both viral loads and CD4 count. Finally, for each best fit parameter estimate, we provide a 95% confidence interval (CI) using 200 bootstrap replicates We solved Eqs. (1)–(5) numerically using the Runge-Kutta 4 algorithm in Berkeley Madonna The estimated viral dynamic parameters during ENF interruption along with their mean and sample standard deviation, and their 95% confidence interval are summarized in −5 and 1.73±0.30×10−5, respectively. Even though the backward mutation rate is slightly lower than the forward mutation rate, early after ENF interruption the ENF-resistant virus population is significantly larger than the ENF-sensitive virus population, and consequently the amount of backward mutation during the early post ENF interruption is usually higher than the amount of forward mutation. Although there is a continued evolution in gp41 after ENF interruption, our results show that the rate of on-going evolution including backward mutation accumulation is not sufficient to explain the rapid waning of ENF-resistant virus. For example, during the first week (month) post interruption, the contribution of ongoing evolution and backward mutation to the loss of cells carrying a drug-resistant proviral genome is only about 0.2 (0.4) cells per ml, which corresponds to the loss of 26 (70) drug resistant virions per ml per week (month). Since the contribution of these de-novo mutations is small, we also fitted the data using the model without de-novo mutation, i.e., sμ = rμ = 0, and found that the changes in estimated parameter values lie within a range of 0–6%. As the loss of resistant virus due to backward mutation is negligible, we find the fitness cost, i.e., the reduction of the infectivity of the resistant virus compared to the wild-type virus, plays a more important role in the decay of ENF-resistant virus and the increase of ENF-sensitive virus. Fitting our model to the data suggests that ENF-resistant virus is 17±3% less fit  and (7) for the steady state level of total virus, which to a good approximation are equal to, pλ/(cδ), during ENF interruption and re-administration.Marked differences in the level of plasma viral load is seen when the target cell recruitment rate, rruption  and re-arruption . After eoad on λ  and 5d, + T cell counts (mean change: 0.13 cells/µl/week) suggesting that steady state was reached by the end of this long-term treatment. They also observed the steady state T cell level after a long period of ENF interruption. Below we calculate from our model the steady state level of uninfected CD4+ T cells to understand how the uninfected target cell level differs between long-term ENF interruption and long-term ENF re-administration.The changes over time of the CD4 count, and of the proportion of uninfected cells, cell infected with sensitive virus, and cell infected with resistant virus are shown in ET, and ENF re-administration, rε = 0, as no drug is present. After drug is given, rε>0 and Eq. (8) shows that the target cell level should increase. Furthermore, in addition to the efficacy of the drug against resistant virus, rε, the fitness cost, α, also contributes to the maintenance of a higher level of uninfected target cells during ENF re-administration. In fact, even if the drug is completely ineffective against resistant virus , and the viral load is approximately equal during both ENF interruption and ENF re-administration as shown above, HIV infected patients with ENF re-administration will still have a higher uninfected target cell level due to the fitness loss of resistant virus . Above we showed that during re-administration the total viral load, and hence the total number of infected cells also stays approximately constant. Hence the CD4+ T cell count, which includes both uninfected and infected CD4+ T cells, is expected to increase with the increase in target cells. This is an important result as it shows that even though the resistant virus becomes dominant during ENF re-administration  = rV(t)/sV(t), the ratio of the two strains:r(0)≈100, i.e. resistant virus is approximately 100-fold more plentiful than sensitive virus. As time off therapy increases the level of resistant virus falls and r(t) decreases. When r(t)<1, the sensitive virus is the dominant strain. The time, θt, for the proportion of resistant virus to reach r(θt) during ENF interruption isα, causes the ENF-sensitive virus to be dominant sooner, while an increase in initial ENF-resistant virus proportion, r(0), results in a longer time for the ENF-sensitive virus to be dominant. Varying the T-cell count at the time of interruption from 50 to 250 or 500 µl-1 or increasing the T cell source rate, λ, does not significantly impact the proportion of ENF-resistant virus  + T cell counts is unclear. At comparable plasma viral loads, drug resistant HIV can be associated with more sustained CD4+ T cell gains and reduction of the risk of morbidity and mortality + T cell counts have been observed to increase during therapy as the ENF resistant virus with less capacity to infect T cell replaces the ENF-sensitive virus. A large prospective study has recently been completed in which ENF was given as a “pulse” to determine if the expansion of ENF resistance positively affects CD4+ T cell counts. Preliminary data from 3 individuals has previously been published The impact of antiretroviral drug-resistance on viral load, CD4Interruption of ENF after the emergence of ENF resistance results in a rapid decay of the resistant variant + T cell count from HIV-1 infected patients, who underwent ENF interruption and subsequent re-administration while continuing to receive the other drugs in their regimen Our model, which describes the dynamics of ENF-sensitive virus, ENF-resistant virus, target cells, cells infected by ENF-sensitive virus and cells infected by ENF-resistant virus, includes the fitness cost of ENF-resistant virus as well as forward and backward mutations. The model was used to fit data concerning the level of ENF-sensitive viruses, V38A ENF-resistant viruses and the CD4Our parameter estimates, model analysis and numerical simulations produced several interesting observations. First, the magnitude of the backward mutation rate of V38A is approximately the same as that of the forward mutation rate. This indicates that ongoing viral evolution might have some contributions to the loss of ENF-resistance during ENF interruption, supporting the results of phylogenetic analysis in Kitchen et al. Second, we found that the fitness