Beet black scorch virus (BBSV), a member of the genus Necrovirus, can be phosphorylated at threonine-41 (T41) by cAMP-dependent protein kinase (PKA)-like kinase in vivo and in vitro. Mutant viruses containing a T41A non-phosphorylatable alanine substitution, and a T41E glutamic acid substitution to mimic threonine phosphorylation were able to replicate but were unable to move systemically in Nicotiana benthamiana. Interestingly, the T41A and T41E mutants generated unstable 17 nm virus-like particles that failed to package viral genomic (g) RNA, compared with wild-type BBSV with 30 nm virions during viral infection in N. benthamiana. Further analyses showed that the T41 mutations had little effect on the gRNA-binding activity of the CP. Therefore, we propose a model whereby CP phosphorylation plays an essential role in long-distance movement of BBSV that involves formation of stable virions.Plant virus coat proteins (CPs) play a fundamental role in protection of genomic RNAs, virion assembly, and viral movement. Although phosphorylation of several CPs during virus infection have been reported, little information is available about CP phosphorylation of the spherical RNA plant viruses. Here, we demonstrate that the CP of  Beet black scorch virus (BBSV) belongs to the genus Necrovirus in the family Tombusviridae, and was first reported in the late 1980s in China2345Olpidium brassicaeChenopodium amaranticolor, Tetragonia expanse, and Nicotiana benthamiana, can be mechanically infected by BBSV79N. benthamiana, but is dispensable for cell-to-cell movement in C. amaranticolor1112BBSV is a small, approximately 30 nm icosahedral virus, consisting of a 3644 nucleotide (nt) plus-sense, single-stranded (ss) genomic (g) RNA lacking a 5’-cap structure and a 3’-poly(A) tail that encodes six functional proteins. The gRNA serves as the mRNA for the 5’ proximal ORF, which encodes a 23 kDa protein (p23), and an 82 kDa readthrough protein (p82). P23 and p82 interact to form the replicase complex, which is required for gRNA replication and synthesis of two subgenomic RNAs (sgRNAs). Each of the three proteins  translated from sgRNA1 are required for viral cell-to-cell movement and the 24.5 kDa coat protein (CP), which is translated from sgRNA2, is required for systemic infection of Tombusviridae are composed of an RNA binding domain (R-domain), a shell domain (S-domain), a hinge domain (H-domain) and a protruding domain (P-domain)14161718204KRNKGGKKSR13) is involved in CP nuclear localization and gRNA binding, and that lysine and arginine at positions 4 and 5 are key amino acid requirements for gRNA binding16necrovirus systemic movement16The CPs of the Cauliflower mosaic virus (CaMV), Potato virus A (PVA), Plum pox virus (PPV), Bamboo mosaic virus (BaMV), Potato virus X (PVX), Groundnut bud necrosis virus (GBNV), and Brome mosaic virus (BMV), are regulated by phosphorylation2425262728293031in vitro, but when the N-terminal CP residues are phosphorylated by protein kinase C (PKC) or by a casein kinase mixture (CKI and CKII), the RNA is converted to a translatable formet al. (2014) and Ivanov et al. (2001 and 2003) hypothesize that CP phosphorylation promotes virion or ribonucleoprotein (RNP) disassembly to release gRNA for replication or translation27282728A number of studies have shown that CP stability and functions of diverse plant viruses, such as et al. (2007) have reported that BBSV virions can be phosphorylatedN. benthamiana. We have also conducted comprehensive biochemical approaches, including mass spectrometry and phosphorylation assays that demonstrate that the CP is phosphorylated at threonine 41 (T41) in vivo and in vitro by PKA-like kinases. Our results also indicate that CP T41 mutants form incompletely-assembled virus-like particles (VLPs) that are unable to package gRNA and are less stable than wild-type (wt) virions, and that these mutants compromised in systemic movement during infection of N. benthamiana. In summary, our results reveal a correlation between CP phosphorylation, virion assembly and long-distance movement, which has been largely unexplored for spherical RNA plant viruses.Although several models have suggested that CP phosphorylation modulates plant virus or RNP disassembly after cell entry, regulation of virion assembly by phosphorylation remains elusive. Weiland N. benthamiana leaf tissues was separated by SDS-PAGE  and the online servers KinasePhoshttp://KinasePhos2.mbc.nctu.edu.tw/), the 38IRST41 region in the N-terminus of BBSV CP is similar to the R-R-X-[S/T] motif, which is a conserved substrate recognition site of cAMP-dependent protein kinase (PKA). To determine whether BBSV CP is phosphorylated by PKA, total protein was extracted from BBSV-infected-N. benthamiana leaf tissues at 14 dpi, and subjected to Western blot with a phospho-(Ser/Thr) PKA substrate antibody (α-pS/T) and a CP-specific antibody (α-CP). A weak phosphorylated band corresponding to the CP position was present in the crude extracts from BBSV-infected plants  fused with an N-terminal hexahistidine tag was expressed in E. coli and affinity purified over Ni-NTA agarose . As kinase specificity controls, we also used the recombinant N. benthamiana CKII (NbCKII) purified as previously describedin vitro.To provide additional evidence to confirm that CP is a substrate for PKA, and that T41 is the phosphorylation target site, we performed a series of  agarose  and usedin vitro PKA phosphorylation of rCPwt. Three independent experiments consistently revealed that rCPT41A, rCPR39A, and rCPT41E displayed approximately 50, 67 and 52% phosphorylation efficiency compared with CPwt , total RNA and protein were extracted from the infected protoplasts and subjected to Northern and Western blot analyses. The accumulation levels of the progeny RNAs   , A, wheriameters , B and E n = 50, , C and Dwt and mutant viruses, particles obtained from the partial and refined purifications were subjected to Western blot analyses using CP-specific antibodies. The results verified that BBSVwt inoculations contained high amounts of CP, whereas the partially purified T41 and R39 mutant BBSV preparations contained lower amounts of CP  was less than half the amount recovered from BBSVwt samples (1.35 mg per 10 g leaf tissue) after the refined purification  and Tobacco mosaic virus (TMV)414243Alfalfa mosaic virus (AMV), and Cucumber mosaic virus (CMV), which belong to the family Bromoviridae45Potato virus X (PVX), in the family AlphaflexiviridaePotato mop top virus (PMTV) and Poa semilatent virus (PSLV) in the family Virgaviridae484450Protein phosphorylation, which regulates signal transduction, biological activity, molecular interaction and subcellular localizationIn addition to the nonstructural MPs, viral coat proteins (CPs) involved in virion or RNP assembly can also affect virus movement through phosphorylation with host serine (S)/threonine (T) kinases. As examples, phosphorylation of CPs of helical PVA and PVX regulates disassembly of RNP or virion to permit replication and translation of viral genomic RNA262728N. benthamiana at four residues, of which only T41 plays a dominant role in viral long-distance movement   region4KRNKGGKKSR13) domainWe initially hypothesized that the failure to encapsidate RNA might be a consequence of RNA binding defects of the CP mutants. However, the negatively charged mutant (rCPT41E) , lane 3 Sesbania mosaic virus (SeMV), Tomato bush stunt virus (TBSV), CNV, and Hibiscus chlorotic ringspot virus (HCRSV) affect the size of the virus particles545556T = 3 virion assembly of CNV, and deletion of these important regions results in the formation of T = 1 virus-like particlesT = 1 and T = 3 virus particles reveal that T = 3 intact virion is composed of two types of CP dimers which are C-C and A-B subunit dimers. However, T = 1 virus-like particle only contains a single subunit dimer that is very similar to the A-B subunit dimers found in T = 3 particles. Further analyses demonstrate that the S domain of the C-C dimer have a flat conformation with an ordered N-terminal ARM and β-annulus at the internal surface of particle for RNA packaging, whereas the S domain of A-B dimer is more angled and provides limited space at the N-terminus, which tends to be disordered and limits RNA binding. Although RNA binding plays an important role in regulating the A-B and C-C dimers conformations of CNV, TBSV, and