cost of ENF-resistant mutations has a major role in the loss of ENF-resistant virus and the turnover of the virus population during ENF interruption. We estimated that the ENF-resistant virus is 17±3% less fit than the ENF-sensitive virus. This reduced fitness of V38A ENF-resistant virus agrees with the experimental finding of reduced fitness of the V38A mutant virus compared to wild-type virus in vitro λ, the virus production rate, p, the infected cell death rate, δ, and the virus clearance rate, c. A higher value of p or λ and/or a lower value of δ or c give rise to a higher plasma HIV RNA level.Third, we found that the fitness cost and the initial proportion of the ENF-resistant virus do not have any observable role in defining plasma HIV RNA levels. Neither does ENF efficacy during ENF re-administration contribute to setting plasma viral levels. The plasma viral RNA level is determined mainly by the target cell generation rate, Our next observation is related to the advantage or disadvantage of re-administrating the drug following an interruption. Once the drug is re-administered, the resistant virus rapidly reemerges and becomes dominant over the sensitive virus. This indicates a strong selective pressure of the drug on the virus population. The turnover rate of the virus population is more rapid during the drug re-administration than during the drug interruption. This shows that the advantage of the drug-resistant virus over drug-sensitive virus during the drug re-administration is greater than the disadvantage of the drug-resistant virus over the drug-sensitive virus during the drug interruption. The rapid reemergence of the resistant virus also indicates the persistence of actively replicating resistant virus as suggested in + T cells (+ T cell count over the period of 3 months compared to the same patients during ENF interruption (+ T cell level during the drug re-administration increases as the efficacy of the drug against resistant virus increases or/and the fitness cost of resistant virus increases.One of the most interesting results demonstrated by our model is that despite sustained high levels of viral load, re-administration of ENF helps in maintaining a significantly higher level of CD4 T cells  and 6. Drruption . The CD4rruption  and 6c. There are several limitations of this study. The results are based on limited data from only three subjects. Moreover, there are fewer data points available during ENF re-administration, which might produce more uncertainty in the results derived from ENF re-administration. To gain more confidence in the results obtained here, extensive studies with more data are necessary. We have considered the V38A mutant virus as a representative of all ENF-resistant viruses. However, there are many other mutant viruses, which may possess different fitness costs and different mutation rates. It should be noted that in the experiment only the proportion of V38A was measured, and so there might be other mutant virus resistant to ENF that would have been included in the ENF-sensitive viral load. A detailed quasi-species model, as in Murray and Perelson + T cell levels.In summary, we have used mathematical models to help explain the viral dynamic properties of drug sensitive and resistant viruses in the presence and the absence of the drug ENF. Our results show that even though forward and backward mutations occur during therapy interruption, the primary factor leading to the loss of resistant virus during therapy interruption is the fitness cost of the resistant virus. In the presence of drug, the efficacy of drug against resistant virus is also one of the main factors determining dominance of the drug resistant virus in the population. More importantly, even though the drug is ineffective in suppressing plasma viral load due to the presence of resistant virus, our results support the concept that continued therapy may have a residual immunologic benefit by preserving peripheral blood CD4


A small focus of hemorrhagic fever (HF) cases occurred near Cochabamba, Bolivia, in December 2003 and January 2004. Specimens were available from only one fatal case, which had a clinical course that included fever, headache, arthralgia, myalgia, and vomiting with subsequent deterioration and multiple hemorrhagic signs. A non-cytopathic virus was isolated from two of the patient serum samples, and identified as an arenavirus by IFA staining with a rabbit polyvalent antiserum raised against South American arenaviruses known to be associated with HF . RT-PCR analysis and subsequent analysis of the complete virus S and L RNA segment sequences identified the virus as a member of the New World Clade B arenaviruses, which includes all the pathogenic South American arenaviruses. The virus was shown to be most closely related to Sabiá virus, but with 26% and 30% nucleotide difference in the S and L segments, and 26%, 28%, 15% and 22% amino acid differences for the L, Z, N, and GP proteins, respectively, indicating the virus represents a newly discovered arenavirus, for which we propose the name Chapare virus. In conclusion, two different arenaviruses, Machupo and Chapare, can be associated with severe HF cases in Bolivia. Four rodent-borne arenaviruses are known to cause hemorrhagic fever (HF) in the New World. These include Junín, Machupo, Guanarito, and Sabiá viruses, which are found in rural areas of Argentina, Bolivia, Venezuela, and Brazil, respectively. In December 2003 and January 2004, a small number of HF cases were reported in rural Bolivia in an area outside the known Machupo HF endemic zone, and sera from one fatal case was available for laboratory testing. The man had symptoms similar to those seen with other arenaviral HF cases—acute febrile illness beginning with headache, joint and muscle pain, and vomiting—and rapidly progressed to shock, bleeding, and death at 14 days post onset of illness. Virus was isolated from two of the patient's serum samples and identified as an arenavirus by reaction of virus infected cells with arenavirus-specific antibodies and by genetic detection techniques (PCR). Subsequent complete genome analysis of the virus showed the virus to be a distinct newly discovered member of the arenavirus family, and the name Chapare virus was proposed. The virus is phylogenetically related to other arenaviruses that naturally cause hemorrhagic fever in South America, particularly Sabiá virus. Physicians should consider Chapare virus as a potential etiologic agent when encountering HF cases in the region. Arenaviridae is composed of largely rodent-borne viruses which are divided into Old World and New World complexes Calomys callosus rodents Calomys callosus in Bolivia The family rd, 2004. In addition, no members of case household or other close contacts were affected. His clinical course included fever, headache, arthralgia, myalgia and vomiting with subsequent deterioration and multiple hemorrhagic signs and death on January 17th, 2004 (14 days post onset). Based on these symptoms, the patient was initially suspected of having yellow fever or dengue HF. However, initial tests for these agents were negative. Initial IgM, IgG, antigen capture and RT-PCR testing for Machupo virus or related arenaviruses were also negative.In late 2003 reports were received of a small cluster of HF cases in a rural area near the Chapare River, close to Cochabamba, Bolivia in the eastern foothills of the Andes . Exact dPatient specimens were sent to the biosafety level 4 (BSL4) containment laboratory at the Special Pathogens Branch in Atlanta where virus isolation attempts could be performed. These specimens consisted of 4 acute phase sera, collected on days 4, 7, 9 and 14 post onset of disease. Both the day 7 and day 9 sera yielded a non-cytopathic virus by growth in Vero E6 cells. These were identified by immunofluorescent antibody (IFA) staining with rabbit polyvalent hyperimmune serum raised against South American arenaviruses previously known to be associated with HF . RT-PCR analysis of the virus isolate RNAs amplified a 481 bp fragment which yielded nucleotide sequence related to known New World Clade B arenaviruses . Full length virus genome sequences were successfully determined for the virus isolated from the day 9 post onset bleed (designated strain 810419) by RT-PCR and sequence analysis followed by primer walking utilizing newly derived sequence information. The full length S segment was amplified by using the 19C primer designed based on the conserved RNA termini of New World Arenaviruses Chapare virus was found to be monophyletic with Sabiá virus on phylogenetic analysis of the nucleotide or encoded amino acid sequences of the complete S or L segment , or NP, The pathogenicity of the New World Clade B viruses correlates with the efficient interaction of their GP1 surface glycoproteins with the human cellular receptor, transferring receptor 1 (TfR1) The relationship of Chapare virus from Bolivia to Sabiá virus from Brazil is intriguing. Both these virus clearly cause HF similar to that seen with Junín, Machupo and Guanarito viruses. The single HF case associated with a naturally acquired Sabiá virus infection was reported in the community of Sabiá, in Sao Paulo, Brazil in 1990 Calomys callosus) and Chapare viruses (reservoir unknown). Furthermore, both Machupo and Chapare viruses are agents of fatal hemorrhagic fever in Bolivia.Due to the difficulties of working in this resource poor rural region, initial follow up efforts in the Chapare area, did not yield a more precise description of the reported cluster of cases with similar illness, and a limited ecological study did not reveal the rodent reservoir of this virus. It is hoped that more extensive studies in the area will reveal the extent to which Chapare virus poses a public health problem in this area, and shed light on the source of human infection. In summary, three arenaviruses are now known to be present in Bolivia, namely Machupo and Latino viruses  GAC RTG CCW GGI GGI TAY TG; GP1126-, TAC CAA AAT TTG TGT ART TRC ART AIG G; GP1153+, CCT TAY TGY AAY TAC ACI AAA TTT TGG T; GP1396-, ATG TGY CTR TGI GTI GGI AW).Initial virus genetic detection and analysis was conducted on total RNA extracted from infected Vero E6 cells, using Tripure Isolation Reagent  in a ratio of 1∶5 and incubated at room temperature for a minimum of 10 min. Total RNA was isolated by using the RNaid Kit following the manufacturer's recommendations , and the extracted RNA was reconstituted in 50 µL HReverse Transcription (RT) was done using 2.5 µL of RNA in a 25 µL total reaction volume and AMV RT  at 42°C for 1 hr. Subsequent PCR amplification using FastStart Taq DNA Polymerase with GC-rich solution (Roche) was performed using 5 µL of the completed RT reaction in a 25 µL reaction volume with the following cycling conditions: 2 min at 95°C, , and a final elongation of 10 min at 72°C. Resulting DNA products were visualized and purified using a 1% agarose gel, and the Qiagen Gel Extraction Kit . PCR products were sequenced directly (without cloning) using the corresponding primers in a BigDye Terminator v3.1 reaction on a 3100 Genetic Analyzer . Sequence was further analyzed using Sequencher .To obtain full length sequence for each segment, alignments of all New World arenavirus complete genomes were used to design primers for the conserved regions (available upon request). The full length S segment was generated following the Thermoscript RT-PCR system's directions  and using the 19C primer Different fractions of the full-length L RNA were amplified using 2-step or 1-step RT-PCR protocols and following the manufacturer recommendations. Briefly, cDNA was synthesized in the first approach using 10 µl of purified RNA, specific primers, dNTPs and Superscript III (Invitrogen) in 20 µl reactions. Amplification reactions were done using 5 µl of cDNA, specific primers, dNTPs and Platinum Taq DNA polymerase High Fidelity (Invitrogen) in 50 ul reactions. Alternatively, 1-step RT-PCR were performed using 5 µl of RNA, dNTPs and the enzyme blend provided by the SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity (Invitrogen) in a 50 µl reactions. Amplification reactions were analyzed in TBE/agarose gels and DNA bands purified using QIAquick Gel Extraction Kit (Qiagen). Sequencing reactions were done as described above.All full length S and L segment sequences available in Genbank were used to compute pairwise uncorrected genetic distances using PAUP 4.0b10 (Sinauer Associates) for the following viruses: Allpahuayo, Amapari, Chapare, Flexal, Guanarito, Junín, Machupo, Paraná, Pichindé, Pirital, Sabiá, Tacaribe, Tamiami, and Whitewater Arroyo.A representative sub-set of full length sequences  were included in a Bayesian phylogenetic analysis. Sequence alignments were done with ClustalX Table S1Amino acid distances for complete L and Z genes and nucleocapsid and glycoprotein genes of the New World arenaviruses(0.34 MB PDF)Click here for additional data file.