hepatitis E virus (HEV) particles5256T = 3 particle assembly. Support for this hypothesis comes from several recent findings with hepatitis B virus (HBV)59605960Several studies have provided evidence that the N-termini of the CPs of some viruses, such as T = 1 virus-like particle assembled from rCP purified from E. coli (unpublished data). Consistent with the CP of another necrovirus TNVT = 1 virus particles in Tombusviridae5256T = 1 particle are flexible and disordered, so can not be visualized. However, the T41 residue is visible and is located at a loop region in the H domain that connects the S domain and the β-annulus. We speculate that in the host environment, phosphorylation at T41 might provide a molecular switch that participates in conformation changes of the N-terminus from a disordered state (A-B dimer) to an ordered state (C-C dimer), and that these changes facilitate RNA binding by the N-termini of the C-C dimers and permit final T = 3 virion morphogenesis via integration with A-B dimers. It is easy to image that if all of the dimers have the C-C conformation, the redundant ordered N-termini would induce collision in the nascent particles and might interfere with RNA-binding and assembly, as well as steps involved T = 3 virion morphogenesis. This hypothesis is supported by the results that both the T41A and T41E mutants formed incompletely-assembled particles that resemble T = 1 virions in inoculated leaves and these enriched particles did not contain gRNA. Hence, it is tempting to speculate that dynamic phosphorylation and de-phosphorylation events are required for morphogenesis of T = 3 virions.With BBSV, we have carried out an analysis of the crystal structure of an unphosphorylated in vitro particle assembly assay in preliminary attempts to verify our hypotheseswt  of IP buffer . The homogenate was centrifuged at 4 °C for 30 min at 10,000 g, and the supernatant was subjected to two additional centrifugation steps. The final supernatant was incubated with CP-specific antibody (1:2000) at 4 °C overnight, and further enriched by addition of protein G agarose (Millipore) at 4 °C for 4 hours. The immunoprecipiated CP was used for liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunoblot analyses.Two grams of inoculated in vivo phosphorylation of CP.For LC-MS/MS identification, immunoprecipitated CP extracted from BBSV inoculated leaf tissues was separated by 12.5% SDS-PAGE and stained with Coomassie brilliant blue R250 (Sigma). After in-gel trypsin digestion and enrichment, the phospho-peptides were separated by nanoscale C18 reverse phase liquid chromatography (Waters), and then electro-sprayed into a Q-Exactive mass spectrometer (Thermo) at the Mass Spectrometry Facility of China Agricultural University. For immunoblot analyses, immunoprecipitated CP was separated by SDS-PAGE and transferred to nitrocellulose membranes (Amersham). A phospho-(Ser/Thr) PKA substrate antibody  was used to detect in vitro phosphorylation assays, wt and mutant fragments were amplified with appropriate primers listed in Several infectious clones used in this study were derived from the infectious cDNA clone of BBSV Ningxia isolate (pUBF52)N. benthamiana plants was performed according to a previous studyin vitro transcripts were adjusted to equal amounts by UV spectrophotometry to ensure uniformity of the experiments. At 12 and 24 dpi, total RNA and protein was extracted from infected leaves and prepared for further analyses.Mechanical inoculation of various mutants onto 2-week-old N. benthamiana6 isolated protoplasts were transfected with 20 μg of purified transcripts according to a PEG-calcium-mediated transfection methodFor protoplast transfections, mesophyll protoplasts were prepared from the second and third true leaves of 3 to 4-week-old Northern blots were conducted according to the manufacturer instructions  and DIG-labeled probe was prepared from a DNA fragment corresponding to the 3¢ UTR of the BBSV genomeE. coli strain BL21 (DE3) pLysS cells (Novagen) were transformed with pET30a-CPwt or phosphorylation mutant constructs by heat shock, and cultured at 37 °C until the OD600 reached 0.4. Then, 0.2 mM IPTG (Sigma) was used to induce protein over-expression at 18 °C for another 18 hours. Bacteria cells were then harvested, resuspended in T buffer  containing 0.1% Triton X-100 and disrupted by ultrasonication. After centrifugation for 20 min at 20,000 g, the supernatant was collected to purify the N-terminal hexahistidine tagged CP by Ni-NTA agarose affinity (Bio-Rad). After washing with increasing step-wise concentrations of imidazole, proteins eluting in T buffer containing 400 mM imidazole were concentrated with 30 kDa Amicon-Ultra-15 filters (Millipore).E. coli strain BL21 (DE3) pLysS cells. After culturing and induction, proteins were purified from the cells by affinity chromatography on glutathione affinity sepharose columns . GST-tagged proteins were eluted by T buffer containing 2 mM DTT and 60 mM L-Glutathione and concentrated with Amicon-Ultra-15 filters (30 kDa). Purified protein concentrations were determined by measuring the OD280 with a NanoDrop ND1000 spectrophotometer, and proteins were assessed by SDS-PAGE.Three pGEX-KG32P-ATP (Perkin Elmer) and 200 μM unlabeled ATP. To inhibit PKA phosphorylation, 1 μM of PKA specific inhibitor H-89  was added. After incubation at 30 °C for 30 min, the reactions were quenched by addition of 5X SDS sample buffer  and boiled for 5 min. Then samples were separated by 12.5% SDS-PAGE followed by autoradiography.Five μg of rCP or kinase specific substrates and 0.5 μg of kinase [purified NbCKII or commercial mammal kinases PKA or CaMKII (New England Biolabs)], were incubated in 10 μl reactions containing 1X kinase reaction buffer (New England Biolabs), 3 μCi γ-wt and mutant viral particles for Transmission electron microscopy (TEM), Western blots and agarose gel electrophoresis analyses.Three procedures were used to obtain BBSVN. benthamiana leaves inoculated with BBSVwt or mutant viruses at 14 dpi by grinding in 200 μl of 20 mM PBS (pH 7.0) followed by centrifugation at 4 °C for 30 min at 6,000 g. The supernatants were collected and used for various analyses described in the Results.To rapidly obtain viral particles, virus particles in clarified leaf sap were extracted from one gram of g. Then the supernatant was adjusted to 8% PEG6000 and gently shaken for 30 min at 4 °C. The homogenate was centrifuged at 4 °C for 30 min at 13,000 g, and the pellet containing viral particles were resuspended in 50 μl ddH2O. For subsequent analyses to visualize virus particles, 5 μl of partial purified particles were subjected to Western blots with a CP-specific antibody. To detect viral gRNA, 2 μg particles were separated by 1% agarose gel in electrophoresis buffer  and stained with EtBr.A previously described partial purification procedure was used to concentrate virus particlesg to remove the insoluble material. The supernatant was collected, mixed with 15% chloroform, and centrifuged at 4 °C for 30 min at 13,000 g. Then the supernatant was collected, mixed with 6% PEG6000 and 3% NaCl, stirred for 2 hours and stored overnight at 4 °C. For virus precipitation, the homogenate was centrifuged for 30 min at 13,000 g and the pellet was thoroughly resuspended in 0.2 M sodium phosphate buffer (pH 7.0) containing 0.1% TritonX-100. The clarified supernatant was centrifuged at 4 °C for 90 min at 32,000 g and the final virion pellet was resuspended in 100 μl ddH2O.To obtain more entensively purified viral particles, a conventional and refined purification procedure was performed as described earlierTEM was carried out according to methods described previouslyFive μg of purified rCPs or BSA, which served as negative control, were separated by 12.5% SDS-PAGE and transferred to nitrocellulose membrane. The proteins were renatured by incubating the membrane overnight at 4 °C in 20 ml of renaturation buffer . Then, a digoxigenin (DIG)-labeled North-western blot procedure was performed as previously describedThe stability of virus particles to endogenous RNases in leaf sap was determined by an RNase-sensitivity assay as previously reported16How to cite this article: Zhao, X. et al. Phosphorylation of Beet black scorch virus coat protein by PKA is required for assembly and stability of virus particles. Sci. Rep.5, 11585; doi: 10.1038/srep11585 (2015).