Tadarida brasiliensis. This is the first human rabies case caused by an insectivorous bat rabies virus variant reported in Latin America.The first human rabies case in Chile since 1972 occurred in March 1996 in a patient without history of known exposure. Antigenic and genetic characterization of the rabies isolate indicated that its reservoir was the insectivorous bat  In Latin America, rabies in bats was suspected during the 1910s in Brazil and was definitively diagnosed for the first time in Trinidad in 1931 . A rabies antibody titer of 1:625 was found in the serum specimen by using the indirect fluorescent-antibody (IFA) technique The patient died on March 5, 1996, when artificial respiratory support was disconnected. Postmortem tissue samples of cerebral cortex, hippocampus, cerebellum, and nuchal skin biopsy were sent to the ISP Rabies Laboratory for diagnosis. The cerebellum and skin specimens were positive for rabies virus antigen by DFA assay.Rabies postexposure prophylaxis with the suckling mouse brain Fuenzalida-Palacios vaccine was administered to the victim’s mother and to 10 health-care providers who had possible contact with the patient’s saliva. The rabies postexposure prophylaxis schedule used was 2 mL of vaccine, subcutaneously, on each of days 1, 2, 3, 4, 5, 6, 21, and 90. Blood samples were taken from vaccinees on day 14 after the initial dose of vaccine; IFA assay showed that adequate immune responses had developed.Tadarida brasiliensis (free-tailed bat) in Chile, which had been designated as antigenic variant 4 (AgV4) (The virus was isolated from the patient’s brain tissue by intracerebral inoculation of suckling mice Genetic characterization was done by sequencing a 320-bp portion of the rabies virus nucleoprotein gene from nucleotide position 1,157 to 1,476, as compared with the SADB 19 strain (This human rabies virus isolate was compared with viruses obtained from domestic animals and insectivorous bats in urban centers in Chile from 1977 to 1998 T. brasiliensis (Lasiurus sp. (T. brasiliensis is the likely reservoir of the rabies virus isolated in this case.Although five genetic variants of rabies virus are found in Chile iliensis , group Durus sp. , group Eurus sp. . The higThe absence of a history of an animal bite, the clinical presentation of the disease without the classic signs of hydrophobia or aerophobia, and the absence of any human rabies cases for a period of 24 years in Chile were the primary reasons that rabies was not first suspected and a definitive diagnosis was delayed in this case. Retrospective studies of human rabies epidemiology have demonstrated that it is not uncommon to observe rabies cases in which there is no history of a bite, mainly in situations involving insectivorous bat rabies variants. For example, of the 17 human rabies cases associated with insectivorous bats reported in the United States from 1980 to 1996, only one had clear documentation of a bite In cases in which a patient shows clinical signs of central nervous system involvement of unknown or suspected viral origin, health-care providers should be aware of the importance of conducting a thorough medical history to appropriately assess the possibility of rabies. With the important changes in the epidemiologic patterns of rabies in Latin America, this disease should be included in the differential diagnosis of neurologic diseases characterized by acute encephalitis and progressive paralysis, even when no previous history of an animal bite exists and even in regions where canine rabies has been eradicated.


Since the time of Charles Darwin, studies of interspecific hybridization have been a major focus for evolutionary biologists. Although this phenomenon has often been viewed as problematic in the fields of ecology, taxonomy and systematics, it has become a primary source of data for studies on speciation and adaptation. Effects from genetic/evolutionary processes, such as recombination and natural selection, usually develop over extended periods of time; however, they are accelerated in cases of hybridization. Interspecific hybrids exhibit novel genomes that are exposed to natural selection, thus providing a key to unravel the ultimate causes of adaptation and speciation. Here we provide firstly a historic perspective of hybridization research, secondly a novel attempt to assess the extent of hybridization among animals and thirdly an overview of the reviews and case studies presented in this theme issue. Research on interspecific hybrids has played a major role in the field of evolutionary biology, since deciphering the mechanisms preventing or allowing interbreeding of species helps to answer general questions such as: How are reproductive barriers between species evolving? Which genes contribute to reproductive isolation? Is gene flow between species (introgression) a creative, additional force in adaptation? During the past 150 years research on interspecific hybridization went through three main phases : the firThose forms which possess in some considerable degree the character of species, but which are so closely similar to some other forms, or are so closely linked to them by intermediate gradations, that naturalists do not like to rank them as distinct species, are in several respects the most important to us.Since Darwin first described hybrid formation in the context of speciation, interspecific hybridization has attracted the attention of many evolutionary biologists. Hybrids are formed when different species interbreed, resulting in the combination of genetic material from previously isolated gene pools. Dacus) suggested that the introduction of genes from another species could serve as the raw material for an adaptive evolutionary advance even though the original hybridization was disadvantageous. During the 1950s and 1960s a number of empirical cases of plant and animal hybridization led to the development of models of hybridization, which were then discussed in the seminal review by However, until the 1930s the common opinion was that sterility of interspecific hybrids caused the lack of evident hybridization. In the 1930s and 1940s a number of botanists started to experimentally study interspecific hybridization, either using crossing experiments  or fieldAn overview of the past 150 years of hybridization studies illustrates two aspects that are most likely apply to many other fields. Firstly, scientific progress is largely driven by technological advances. Secondly, questions raised 100 years ago are still in the focus of current research projects. The latter is most likely explained by the remaining gap in the recent integration of genetics and ecology. Although we have experienced a fruitful exchange of ideas among both fields of research, the field of molecular ecology has been mainly characterized by the application of selectively neutral markers, such as mitochondrial or microsatellite DNA, to address ecological questions. Mainly, these studies used genetic information to identify species to measure the effect of gene flow or to reconstruct phylogenetic relationships or phylogeographic patterns. However, the genetic basis of natural selection and reproductive isolation were examined only recently, and thus there are still relatively few ecological genetic analyses that have gathered data concerning functional genes, neutral loci and hybrid fitness. Yet, these few studies have illustrated, for example, how introgressive hybridization contributes to the evolution of interbreeding species .We would also like to stress two other, more general, aspects emerging from studies using literature or other databases. On the one hand, they illustrate the additive value of multiple studies of the same phenomenon over a broad taxonomic range, such as pure taxonomic or barcoding studies . This stDuring the recent past, a number of reviews on interspecific hybridization illustrated an unexpectedly wide taxonomic range and, depending on the group of organisms, relatively high frequencies of interbreeding species, e.g. among vascular plants (25%), European butterflies (12%), fruitflies (1%), birds (10%), British ducks (25%) and European mammals 6%; . These fR2=0.29, p=0.02). The deviations of the regression line  are composed of differences in study intensity and taxonomic practice in the respective research community, true differences among taxonomic groups and random error. From a second correlation between all records used in our study and the number of described species per taxonomic group, we isolated the residuals as an indicator of a study bias . Some taxa, especially vertebrates and the economically important pests and crops, have received far greater attention from taxonomists and population geneticists than most other taxa (e.g. marine invertebrates). This parameter explained a large part of the residual variation derived from the first regression . This pattern indicates that the rate of interspecific hybridization is relatively homogeneous among animal groups and only a few groups deviate from this pattern. Thus, the level of interspecific hybridization might represent an inherent and neutral parameter of evolutionary change. This analysis does not reveal anything about the potential significance of hybridization, or the frequency of backcrossing and introgression. However, it implies that in each taxonomic group we have a priori similar chances for gene exchange among species. We do not know how accurately the hybrid records reflect the actual number of interspecific hybrids. However, if the proportion of multiple hits in the number of hybrid records and the total number of records is roughly the same, we estimate the average hybridization rate to be approximately 1% (range 0.1–3%). This relatively low frequency of hybridization in animals is certainly at odds with previous estimates, which have predicted a frequency of approximately 10% . We found 21 972 publications concerning hybrid animals within the field of ecology, evolution and systematics . In ordetely 10% . This diBut even if the hybridization frequency is low, we would argue that the studies of interspecific hybridization should remain a major focus of evolutionary biology for two reasons. Firstly, although the frequency of individuals which actually interbreed with other species might be rare, the occurrence of occasional hybridization events might be sufficient to cause long-lasting changes in the genomic architecture of species. Thus hybridization might be rare but if it occurs it has a major impact on the origin and fate of evolutionary lineages. Secondly, hybridization provides a natural setting to study efficiently the interaction between genetic and ecological differentiation. In an attempt to justify the study of rare phenomena, By combining previously isolated gene pools, interspecific hybridization results in the origin of new genotypes. If this phenomenon is viewed at the level of populations, then interspecific hybridization can also result in significant shifts of allele frequencies. When compared with mutation and recombination within species , interspecific hybridization can result in rapid and long-lasting changes among interbreeding species. The evolutionary change due to hybridization can occur within one generation, thereby exposing new gene combinations to natural selection. Furthermore, by compressing the time scale over which evolutionary processes occur, and by allowing the formation of new gene combinations, interspecific hybridization enables investigations into the processes of natural selection. Owing to these properties, examples of interspecific hybridization have been described as ‘natural laboratories for evolutionary studies’ , ‘windowGeospiza) have revealed, for example, the effect of introgressive hybridization on developmental genetic pathways of phenotypic traits  that reduce heterospecific matings. This process will subsequently enhance reproductive isolation between species and result in reinforcement. If hybridization is less costly, or provides fitness advantages, prezygotic isolation might be weak and allow hybridization events. Even though these events might occur at a low frequency they are bound to have long-lasting consequences for the genetic architecture of populations due to gene flow among species. How behaviour of males and females determines the outcome of hybridization is illustrated in the studies of Many studies on interspecific hybridization have focused on aspects of mating biology, since parental taxa and interspecific hybrids are often characterized by significant shifts in reproductive modes  and ploidy levels . In thisOne of the main topics in the field of interspecific hybridization is related to the question of whether the selection against hybrids is environmentally independent or dependent . The spatio-temporal distribution of plankton species and hybrids along environmental gradients suggests the action of exogenous selection (Although previous studies identified certain taxonomic groups that are known to have accelerated levels of interspecific hybridization, our literature survey suggests that hybridization represents a phenomenon distributed uniformly across the animal kingdom. As the historical overview, the reviews and the case studies presented in this issue demonstrate, interspecific hybridization remains a growing and exciting field in evolutionary biology.