Pulmonary arterial hypertension (PAH) is a rare condition that can ultimately lead to right heart failure and death. In this study we estimated the health care costs and resource utilization associated with PAH in a large US managed care health plan.Subjects with claims-based evidence of PAH from 1/1/2004 to 6/30/2010 (identification period) were selected. To be included in the final PAH study sample, subjects were required to have ≥2 claims with a primary PH diagnosis; ≥2 claims with a PAH related-diagnosis ; and ≥1 claim with evidence of a PAH-indicated medication. The earliest date of a claim with evidence of PAH-indicated medication during the identification period was set as the index date. Health care costs and resource utilization were compared between an annualized baseline period and a 12 month follow-up period.504 PAH subjects were selected for the final study cohort. Estimated average total health care costs were approximately 16% lower in the follow-up period compared to the baseline period , but substantively high in each period relative to costs reported for other chronic diseases. Pharmacy costs were significantly higher in the follow-up period vs. the baseline period,  but medical costs were significantly lower in the follow-up vs. baseline . These costs were mirrored in health-care resource utilization estimates. The average counts of ambulatory visits and inpatient stays were lower in the follow-up vs. the baseline (both p < 0.001). Results varied in exploratory analyses when less restrictive subject identification algorithms were used.Subjects with evidence of PAH had substantively high health care costs. Medical costs appeared to decrease following PAH medication use, but with a concomitant increase in pharmacy costs.The online version of this article (doi:10.1186/s12913-014-0676-0) contains supplementary material, which is available to authorized users. Pulmonary arterial hypertension (PAH) is one of five groups of pulmonary hypertension (PH) classified by the 2009 European Society of Cardiology and European Respiratory Society guidelines . PAH is Symptoms of PAH include dyspnea on exertion, fatigue, chest pain, and fainting . AlthougPAH is a rare but costly disease. The prevalence of PAH in the US has been estimated at 109 per million among individuals under 65, and 451 per million among individuals 65 and over . LimitedThe objective of this study was to explore the health care costs and resource utilization associated with PAH in a large, national US managed care health plan using contemporary real-world data with a substantial time frame . In this study, we required that PAH subjects have claims-based evidence of PAH-indicated medications, PH diagnoses, and PAH-related diagnoses in order to make subject selection as specific as possible. By setting the index date as the earliest date of evidence for a PAH-indicated medication (and then performing comparisons between the baseline and follow-up periods), we hoped to gain additional insight into how PAH medication use may influence total health care costs among PAH patients. Additionally, in this study we examined both health plan and patient paid costs, to better understand the economic burden on payers and patients, respectively.This study used administrative claims of commercial and Medicare Advantage enrollees with Part D prescription drug coverage from a large national managed care organization. During the period of the study, the database contained approximately 41.7 million members with continuous medical and pharmacy benefits . The geographic makeup of individuals covered by the health plan was diverse, with services rendered in all major regions of the United States. No identifiable protected health information was extracted or accessed during the course of the study. Pursuant to the Health Insurance Portability and Accountability Act , the useSubjects were initially identified from the database who had at least 1 claim during the period from 01 January 2004 to 30 June 2010 (defined as the identification period) for either a PAH-indicated study medication ; an ICD-9-CM diagnosis code in any position for primary pulmonary hypertension (416.0) or other chronic pulmonary heart diseases (416.8); or a diagnosis code in any position for a PAH-associated condition  were captured from enrollment data based on the claim used to identify the index date. Comorbidities were identified based on the presence of diagnosis and/or service codes on medical claims during the baseline and follow-up periods, using AHRQ classifications . Each AHHealth care resource utilization and costs were captured in the baseline and follow-up periods. The baseline continuous measures were annualized, to provide comparable metrics to the follow-up period. Health care costs were computed as the combined health plan and patient paid amounts; in addition, payments from Medicare (or other payers) were estimated based on coordination of benefits information obtained by the health plan, and these estimates were incorporated into the total paid amount. The following cost variables were calculated: total costs, pharmacy costs, and medical costs. Medical costs were further broken down into ambulatory costs, emergency services costs, inpatient costs, and other medical costs. Costs were presented as total , health plan paid and patient paid. Patient paid costs were comprised of the fixed amount that a member paid for a specific service as defined in their benefit plan, the amount that was applied towards the member’s deductible, and any additional dollars paid by a member at the pharmacy . Procedures, drug dispensings, and doctor visits that occurred during an inpatient admission were included in the costs for that admission (the same applied to costs incurred during an emergency department or ambulatory visit). Costs were adjusted to 2011 US$ using the annual medical care component of the Consumer Price Index. Health care resource utilization was operationalized as the numbers of the following: ambulatory visits, emergency department visits, and inpatient admissions. Comparisons of continuous variables were made using signed rank test. All software analyses were conducted in SAS 9.2.Medications that could be used to treat PAH were identified from pharmacy claims  and medical claims . Medication use was captured separately in both the baseline and follow-up periods.Medication treatment patterns were captured across follow-up period pharmacy claims, and only for those subjects who had evidence of a single PAH-indicated study medication in the first 30 days of the follow-up period. Discontinuation of index therapy was defined as the earliest gap (if multiple exist) in therapy of >60 days between the “run out” of days supplied from the previous prescription  to the fill date of the next prescription or the end of follow-up. If a subject had a 60-day gap in index therapy, the discontinuation date was noted as the run out date. Subjects whose last run out date in a series of continuous use was within 60 days of the end of the study period were considered continuous users of therapy with no evidence of discontinuation. Switches from index PAH treatment at the time of discontinuation were assessed. A switch in therapy was defined as a filled prescription for a new PAH medication that occurred within 60 days prior to or following the date of index PAH medication discontinuation. PAH treatment add-ons were identified for patients adding new PAH therapies to their index therapy while continuing use of index therapy. Add-on was defined as the addition of a new PAH medication other than the index medication at least 60 days prior to the date of discontinuation or 60 days prior to the end of the study period if there was no evidence of discontinuation. Subjects who did not discontinue, switch, or add-on to their index therapy were classified as having maintenance of index therapy. Finally, the medication possession ratio (MPR) was captured and reported separately for each PAH index therapy. MPR was defined as the proportion of follow-up days with the index therapy available based on fill dates and day supply given on the pharmacy claims. MPR was calculated as the number of non-inpatient days where the medication of interest was available (numerator) divided by the total number of non-inpatient days (denominator). MPR was captured until discontinuation for subjects who discontinued; for those who didn’t discontinue, MPR was captured during the entire 12 month follow-up period.In total, there were 41,666,589 enrollees in the health plan during the study period. Of these, 504 PAH subjects who met continuous enrollment criteria and had claims-based evidence of PAH-indicated medications, PH diagnoses, and PAH-related diagnoses were selected for the final study sample , the average of the total annual health care costs were higher in the baseline period  vs. the follow-up period  (p < 0.001)  vPatterns of health care resource utilization between the baseline period and follow-up period were consistent with the patterns observed for health care costs. Average annual counts of inpatient stays , ambulatory visits , and ER visits  were higher in the baseline period vs. the follow-up period , and 1,719 subjects were selected who met continuous enrollment criteria and had claims-based evidence of a PAH-indicated medication (PAH medication group) Figure . Trends In this study we investigated the health care costs and resource use associated with PAH using a large, national US managed care health plan database. The index date in this study was set as the earliest date of evidence for a PAH-indicated medication, and health care costs were compared before and after the index date. Cost estimates for PAH in the final study sample were substantively high, and were calculated to be $116,681 in the baseline period and $98,243 in the follow-up period. We found that although pharmacy costs increased significantly from the baseline to follow-up period, medical costs decreased significantly from the baseline to follow-up period. The decrease in medical costs appeared to be driven primarily by inpatient costs, which decreased by more than two-thirds from baseline to follow-up. Importantly, the index date for the final PAH study cohort was set as the earliest date of a claim with evidence of PAH-indicated medication during the identification period. A possible interpretation of these trends is that although medication costs for PAH subjects are high, pharmaceutical management with a PAH-indicated medication is associated with a reduction in medical costs among subjects with disease. However, due to the observational study design, it was not possible to establish a direct causal relationship between PAH medication use and pre-index/post index changes, such as a reduction in medical costs.The differences observed for health care costs were reflected in health-care resource utilization estimates, as average counts of ambulatory visits, inpatient stays, and ER visits were lower in the follow-up period vs. the baseline period. Sildenafil (Revatio®) and bosentan were the most commonly prescribed PAH medications during the follow-up period among subjects in the final PAH study sample. Although evidence of PAH-indicated medication use appeared to offer benefit in this sample , only about half of PAH subjects maintained their index medication regimen. Discontinuation of the index regimen was observed for about 38% of subjects, and about 11% of subjects had evidence of augmentation. Future research could investigate PAH costs and treatment patterns among specific subgroups of patients, such as a patient population stratified by age or a population stratified by gender.Several prior studies have investigated