The paper of When gentamicin-loaded bone cement  became available in Germany in the 1970s, efforts to treat osteomyelitis by filling the debrided cavity with solid plugs of antibiotic-loaded bone cement were not successful in patients in France and Germany, nor in animal experiments in the Mayo Clinics . Thus, KOne of the arguments against the use of antibiotic-loaded beads is the lack of proof of effectivity in good randomized clinical trials (RCTs). The most cited RCT was performed in the US in an attempt to get FDA approval, in 380 patients with osteomyelitis: systemic antibiotic treatment versus gentamicin beads. However, the protocol was violated because surgeons worried about withholding systemic antibiotics from patients who were treated with beads. In this trial, however, the costs were lower when beads were used, and there were less systemic adverse events with antibiotics when local antibiotics were used (as compared to treatment with systemic antibiotics) . AnotherAn RCT was even performed in a developing country. This rarely cited study was done in an isolated Himalayan region of Nepal. Lars Lindberg  and Margareta Höök treated 45 patients with debridement followed by the use of gentamicin beads or open treatment package of the debrided cavity . The ranIn the developing countries surgeons also became aware of the benefit of local antibiotic treatment of bone infections. The Septopal beads were, however, too expensive and surgeons had to look for alternative solutions. Beads made of plaster of Paris were an important alternative in the beginning.The use of plaster of Paris in bone defects had been known since 1892, when Trendelenburg in Bonn (Germany) filled bone defects . AdmixtuFor many years, a group of Dutch orthopedic surgeons used preformed gentamicin-containing plaster of Paris beads during their visits to Indonesia and Africa; these were made by a pharmacist at the hospital in Arnhem . So gent-1) than Septopal beads (8.6 cm -1), so the release is worse. The paper of Rasyid et al. describes how a method can be developed to create hand-made gentamicin-loaded PMMA beads with very good pharmacokinetic properties in combination with substantially reduced costs when compared with the commercially available beads : 112 USD instead of 350 USD.The production by orthopedic surgeons in Indonesia of hand-made beads was originally done by mixing bone cement with fosfomycin, an antibiotic often used in developing countries . They foThese authors used an elegant method to increase the antibiotic release: simply the reduction of the monomer content by 50%, which causes large pores in the incompletely polymerized polymer particles. Porosity is one of the most important factors in the process of sustained release of bone cement . In SeptIn western countries, improvement of antibiotic release from bone cement is also important. Spacers have a low surface area to volume ratio, resulting in a limited and often insufficient release of antibiotic . In the Staphylococcus aureus and S. epidermidis, possibly in combination with gentamicin  that made it possible to order vancomycin-gentamicin PMMA beads for an individual patient, based on the resistance pattern of a causative agent . For 5 yFinally, molds are helpful in the production of hand-made beads. Surgeons should be aware that beads or spacers that they make themselves must have good pharmacokinetic properties to be effective, and that not all kinds of antibiotics may be released properly from bone cement. With the special technique for improvement of antibiotic release properties of bone cement described in the article of Neut et al., and the use of a mold, it is now possible for the surgeon to make customized antibiotic-loaded PMMA beads with different antibiotics and with an improved antibiotic release. We will need this technique increasingly for prosthetic joint infections and osteomyelitis, since resistant bacteria such as CNS, MRSE and MRSA are becoming ever more frequent. It is remarkable that the scientific help of a western university (Groningen) to solve a problem in a developing country may improve the treatment of bone and joint infections in many centres in the western world itself.


Cryptococcus gattii was reported in the state of North Carolina, USA. An otherwise healthy HIV negative male patient presented with a large upper thigh cryptococcoma in February, which was surgically removed and the patient was started on long-term high-dose fluconazole treatment. In May of 2007, the patient presented to the Duke University hospital emergency room with seizures. Magnetic resonance imaging revealed two large CNS lesions found to be cryptococcomas based on brain biopsy. Prior chest CT imaging had revealed small lung nodules indicating that C. gattii spores or desiccated yeast were likely inhaled into the lungs and dissemination occurred to both the leg and CNS. The patient's travel history included a visit throughout the San Francisco, CA region in September through October of 2006, consistent with acquisition during this time period. Cultures from both the leg and brain biopsies were subjected to analysis. Based on phenotypic and molecular methods, both isolates were C. gattii, VGI molecular type, and distinct from the Vancouver Island outbreak isolates. Based on multilocus sequence typing of coding and noncoding regions and virulence in a heterologous host model, the leg and brain isolates are identical, but the two differed in mating fertility. Two clinical isolates, one from a transplant recipient in San Francisco and the other from Australia, were identical to the North Carolina clinical isolate at all markers tested. Closely related isolates that differ at only one or a few noncoding markers are present in the Australian environment. Taken together, these findings support a model in which C. gattii VGI was transferred from Australia to California, possibly though an association with its common host plant E. camaldulensis, and the patient was exposed in San Francisco and returned to present with disease in North Carolina.In 2007, the first confirmed case of  Significant medical advances have been achieved in the fields of antimicrobial agents and vaccine development, yet both newly emerging and re-emerging infectious diseases in humans, livestock, and plants remain serious global health and economic burdens Cryptococcus species complex Cryptococcus gattii clinical and environmental isolates.An essential component for tracking emergence and epidemiology of bacterial, parasitic, and fungal infections is molecular strain typing. In the genomic era, whole genome sequences have allowed comprehensive typing though sequence-based methods, including multilocus sequence and variable number of tandem repeat (VNTR) typing approaches C. gattii is a basidiomycetous yeast closely related to other members of the Cryptococcus pathogenic species complex, including C. neoformans var. grubii and var. neoformansC. gattii has often been associated with tropical and subtropical climates including Australia and South America C. gattii can be subdivided into serotypes B and C based on unique capsular antigenic determinants Cryptococcus species complex and usually results in pulmonary infection/pneumonia, central nervous system (CNS) dissemination, and