the economic burden of PAH in the US. The total health care costs among PAH subjects reported in this study are higher than those reported by Said et al. In a retrospective study using administrative claims data, Said et al. found that the total health care costs of PAH patients identified from 2004–2009 were $4,021 per-patient-per-month (PPPM) . By compHowever, the costs reported in the present study are similar to costs for PAH determined by Copher et al. In a retrospective claims-based analysis, Copher et al. reported that the total health care costs of PAH patients identified from 2004–2008 were $9,295 per-patient-per-month , which iCertain data limitations that are inherent to claims-based analyses should be considered when interpreting the results of this study. Claims data are collected for the purpose of payment, not research, and therefore capture an individual’s medical history to a limited degree. Claims data do not contain detailed information on symptoms, no self-reported burden of illness measures were obtained in this study, and quality of life measures were not available. Importantly, existing ICD-9-CM codes may not be specific enough to distinguish PAH from other related conditions, and it is therefore difficult to locate subjects with PAH in administrative claims. Based on exploratory analyses, we found that requiring evidence of a PAH-indicated medication appeared to be a more specific inclusion criterion than requiring PH/PAH-associated diagnosis codes. Average total health care costs among the PAH medication group and PAH diagnosis group were both lower than was observed among subjects in the final study cohort. This suggests that some of the subjects selected in these exploratory analyses may have had earlier/pre-treatment stages of PAH, and/or perhaps suggests that the selection criteria for these analyses were not specific enough, in comparison to the final algorithm. It is important to keep in mind that the PAH medication group and the PAH diagnosis group were performed as exploratory analyses; they each incorporated different subsets of selection criteria used for the final study cohort, but were not intended to be as specific as the final study cohort. For inclusion in the final study cohort, we used a claims-based algorithm requiring evidence of a PAH-indicated medication, PH diagnosis codes, and PAH-associated condition diagnosis codes, in order to increase specificity of selection. However, further research, such as a medical chart review, would be needed to validate the claims-based patient identification algorithm used here.No comparisons were performed in this study using a control group that did not have PAH, and this is a further study limitation. As we required a period of 12 months of continuous enrollment following the index date for study inclusion, patients who died shortly following the index date would have been excluded from the study, and this may have impacted cost estimates. Also, measures from the 180 day baseline period were annualized, and this may have introduced some bias into the results. Cost and resource utilization estimates may also have been impacted by comorbid diseases. We observed high rates of some comorbid diseases in this population, such as systemic lupus erythematosus and respiratory infections, and this may have led to higher costs and utilization in some patients. Finally, the results observed in this study are most applicable to a managed care population diagnosed with and being treated for PAH, and may not be generalizable to other populations or to the US population in whole.Additional limitations regarding pharmacy claims should be considered. For example, it is not possible to determine whether subjects consumed their medication as prescribed. In addition, over-the-counter medications and medications provided as samples by physicians cannot be located in claims data. A small proportion of study subjects (6%) had evidence of PAH medication use prior to the index date, and this may have affected comparisons of costs between the baseline and follow-up period . We assumed that pharmacological treatment for PAH was a strong indication that physicians believed their patients to experience PAH (regardless of diagnosis coding), as the medications used are indicated only for the treatment of PAH . However, this assumption is most at risk in the case of tadalafil and sildenafil, which are branded as Adcirca® and Revatio® for treatment of PAH only, but which are also formulations of generics that are branded as Cialis® and Viagra®, respectively, which are not indicated for PAH. Likewise, another caveat is that some off label use of Cialis® and Viagra® may occur, and patients who used these medications to manage PAH were not included in this study. Pharmacy claims for the managed Medicare population also present certain limitations. Prior to 2006, managed Medicare enrollees had pharmacy covered through their health plan, one of the benefits of enrollment in certain managed Medicare plans. However, for a portion of these enrollees, these pharmacy benefits had caps on expenditures. Enrollees with substantial pharmacy expenditures may have exceeded their cap in certain years. For these members, additional filled prescriptions may not be observed in the claims data, and it would appear that these individuals ceased to fill prescriptions. This limitation may have impacted the findings we report for treatment patterns.The results from this study indicate that medical costs decreased by nearly half from the baseline to the follow-up period (following evidence of use of PAH medication). However, annual total follow-up costs among PAH patients reported here were still substantively high on a per subject basis. Further, this study found that about half of subjects with PAH discontinued, switched, or augmented their index PAH medication. Further research is needed to examine whether the treatment patterns revealed in this study represent clinically optimal or cost-effective care.


Guidelines for the treatment of psychoses recommend antipsychotic monotherapy. However, the rate of antipsychotic polytherapy has increased over the last decade, reaching up to 60% in some settings. Studies evaluating the costs and impact of antipsychotic polytherapy in the health system are scarce.To estimate the costs of antipsychotic polytherapy and its impact on public health costs in a sample of subjects with psychotic disorders living in residential facilities in the city of Sao Paulo, Brazil.A cross-sectional study that used a bottom-up approach for collecting costs data in a public health provider´s perspective. Subjects with psychosis living in 20 fully-staffed residential facilities in the city of Sao Paulo were assessed for clinical and psychosocial profile, severity of symptoms, quality of life, use of health services and pharmacological treatment. The impact of polytherapy on total direct costs was evaluated.147 subjects were included, 134 used antipsychotics regularly and 38% were in use of antipsychotic polytherapy. There were no significant differences in clinical and psychosocial characteristics between polytherapy and monotherapy groups. Four variables explained 30% of direct costs: the number of antipsychotics, location of the residential facility, time living in the facility and use of olanzapine. The costs of antipsychotics corresponded to 94.4% of the total psychotropic costs and to 49.5% of all health services use when excluding accommodation costs. Olanzapine costs corresponded to 51% of all psychotropic costs.Antipsychotic polytherapy is a huge economic burden to public health service, despite the lack of evidence supporting this practice. Great variations on antipsychotic costs explicit the need of establishing protocols for rational antipsychotic prescriptions and consequently optimising resource allocation. Cost-effectiveness studies are necessary to estimate the best value for money among antipsychotics, especially in low and middle income countries. In the last decade, antipsychotic polytherapy has been subject of controversy and criticism in the treatment of schizophrenia and related psychoses . DiffereIn terms of efficacy, there is no antipsychotic, whether typical or atypical, with a clear superiority over the others, with the exception of clozapine, which is more efficacious in cases of treatment-resistant schizophrenia ,13. AntiGiven the fact that, with the exception of clozapine, all antipsychotics do not differ substantially in terms of efficacy and virtually all guidelines recommend antipsychotic monotherapy for the treatment of psychoses, it is noteworthy that the use of antipsychotic polytherapy has increased –17.According to a study based on chart review, most polypharmacy prescriptions have justifiable clinical rationales, such as refractoriness, problems of adherence, poor control of symptoms and use of a second antipsychotic for other reasons . In otheWhether clinically justifiable or not, antipsychotic polypharmacy has not proven superior efficacy over monotherapy, and because it is so widely used, it is important to evaluate the economic impact of this practice.The costs and economic impact of antipsychotic polypharmacy are relevant for the budget in mental health services . AntipsyIn the State of Sao Paulo, Brazil, six atypical and eight typical antipsychotics (including two depot) are freely available in the public health system to all citizens. Despite some governmental initiatives to elaborate treatment guidelines, the antipsychotic costs were not considered in the recommendations and no treatment algorithm was effectively implemented in the mental health care system. There is a huge variation of costs among antipsychotics and a lack of information about the costs of different patterns of antipsychotic prescription. Accordingly, we aimed to estimate the costs of antipsychotic polypharmacy and its impact on public health care costs in a sample of patients with psychotic disorders living in residential facilities in the city of Sao Paulo.A cross-sectional study was carried out in the city of Sao Paulo, from February 2011 to May 2012. This study is part of a research project that evaluated the direct costs of health services for a sample of subjects discharged from long-term psychiatric hospitals.Setting: Residential facilities were created in Brazil under public government funding in 2000, to support people discharged from long-term psychiatric hospitals. There are two types of services: Residential facilities type 1, where six to eight people with mental disorders can live independently, sharing a house in the community, with an off-site carer available in an on-call basis, and Residential services type 2, for people with lower level of autonomy, in need of permanent care and with no family relationships. Type 2 is a fully-staffed home, with up to eight residents living there under 24-hour supervision of two carers taking turns working in 12 hours daily-basis regime. Carers should support all residents' needs and also help on the psychosocial rehabilitation process, in order to improve their autonomy and social skills. These services are linked to a Center of Psychosocial Rehabilitation (CAPS), a mental health center with specialists on clinical and psychosocial treatment for moderate and severe psychiatric disorders. Carers are also responsible to dispense all the medicines prescribed by psychiatrists in CAPS. They record all the activities related to the residents, in files kept in the residential facilities type 2. Until 2010, approximately 9,000 people were living in psychiatric hospitals in Brazil , and of Sampling: In 2008, two psychiatric hospitals were closed, and all the 160 inpatients were transferred to the 20 existing residential facilities in the city of São Paulo or to a psychiatric hospital in another city. No hospital records of these patients were available for data collection. At the beginning of this study sampling, in 2010, there were 151 people discharged from the two hospitals living in the residential facilities, and they were all selected to participate, according to the following inclusion criteria: being a former resident of a psychiatric hospital for one year or more and being able to understand and answer to the questions in the interviews.This study was approved by the Ethical Committee of the Secretary of Health of the city of Sao Paulo. Carers and residents were adequately informed of all aspects regarding the participation and the purpose of the study, providing a written consent prior to the interviews.Seven trained researchers interviewed the carers and residents. Instruments used to assess demographic and clinical characteristics of the sample were: the Mini International Neuropsychiatric Interview (MINI)  for psycA validated Brazilian version 5.0 of Mini International Neuropsychiatric Interview  was usedIn order to assess the severity of psychiatric symptoms, a validated Brazilian version of Clinical Global Impression—Severity (CGI-S)  was applThe Brazilian version of the Quality of life Scale , was useThe Independent Living Skills Survey , BraziliThe Social Behavior Scale (SBS) , BraziliInventário Sociodemográfico de Utilização e Custos de Serviços—ISDUCS) [The Client Sociodemographic and Service Receipt Inventory (CSSRI)  is a sem—ISDUCS) . The CSSData were collected by semi-structured and structured interviews conducted by trained psychiatrists, directly with residents and carers; some additional information could be obtained from the records available in the residential services. The SBS and the ILSS were applied with the carers, as such scales rely on the information obtained from the carers' through the observation of the residents' behavior.The QLS, the MINI and the CGI-S were applied by psychiatrists directly with the residents. The CSSRI was used to assess sociodemographic characteristics, previous and current occupational status, pattern of service uses, medicines and treatment received. Input to CSSRI were obtained from the interviews with both carers and residents, as well as from the records.In this study we considered as antipsychotic polytherapy only those residents who were regularly taking two or more antipsychotics  in the last 30 days previous to the interview. Data related to occasional antipsychotic use were also collected, but was not considered polytherapy. Antipsychotic monotherapy was defined as the use of only one antipsychotic, even when other non-antipsychotic drugs were taken concomitantly. The combination of one antipsychotic with other drug classes (e.g. benzodiazepines or antidepressants) was not considered an antipsychotic polytherapy in this study.Carers were responsible to dispense and supervise all the medicines taken by the residents, and all the information was daily recorded. The antipsychotics were freely provided by the public health system. The typical antipsychotics included haloperidol, chlorpromazine, levomepromazine, periciazine, thioridazine, pimozide, pipotiazine depot and haloperidol depot. Atypical antipsychotics included risperidone, quetiapine, olanzapine, ziprasidone, aripiprazole and clozapine.A bottom-up approach was used for the estimation of direct costs, according to the public health service provider perspective.The CSSRI allows calculating health care costs using the modality of service or intervention and the frequency of use. Direct costs of residential care, health care and treatments were calculated for each resident, for the year 2011.Direct costs of the public health services for a 30-day period included: inpatient costs + outpatient costs + emergency costs + medicine costs + psychosocial interventions  costs + transport costs (ambulance).All costs were estimated for the year 2011 and the estimation of unit cost for medicines was based on Brazilian Government Drug Price Database . This daThe estimation of cost for accommodation services was obtained directly from the residential managers. Information about residential costs included: electricity, gas, water, rent, repairs, transport, human resources, food and house supplies and overhead. The estimation of costs for public health services included hospital, emergency, outpatient and community services and transport costs. The estimation of health services costs was based on the frequency of service use in the previous month and on the calculation of the unit of cost: per visit (consultations) and per day . Units of costs for health services were directly obtained from service managers.Two groups (antipsychotic polytherapy group and monotherapy group) were compared through non parametric test  in terms of social and demographic characteristics, severity of psychiatric symptoms, psychiatric diagnoses, medicines, drug association with benzodiazepines, and type of antipsychotic used.A multivariate linear regression analysis was fitted with direct costs of public health care per person per month as dependent variable, and the number of antipsychotics, geographical location of the facility, olanzapine use, and the length of time living in the residential facility were included as covariates. Direct costs of public health care included costs with inpatient, outpatient, emergency and community health services and treatment received in the previous month .Firstly, univariate linear regressions were built considering, individually, different covariates, exploring the predictive of each measure on the costs of the residential facilities; then a multivariate linear regression analysis was fitted only with those covariates statistically significant in the univariate analysis (adopting significance level at 0.05).Four out of 151 residents refused to participate to this study. One hundred forty seven subjects were included in the sample; 134 used antipsychotics regularly; the rate of antipsychotic polytherapy (two or more antipsychotics in daily use) was 38% (N = 56) . DemograOverall, the mean (SD) length of time living in residential service was 35 (15.4) months. The mean length of time living in residential service was significantly lower in polypharmacy group than in monotherapy group (p = 0.011). The mean (SD) length of time living in psychiatric hospitals were 115.39 (106.02), with no significant difference among them.The mean (SD) number of individual psychotropics use was 3.37 (1.4), range 0 to 7, and the mean (SD) number of individual antipsychotics was 1.45 (0.9), range 0 to 5.Patterns of antipsychotic prescriptions were: 45.5% (n = 67) used at least one atypical antipsychotic, 64.6% (n = 95) used at least one typical antipsychotic, 14.2% (n = 21) used one typical and one atypical antipsychotic, 13.6% (n = 20) used two typical antipsychotics, and 10.8% (n = 16) used three or more antipsychotics. The use of clozapine use was below 5% .Antipsychotics agents were also frequently associated with benzodiazepines, carbamazepine, valproic acid, biperiden and promethazine . The useThe total direct costs of public health care  for a 30-day period were R$ 529,425.26. The total costs of accommodation were R$ 470,213.78, the total costs of health care were R$ 24,058.45 and the costs of medicines were R$35,153.03. The total costs of psychotropic were R$31,658.29. The costs of antipsychotics corresponded to 94.4% of the total psychotropic costs and to 49.5% of all health services use when excluding accommodation costs of the residential facilities.The mean monthly cost of antipsychotic polypharmacy per person varied according to the type of antipsychotics combination: typical- atypical costs were R$515.64 (R$457.1), while two-typical antipsychotics combination costs were R$8.72 (R$8.1). Olanzapine costs (n = 18) corresponded to 51% of all psychotropic costs and 53% of the total antipsychotic costs.A regression analysis was performed to explore a model in which direct public health care costs  was the dependent variable.The outcome  and its residual distributions were non-normal, as usually cost data are. However, even under non-normal error distribution linear regression and its estimates are BLUE (best linear unbiased estimator), being robust in a technical sense; in other words, it is computable under almost any circumstance.In The rate of antipsychotic polypharmacy observed in our sample (38%) is within the range observed in other studies ,19,34,35Studies estimating the costs of antipsychotic polypharmacy are scarce. Instead, most studies estimated the costs and cost-effectiveness of a specific monotherapy treatment. In this regard, there is no evidence on the clinical superiority and on cost-effectiveness among antipsychotics, with the exception for clozapine for severe psychotic disorders .The choice of an antipsychotic therapy is strongly associated with health services costs. Atypical antipsychotic monotherapy costs can be 167 times greater than typical antipsychotic monotherapy costs . A BraziThe lower rate of clozapine use is not justified by its cost; assuming the unit costs used in this study, the cost of clozapine therapy (450mg/daily) plus four complete blood counts (CBC) per month would be 50% of the costs of olanzapine therapy with a daily dose of 10 mg. Therefore, protocols for rational prescription of antipsychotics might be crucial for optimizing budget allocation. The reason for this low clozapine use should be better investigated, but one hypothesis is the difficulty to guarantee the weekly blood counts needed with this drug, or maybe the lack of training on its use. In Brazil, the choice of medicines is usually based on the physician’s preferences and the majority of professionals don’t take the costs and price into account.A North-American study demonstrated that the combination of two antipsychotics added annually U$4,244.00 per person when compared with monotherapy . In our The present study has some limitations. This was a cross-sectional study and data was collected retrospectively, considering only the month previous to the interview; as a result, variations in the treatment over the year could not be accurately captured. Although the sample size may be considered small, it is not a comparative study, and almost all residents discharged from two psychiatric hospitals in the city of Sao Paulo were included. It was not the aim of the study to compare the rate of antipsychotic polytherapy in the residential facilities with the rate found in other settings, such as hospitals and outpatients services.The unit of costs for psychotropic was based on Federal Government database , and it In summary, antipsychotic polytherapy adds a huge economic burden to health care systems, and the choice of the antipsychotic may negatively impact the financial health care management. The routine prescription of antipsychotics should be evaluated in terms of clinical benefits, cost-effectiveness, potential harms and financial burden to health care. More research is needed to explore how to monitor the rates of polytherapy, the costs and clinical consequences, and also to establish clear criteria for the rational use of antipsychotic polytherapy. Such practices could contribute to a better budget planning in mental health care management and, eventually, to better clinical outcomes. Considering the limited budget in low and middle income countries like Brazil, this study raises the attention to the adoption of better strategies on resource allocation, such as establishing a threshold for costs according to the benefits of newer drugs, through cost-effectiveness comparisons.