in some cases cryptococcoma formation C. neoformans and C. gattii share some common virulence attributes but are also distinct. C. neoformans has a global distribution and predominantly infects immunosuppressed hosts, while C. gattii is more geographically restricted and commonly infects immunocompetent individuals Cryptococcosis is the disease caused by the pathogenic C. gattii research has been on molecular type VGII due to the high percentage (∼95%) of clinical cases caused by this molecular type in otherwise healthy individuals, while only ∼5% of cases result from VGI infection C. gattii molecular type VGI, both in California. One case occurred in a male Atlantic bottlenose dolphin (Tursiops truncatus) in San Diego; the other was in a liver transplant recipient in San Francisco Eucalyptus trees imported from Australia to California, particularly due to the strong environmental association between the VGI molecular type and Eucalyptus trees In the United States and Canada, a major focus of C. gattii has never been isolated from environmental sources in the Eastern United States C. gattii in the Southeastern United States, which occurred in an immunocompetent individual residing in central North Carolina. We show that this case, which resulted in large cryptococcomal granulomas in the leg and brain, resulted from infection with a C. gattii VGI type isolate that is distinct from both the common VGII and rarer VGI isolates associated with the Vancouver Island/Pacific NW C. gattii outbreak. The molecular genotype of the isolates from both leg and brain biopsies show that the molecular profile based on multilocus sequence (MLS) analysis of both coding (MLST) and more variable noncoding (VNTR) markers is shared with only two clinical isolates  out of 85 VGI strains typed C. gattii exposure and disease, and leads to acquisition in one region with presentation in a distant location C. gattii in temperate climates in the Western United States, and put forth a model in which an emergence from Australia to California has resulted in travel-associated disease presentation in the Southeastern United States.C. neoformans or C. gattii. Isolates were then examined for resistance to canavanine and utilization of glycine on L-canavanine, glycine, 2-bromothymol blue, (CGB) agar. Growth on CGB agar indicates that isolates are canavanine resistant, and able to use glycine as a sole carbon source, triggering a bromothymol blue color reaction indicative of C. gattii, whereas C. neoformans is sensitive to canavanine, and cannot use glycine as a sole carbon source, resulting in no growth or coloration on this selective indicator medium. Capsule identification was conducted though India ink analysis and microscopy after growth on Dulbecco's Modified Eagle's Medium (DMEM) for 72 hours at 37°C.Melanin production was assayed by growth and the production of dark pigmentation on Staib niger seed agar medium, and urease activity was detected by growth and alkaline pH change on Christensen's Agar. These tests established that isolates were either http://www.medfac.usyd.edu.au/people/academics/profiles/dcarter.php) For multilocus sequence typing analysis (MLST), each isolate was analyzed with a minimum of eight and in some cases eleven unlinked loci Mating analysis was conducted on Murashige and Skoog (MS) media, which contains myo-inositol that stimulates mating Galleria mellonella (greater wax moth) were conducted with a method similar to previous studies 5 cells, and larvae were maintained in a 37°C controlled environment. For each replicate virulence assay, 12–19 larvae were infected for each strain analyzed. Animals were monitored at 24-hour intervals, and mortality was defined as the cessation of movement upon probing and development of a distinctive dark colorization.Infection assays in the heterologous host The patient is a 46-year-old male with an unremarkable past medical history who noticed a hard mass on his medial right thigh. The mass enlarged over the course of three weeks, but was not painful, and the patient had no other symptoms. His primary care physician evaluated the mass with a magnetic resonance imaging (MRI) scan, which showed a 5×4×4 centimeter mass in the inner mid right thigh involving the adductor magnus muscle. The mass had mild heterogeneous enhancement on T2 imaging  and someCryptococcus. Further evaluation demonstrated a serum cryptococcal antigen of 1∶32, negative HIV serologies, and a normal CD4 lymphocyte count.The chest CT scan revealed a spiculated 1.6×2 centimeter left lower lobe lung mass with multiple, bilateral sub-centimeter pulmonary nodules . The patCryptococcus.No other symptoms were revealed by a detailed medical history, but the patient was regularly exposed to birds in the workplace. However, extensive local sampling of the workplace environment and birdcages failed to reveal a local source of infection. The patient was placed on 400 mg fluconazole a day, and did well until three months later when he presented with a tonic-clonic seizure. An MRI scan showed a large, enhancing mass in the left parietal lobe and a similar appearing mass in the right cerebellar hemisphere . A biopsCryptococcus gattii, and serotyping with commercial monoclonal antibodies  revealed the isolate to be serotype B, consistent with C. gattii . The patient had traveled to San Francisco five months prior to his original presentation. He had never been to the Pacific Northwest, Australia, South America or other C. gattii endemic regions and his only foreign travel had been to Western Europe.After diagnosis, the patient was initially treated with amphotericin B and flucytosine, but due to renal toxicity/nephropathy was changed to oral high-dose fluconazole (800 mg daily). The large size of the mass lesions despite antifungal therapy prompted consideration that the infection might be due to Sixteen months after the seizure he was completely asymptomatic on fluconazole 400 mg a day, and showed a significant decrease in the cerebral cryptococcomas upon reevaluation with magnetic resonance imaging . The patC. gattii was completed using several phenotypic and molecular tests. Isolates were confirmed to be C. gattii based on melanin production on Staib niger seed agar, production of urease on Christensen's agar, and resistance to canavanine with glycine utilization on CGB agar . Differences in mating ability were also observed in other similar or identical genotype isolates (B4496 and E296 (high fertility) vs. PAT12ISO1 and E310 (low fertility))  and brain (EJB2-B) isolates were CGB agar . Capsule species . Isolateh EJB1-L , with sitility)) . There wtility)) .C. gattii on Vancouver Island that has now expanded to mainland British Columbia, Washington, and Oregon. Genomic DNA of each isolate was analyzed by MLST at a minimum of eight loci . MLST analysis also showed that all MLST alleles were identical between both the leg biopsy (EJB1-L) and brain biopsy (EJB2-B) isolates, and each typed as C. gattii molecular type VGI , they were identical to only a unique subset in the collection. To examine if these isolates were at all related to the ongoing Eucalyptus tree hollows  analysis with the Tandem Repeat Finder (TRF) software, strains harboring identical MLST profiles to the isolates from the patient in