The present study compared the testicular cytology and histology between crossbred (Holstein–Friesian [HF] × Tharparkar) and purebred (HF and Tharparkar) bulls to find out differences if any.Four peripubertal bulls from each breed were utilized for the study. Through percutaneous needle aspiration biopsy, Sertoli and spermatogenic cells were extracted, and morphometry was studied. For histological studies, testicular tissues obtained through unilateral castration were utilized. Sertoli cells specific GATA4 antibody was used to study the population of Sertoli cells in the seminiferous tubule through immunofluorescence.The testicular weight, volume, and scrotal circumference differed significantly among the breeds. The diameter and area of the seminiferous tubule was high in HF, followed by Karan Fries (KF), and Tharparkar bulls. However, the degree of compactness, based on qualitative evaluation, was high in Tharparkar followed by KF and HF bulls. The intensity of Leydig cells was higher in Tharparkar bulls followed by KF and HF. The proportion of Sertoli cells was higher (p<0.05) in HF and Tharparkar bulls compared to KF bulls.It may be concluded that variations exist in testicular components of the breeds studied and the proportion of Sertoli cells in relation to spermatogenic cells was significantly lower in crossbred bulls compared to purebred bulls. Bos indicus with genetically superior exotic breeds of Bos taurus such as Holstein Friesian (HF) is being practiced in developing countries to improve the milk productivity. However, the males produced through crossing of B. taurus with B. indicus suffer from serious infertility/sub-fertility problems 13] and cUnilateral castration was performed as per the standard procedure and under strict aseptic conditions by veterinarians. Briefly, prior to castration the bulls were sedated with xylazine hydrochloride  at the dosage rate of 0.25 mL/50 kg body weight. The site of surgery was shaved and cleaned thoroughly with antiseptics. After local infiltration of 5-8 mL of 2% lignocaine  at the level of the spermatic cord, an incision was given at lower part of scrotum with the help of a BP blade (No. 23) with knife. One of the testes was exposed and the related spermatic cord was ligated tightly using catgut. After ligation, intact testis was removed and placed in individual sterile containers containing normal saline with penicillin streptomycin. Due post-operative care was given to the animals after castration as per the standard veterinary practice.Testicular tissue fixed in Bouin’s solution , were dehydrated in various grades of ethanol, cleared in benzene, and finally embedded in paraffin wax blocks. Sections (5-7 µm thick) were prepared using rotary microtome and mounted on albumenized glass slides. For studying the histoarchitecture of testicular tissue, the sections were stained using standard protocol of hematoxylin/eosin stain . StretchFrom each section, seminiferous tubule diameter and area  was determined using pre-calibrated measuring eyepiece. The sections were examined at × 400 and ×1000 magnifications  and measurements were made using the software . About 20 sections of seminiferous tubules that were round or nearly round were chosen randomly and measured for each group. The tubular diameter was measured at ×400 magnification. The diameter of the seminiferous tubule was measured across the minor and major axes and the mean diameter was obtained.Immunohistochemistry was carried out as per the procedure given by . Briefly® Software Package .Descriptive statistics was calculated for the tubular dimensions and the results are expressed as mean ± standard error (SE). One-way ANOVA was performed to compare the parameters in different breeds of bulls. When the p<0.05, the means were considered to be significantly different among the groups. The analyses were performed using Sigma Plot 11The testicular weight, width, volume, and scrotal circumference in Holstein bulls was higher (p<0.05) compared to either KF or Tharparkar bulls .The seminiferous tubules appeared as tortuous, round and oblong in outline, varying in appearance due to the complex coiling of the tubules at different angles and levels. The diameter of the seminiferous tubule ranged from 140 µm to 351.5 µm in HF bull calves, from 137 µm to 265 µm in KF bull calves and from 63 µm to 145 µm in Tharparkar bull calves, respectively. The mean (SE) diameter of the seminiferous tubule in HF, KF, and Tharparkar bull was 228.04±8.94, 183.43±4.57, and 111.71±2.53 µm, respectively  and the 5 to 97096 µm2 with a mean of 43309.73±3338.24 µm2 in HF bulls. In KF bulls, the diameter of seminiferous tubules ranged from 14747 µm2 to 57172 µm2 with a mean of 27145.74±1454.68 µm2. However, in Tharparkar bulls, the area of the seminiferous tubule ranged from 3094 µm2 to 16577 µm2 only with a mean (±SE) of 10021.93±436.03 µm2. The degree of compactness, based on qualitative evaluation, was high in Tharparkar followed by KF and HF bulls  did not differ significantly from either KF (15.55±0.82) or HF (15.98±0.62) bulls. However, the area of Sertoli cells was significantly (p<0.05) higher in HF bulls compared to Tharparkar bulls. Unlike in case of spermatogonia and spermatocytes, the number of Sertoli cells differed significantly (p<0.05) among breeds . In HF bSub-fertility/infertility is very high in crossbred bulls compared to either indigenous or exotic bulls ,4. CrossThe diameter of the seminiferous tubule was high in HF bulls, followed by KF and Tharparkar. The diameter of seminiferous tubule observed in the present study is in agreement with those reported earlier -19. It hThe major cell types in seminiferous tubule include spermatogenic cells  and Sertoli cells (supporting/nursing cells). We used GATA4, a specific marker for Sertoli, peritubular, interstitial cells , for locIt has already been reported that the inherent potential of each Sertoli cell is independent of the absolute numerical size of the Sertoli cell population, those bulls with the highest spermatogenic to Sertoli cell ratio would already be functioning closer to their maximum potential, and their opportunity for increased daily spermatozoa production would be more limited. Because of their intimate physical relationship with the germ cells and the variety of supportive roles they perform, it seemed reasonable to speculate that the absolute numerical size of the Sertoli cell population might establish the upper limit for spermatozoal production in any given testis . Thus, dFrom the findings of the present study, it may be inferred that variations exist in terms of seminiferous tubule area and its compactness, spermatogenic cells area, and concentration of Leydig cells in interstitial tissue among the three breeds studied and the proportion of Sertoli cells in relation to spermatogenic cells was significantly lower in KF bulls compared to either HF or Tharparkar bulls.AK designed the work. UKT and SC conducted experiments. MKMA, SKR, and SN helped UKT for microscopic and statistical analysis. AM, TKM, SY, and AK took part in preparing the manuscript. All authors read and approved the final manuscript.


In Australia’s War against Rabbits, Brian Cooke details the emergence of rabbit hemorrhagic disease (RHD) in Asia and Europe and subsequent efforts to introduce RHD virus into Australia and New Zealand for rabbit control in the 1990s. The book documents the ecologic adaption between a virus and its animal host and provides cogent examples of our inability to contain the spread of (what was thought to be) a well-understood virus after it escaped into the environment.The first failed containment effort ensued after the virus jumped from a quarantine compound on Wardang Island to the coast of South Australia 3 km away. Despite a program carefully designed to counter unplanned spread of RHD virus on the Australian mainland (Operation Garter), the virus prevailed. Uncontained spread also followed an intentional and illegal release of RHD virus in New Zealand in 1997 when a farmer with intractable rabbit problems allegedly smuggled the virus in from Australia. After the outbreak started, instead of helping the New Zealand government control RHD, farmers reportedly used blenders to make “rabbit smoothies” and actively spread RHD virus by applying these slurries to carrot baits.The book is generally well written and heavily referenced and contains many anecdotes penned by a scientist obviously passionate about his work. The flow of the narrative can sometimes be erratic, however, veering from amusing personal stories to a didactic recounting of the taxonomy of rabbit parasites or legislation relevant to pest control in Australia. Australia’s War against Rabbits is best suited for professionals with a keen interest in rabbits, fleas, and their pathogens.


Healthcare workers (HCWs) are at particular risk of acquiring pertussis and transmitting the infection to high-risk susceptible patients and colleagues. In this paper, the return on investment (ROI) of preventively vaccinating HCWs against pertussis to prevent nosocomial pertussis outbreaks is estimated using a hospital ward perspective, presuming an outbreak occurs once in 10 years.Data on the pertussis outbreak on the neonatology ward in 2004 in the Academic Medical Center Amsterdam (The Netherlands) was used to calculate control costs and other outbreak related costs. The study population was: neonatology ward staff members (n = 133), parents (n = 40), neonates (n = 20), and newborns transferred to other hospitals (n = 23). ROI is presented as the amount of Euros saved in averting outbreaks by investing one Euro in preventively vaccinating HCWs. Sensitivity analysis was performed to study the robustness of the ROI. Results are presented at 2012 price level.Total nosocomial pertussis outbreak costs were €48,682. Direct control costs  were €11,464. Other outbreak related costs  accounted for €37,218. Vaccination costs were estimated at €12,208. The ROI of preventively vaccinating HCWs against pertussis was 1:4, meaning 4 Euros could be saved by every Euro invested in vaccinating HCWs to avert outbreaks. ROI was sensitive to a lower vaccine price, considering direct control costs only, average length of stay of neonates on the neonatology ward, length of patient uptake restrictions, assuming no reduced work force due to ward closer and presuming more than one outbreak to occur in 10 years’ time.From a hospital ward perspective, preventive vaccination of HCWs against pertussis to prevent nosocomial pertussis outbreaks results in a positive ROI, presuming an outbreak occurs once in 10 years.The online version of this article (doi:10.1186/s12879-015-0800-8) contains supplementary material, which is available to authorized users. Calugar et al., [1,2 for 17 cases in a hospital in the Unites States. From the hospitals’ perspective, Baggett et al., [1,2, respectively. Zivna et al., [1,2 in a tertiary care medical center in the United States. According to Calugar et al., [Pertussis among healthcare workers (HCWs) is of special concern because of the potential for nosocomial exposure to susceptible patients and other HCWs . HCWs ar et al.,  calculat et al., . Dutch i et al., , meaning et al., . A natio et al., ,13. HoweDuring the outbreak period, data were collected by the occupational health service department of the AMC (hereafter referred as “AMC database”) on all control measures undertaken related to newborns, their parents, staff members, and to the organization within the hospital. The study population consisted of: neonatology ward staff members , parents of newborns , neonates (20 infants) and parents of 23 newborns who were transferred to another hospital. As data from on the pertussis outbreak was used in an aggregated way without identifying the individual participant, no written informed consent and ethical approval were required from participants to perform the data analysis.telephone calls made to parents whose children were transferred to another hospital;time spent by the neonatologist working on controlling the outbreak;survey performed by the occupational health service department as this is normal procedure during an outbreak in the AMC;all drug and vaccination administrations;PCRs done on nasopharyngeal swabs and blood samples taken for serology;Due to patient uptake restrictions , we assumed that the following activities were performed during regular working hours :Based on average Dutch working population  we assumNo further transmission of the pertussis infection took place after restrictions on the patient uptake on the neonatology ward were lifted;Reduced work force was required to run the neonatology ward during the period of restrictions on the patient uptake. We assumed that on day 1, 2, 3 and 4 0%, 5%, 10% and 15% reduced work force was required, respectively. On day five and onwards, this assumption was set at 20%.The following assumptions were made in this study as the AMC database did not capture all data on the outbreak:direct control costs (i.e. (i) medical consumption costs containing antibiotics, (ii) laboratory investigation costs, (iii) outbreak control management costs) andother outbreak related costs (i.e. (iv) replacing costs for sick hospital staff members, (v) losses due to restrictions on patient uptake on the neonatology ward and (vi) savings due to a reduced work force required on the neonatology ward during patient uptake restriction period).Total outbreak costs were calculated by considering:Dutch prices were used to derive medication costs and other resource unit costs -18, and catch-up vaccination: vaccination of all HCWs (n = 133) one year after the outbreak based on the list price for Infanrix® IPV  vaccine  ,20;vaccination of newly employed HCWs staff (assumption 10% per year) for a period of 10 years. New HCWs were assumed to be unvaccinated but would be vaccinated upfront when hired at 100% coverage rate;booster vaccination to be provided eight years after first vaccination due to the declining vaccine effectiveness .Vaccination costs were calculated by considering:ROI of preventively vaccinating HCWs was calculated by dividing the return on investment  by the cost of the investment (i.e. cumulative vaccination costs including booster vaccination):and it is presented as a ratio: the amount of Euros saved by averting an outbreak times one Euro invested in vaccinating HCWs. All costs are presented in Euro at 2012 price level and without time-discounting. Discounting is applied in sensitivity analysis.total nosocomial pertussis outbreak costs, split up as costs of antibiotics, costs of laboratory investigations, costs of outbreak control management, costs due to work absence of sick staff members, losses due to restrictions on patient uptake and reduced costs (=savings) due to reduced working force;ROI of preventively vaccinating HCW assuming one outbreak within 10 years’ time.The analysis was conducted in MS Excel, version 2007 based on the study population and the input parameters displayed in Table Univariate and two-way sensitivity analysis was performed on several input parameters to further test the robustness of the outcomes. In Table Total nosocomial pertussis outbreak costs in the AMC in the Netherlands were €48,682. Direct control cost account for less than 25%. The majority of the costs were caused due to patient uptake restrictions on the neonatology ward, including savings due to reduce working force 33%), and due to absenteeism of HCWs (43%). Medical consumption costs were €785, laboratory investigation costs accounted for €6,982, outbreak management control costs were €3,697, costs due to absenteeism were €21,008, and costs due to patient uptake restrictions were €16,210). Cumulative vaccination costs, including boostering, were €12,208. The return on investment of vaccinating HCWs was 1:4, meaning 4 Euros can be saved by investing one Euro in vaccinating HCWs to prevent a nosocomial pertussis outbreak  used in this study. However, using a broader perspective and including additional costs would have led to even a more favourable (i.e. higher) ROI. The assumptions made in this study led to outbreak cost estimates which can be considered as conservative. First, handling costs of several activities  were not considered as it was assumed that these activities were performed by the staff themselves during their regular working hours. Including the costs of these activities would lead to higher total outbreak costs and a higher ROI. Second, it was assumed that no further transmission of the infection took place when patient uptake restrictions on the neonatology ward were lifted. In practice, additional infections could occur after these restrictions would be lifted, which would lead to additional outbreak costs and a higher ROI. Third, additional costs related to the spread of the infection by children who were brought to other hospitals were beyond our perspective  and therefore not considered. But also productivity losses due to work absence of sick parents (i.e. only fathers as mothers would be on maternity leave) were disregarded because of the restricted perspective. Both - negative externalities to the Dutch society - might be omitted if the HCWs would have been vaccinated. Fifth, it was assumed that vaccine uptake, both in existing and newly joined HCWs, would be 100%, which is slightly higher than the observed vaccine coverage in general population (i.e. 96-97%) [In the Netherlands, a national vaccination recommendation of HCWs against pertussis has yet to be made. In the Dutch society, infants are not fully protected against pertussis in the first few months of their life. To provide protection to this vulnerable group, preventive vaccination of HCWs working with vulnerable infants who are not fully protected could be a relevant intervention. Considering the fact that about 76% of the outbreak costs estimated in this study were caused due to patient uptake restrictions on the neonatology ward of the hospital and by absence of the infected HCWs, it shows the importance of preventing nosocomial pertussis outbreaks and their disregarded impact. Also, it could be argued that hospitals as employers should have some responsibility in preventing nosocomial infections and protecting both, patients and staff members. Therefore, within policy decision making on vaccination recommendations, vaccinating HCWs should also be recommended.In conclusion, the current study demonstrated that from a hospital ward perspective, preventive vaccination of healthcare workers against pertussis to prevent nosocomial pertussis outbreaks does result in a positive return on investment (1:4). Therefore, preventive vaccination of healthcare workers can be considered a wise use of healthcare resources enabling the prevention of nosocomial pertussis outbreaks with the tendency to reduce, both the economic and disease burden of pertussis in both, hospital setting and the society.As data from the nosocomial pertussis outbreak in the AMC in The Netherlands was used in an aggregated way to model the return on investment of vaccinating healthcare workers without identifying the individual participant, no written informed consent and ethical approval were required from participants to perform the data analysis.As this study is not a full economic evaluation but a financial analysis based on a mathematical model, the Consolidated Health Economic Evaluation Reporting Standards checklist (CHEERS) was not necessarily suitable to be used in this case. However, while preparing this manuscript the attempt has been to follow where applicable the CHEERS guideline and to meet the reporting standards of a scientific publication.All relevant raw data used in this study are presented in the current manuscript .