this study were analyzed . Analysie A4MR64 . Overalle A4MR64 . These dGalleria mellonella (greater wax moth). The use of non-mammalian hosts to study fungal virulence and host defense has been increasingly applied and is now well established 5 cells, using 12 to 19 larvae per strain per replicate at 37°C. The selected isolates analyzed were all significantly pathogenic compared to PBS control infections (p<0.005)  . There wC. gattii case reported in the Eastern region of the United States. The most parsimonious explanation is that an otherwise healthy patient was exposed in California, resulting in disease presentation in North Carolina approximately 4 to 6 months after travel to an endemic region. This case highlights an overall increase in C. gattii infections in apparently immunocompetent individuals in temperate climates. The patient had no recent travel history to the Pacific Northwest or to other C. gattii endemic regions, and based on molecular typing the infectious isolate is not related to any of the VGII or VGI isolates causing the ongoing Vancouver Island outbreak, and its emergence into mainland British Columbia, Washington, and Oregon C. gattii molecular type VGI has never been reported from the environment in California, but it seems likely to be present there based on successful environmental isolation of C. gattii molecular types VGII and VGIII in regions of California, including San Francisco and San Diego, and the abundant presence of the host Eucalyptus trees throughout the state Our findings document the first C. gattii genome. The VNTR analysis showed that environmental isolates all differ at one or two rapidly evolving genomic regions, but clinical and environmental isolates that are identical at eight MLST loci are quite closely related. All environmental isolates from Australia were collected from a single location, and slight diversity in geographic regions is expected Eucalyptus trees in San Francisco. No isolates of C. neoformans or C. gattii were obtained. Therefore, expanded sampling will be necessary to definitively elucidate the environmental source of the clinical isolates. We therefore posit that the patient in this report traveled to the San Francisco region, and was there exposed to infectious spores or desiccated yeasts, resulting in colonization of the lungs, and dissemination, disease progression, and presentation with systemic infection of the leg and brain  near San Diego, and C. gattii VGIII has been isolated from the natural environment in the San Diego, California area Eucalyptus and other tree species.While many of the studies related to Ustilago scitaminea, was dispersed from Asia to America and Africa due to its association with infected plant material C. gattii has been isolated from E. camaldulensis trees in San Francisco, a common host tree species, and C. gattii was recently found to complete its natural sexual cycle during co-culture with this plant under laboratory conditions E. camaldulensis have also shown that both same-sex and opposite-sex mating is likely occurring in this environmental niche Hypotheses regarding the spread of global fungal pathogens from common associations with plants have been previously reported. It was recently shown that a basidiomycete, the sugarcane fungal pathogen C. gattii it has been postulated that same-sex mating among closely related isolates could have both contributed to the formation of the hyper-virulent VGIIa/major genotype responsible for the Vancouver Island outbreak and to the ongoing production of infectious propagules Toxoplasma gondii, where limited mating among clonal lineages is hypothesized to have resulted in increased virulence, and may also have enabled this pathogenic microbe to be orally transmitted from animals to humans C. gattii case may be genetic, or epigenetic, as all other analysis indicates that these isolates are from a single rather than mixed infection. Although the exact roles of C. gattii sexual reproduction as it relates to virulence remain to be elucidated, the ability of virulent isolates to retain mating ability suggest this process may play a significant role in virulence and possibly also in the production of infectious propagules.Sexual reproduction among closely related genotypes has the potential to increase pathogenicity of fungi and parasites C. gattii continues to emerge in the United States and elsewhere it is clear that the standardization of clinical testing to identify species (C. neoformans/C. gattii) and cryptic species (VGI/VGII/VGIII/VGIV) is essential. As illustrated by this case, C. gattii infections may be particularly complicated to treat due to their predilection to form cryptococcomas, especially in the brain, which are difficult to manage and risk to surgical resection is considerable. Although clinical laboratory testing at Duke University confirmed that the leg and brain biopsy isolates were both fluconazole sensitive, because of disease progression and the occurrence of cryptococcomas, in which drug access is limited, clinical management was difficult. Although the overall incidence of C. gattii disease in the United States remains low, an increasing number of cases in California, Oregon, Washington, and now North Carolina among immunocompetent and immunocompromised individuals raise the possibility that these represent the onset of an emergence in the temperate climate of the Western United States.As Figure S1C. gattii VGI isolates exhibit mating differences. All mating cultures were incubated at room temperature in the dark for 14 days in dry conditions using the mating type a tester isolate B4546 as a partner on Mirashige and Skoog Media. The clinical isolate B4496 (top left) and the environmental isolate E296 (top right) are fertile. There is a marked delay and paucity, or no hyphal growth in matings with the clinical isolate PAT12ISO1 (bottom left), and the environmental isolate E310 (bottom right).Clinical and Australian environmental (51.87 MB TIF)Click here for additional data file.Figure S2C. gattii isolates. In each panels A and B control isolates are as follows: top left, C. albicans control  Isolate; bottom left, C. neoformans control ; bottom right, C. gattii control . In panel A, the experimental isolate (upper right) is EJB1-L. In panel B, the experimental isolate is EJB2-B. Each isolate of C. gattii produced melanin on Staib niger seed agar (brown pigmentation), produced urease on Christensen's agar (pink coloration), and was resistant to canavanine and utilized glycine on CGB agar (growth and blue coloration). C) Isolates EJB1-L (left panel), and EJB2-B (right panel) each show similar capsule sizes when exposed to India ink following growth on DMEM media for 48 hours at 37Â°C.Phenotypic characterization of (5.82 MB TIF)Click here for additional data file.Figure S3C. gattii. The VNTR3 marker discriminates two of the ST12 isolates (WM276 and A4MR64) from the other 16 VGI isolates. VNTR7 shows 100% sequence identity between all 18 isolates examined (red circle indicates SNP).Analysis of VNTR markers. DNA sequence alignment of VNTR markers among 18 VGI isolates of (38.91 MB TIF)Click here for additional data file.Table S1(0.05 MB DOC)Click here for additional data file.Table S2(0.06 MB DOC)Click here for additional data file.