Introduction: An earlier descriptive study exploring the various supports available to Canadian health and social service providers who deployed to the 2010 earthquake disaster in Haiti, indicated that when systems are compromised, professionals are at physical, emotional and mental risk during overseas deployment. While these risks are generally well-identified, there is little literature that explores the effectiveness of the supports in place to mitigate this risk. This study provides evidence to inform policy development regarding future disaster relief, and the effectiveness of supports available to responders assisting with international disaster response.Methods: This study follows Strauss and Corbin’s 1990 structured approach to grounded theory to develop a framework for effective disaster support systems. N=21 interviews with Canadian health and social service providers, who deployed to Haiti in response to the 2010 earthquake, were conducted and analyzed. Resulting data were transcribed, coded and analysed for emergent themes.Results and Discussion: Three themes were identified in the data and were used to develop the evolving theory. The interview data indicate that the experiences of responders are determined based on an interaction between the individual’s ‘lens’ or personal expectations, as well as the supports that an organization is able to provide. Therefore, organizations should consider the following factors: experience, expectations, and supports, to tailor a successful support initiative that caters to the needs of the volunteer workforce. The earthquake destroyed hospitals, clinics, and other infrastructure, leaving many Haitian health and social service workers buried in the rubble.The purpose of this research is to understand and evaluate the lived experiences of health and social service workers deployed to Haiti in response to the 2010 earthquake. Using this data, we will develop a theoretical model highlighting the support systems required to ensure physical, emotional and psychological protection for volunteers responding to international disasters.Twenty-one Canadian health and social service providers who responded to the disaster in Haiti were interviewed by the primary author. Participants for this study were recruited using maximum variation, purposeful sampling. To guarantee a range of experiences were represented in the interviews, participants were intentionally selected to reflect various career backgrounds, years of experience, length of deployment, and date of departure. A list of participant characteristics is included in Table 1. Each key informant was asked to participate in a single audio-recorded interview, approximately one hour in duration. This study was approved by an academic Research Ethics Board.Participants were made aware that their information would remain confidential, and were required to sign a form of informed consent prior to the start of the interview. The participants were asked questions, according to a semi-structured interview guide, regarding their general experiences in Haiti. In addition, responders were asked to answer questions regarding specific elements of the support systems offered to them before, during, and after deployment. Finally, they were asked to reflect on the effectiveness of the existing systems of support, and identify any perceived gaps.,In accordance with Strauss and Corbin’s (1990) approach to grounded theory, the central phenomenon of this study was identified as the “need for supports for Canadian health and social service providers responding to the disaster in Haiti”.Contextual and Intervening Conditions: a description of the societal, organizational andpersonal contexts and conditions that were present during the responseCausal Conditions: reasons driving for the need for supports;Strategies: systems or interventions implemented that influenced the responder’s ability to respond with resiliency to the context and conditions; andConsequences: the “outcomes from using the Strategies in relation to the Context and Conditions" ,Questions pertaining to each of these categories were used to discover how available supports affected the experiences of front-line responders in Haiti. All of the interviews, researcher’s memos and reflections were transcribed, coded and analysed using NVivo9 qualitative software. The process included the development of higher level pattern codes to determine relationships between the identified categories. To remain consistent with structured grounded theory methodology Model of the Context, Strategies, Causes and Consequences of the Need for Supports for Canadian Health and Social Service Providers Responding to the Disaster in Haiti, the contextual, causal and consequential conditions stem out of the central phenomenon of this study. As seen in the data, the Context of the response was a central feature of the aftermath of the earthquake.In keeping with the style of Strauss and Corbin’sThe first contextual factor, the country context, refers to the context within Haiti and the Haitian society at the time. Volunteers were faced with the challenge of performing emergency and medical procedures with little supplies, while coping with crumbling infrastructure, frequent tremors in the ground, and harsh conditions, such as extreme heat. Secondly, the context of the various relief organizations was explored to determine the organizational and coordinating factors that affected the response context. The participant interviews indicate that the organizational context not organized and was often described as a scene of ‘chaos’. As seen in Figure 1, the country and organizational context directly affected the ‘personal context’ of individuals responding to this disaster. The devastation in Haiti was vast and required a special type of volunteer who possessed the physical and emotional capacity to respond with resilience during such a disaster. An individual’s training, past experiences, attitudes and willingness to work in a team were all found to be crucial for their resiliency on-site. These elements are defined as the ‘causes’ of the central phenomenon, or the factors that influence and determine the types of supports required by the responders.In reaction to the contexts, the Causes of the central phenomenon emerged and integrated with the need for supports along two main themes. The first cause for support related to unclear expectations, which led to feelings of uncertainty and confusion, fear, lack of communication and awareness, stress and guilt. These feelings stemmed from a variety of causes, including unclear roles on-site, disorganization within teams and between organizations, and insufficient safety protocols, training, and psychological supports. The ‘personal context’ of an individual, which we will later refer to as the ‘lens’, relates to participants’ perceptions that influence their experiences, attitudes and personal initiatives in preparation for deployment.In response to the contexts and conditions, many organizations employed two main strategies to support their delegates. As seen in the model, the supports were determined based on both the context of the disaster as well as the causes. The first of these strategies, Instrumental Supports, includes safety and security supports, psychological and emotional supports, as well as physical supports. The second category of strategies focuses on Communication and Leadership. These supports include recruitment and screening techniques, creating positive team dynamics, appointing effective team leaders, ensuring clear and consistent communication between Canadian headquarters and the on-site base, and highlighting the need for accurate media coverage of the disaster situation, in order to avoid confusion and unclear expectations. There was no universal standard for supports given to response workers in Haiti. Therefore, responders were heavily reliant on their affiliated organizations to provide them with various types of support pre-departure, on-site and post-deployment. Results from the interviews indicate that the effectiveness of these supports varied greatly by organization.Finally, the Consequences of the response refer to the overall experience and impact of the response, as perceived by the participants. The consequences can be positive or negative, and are determined as a result of an interaction between the context, an organization’s support strategy, and an individual’s expectations and personal lens. According to Strauss and Corbin(1) The “lens” can mediate the effectiveness of a support strategyThe data indicate that a participant’s lens can positively or negatively mediate, either by adding to or taking away from, the effects of a support strategy on the desired outcome . The lens is shaped by background experiences, personal beliefs and attitudes, as well as individual initiatives and preparedness. Thus, achieving a positive experience requires the recruitment of delegates who possess a specific lens, or set of characteristics, making screening and recruitment a critical factor for all organizations. Many participants, particularly those stemming from smaller organizations, reported that there was no formal screening program required, before deploying as a volunteer. Participants agreed that organizations should screen for individuals who demonstrate ongoing interest in disaster relief, and who have taken personal steps, such as familiarizing themselves with a country’s language or completing emergency response training courses, in preparation for deployment. By doing so, organizations can effectively recruit during times of normalcy, thus bypassing the initial ‘disaster tourism’ or ‘response hype’ that accompanies large disasters. As stated by one participant,The idea is that you recruit when there’s nothing traumatic going on, so that way you really have time to do training, answer questions, check out the background of volunteers...but when there’s been a big disaster, you’re always begging people that are like what we’d call maybe walk-up volunteers. And so you have to be prepared to try to do some kind of training or check out before they go.” (Faith-based organization)“In addition to mediating supports, the lens can, in unique settings and situations, compensate for lack of supports or ineffective support strategies. This means that the lens can itself suffice, or can independently serve, as a support mechanism that is innate to the individual. While reactions such as these are ultimately attributed to an individual support mechanism, they can be affected, either positively or negatively, when in combination with support strategies. The data indicate that the perceived level of effectiveness of supports was highly dependent on an individual’s attitude, beliefs, personal background and experiences.(2) Expectations can override the effects of support strategies to affect the outcomeThe data indicate that expectations can override the effects of a support strategy and affect the overall outcome of the response experience. This point differs from item (1) above, in that expectations differ greatly from the lens. The lens is an innate attribute, unique to the individual, and rooted in personal experiences, attitudes and beliefs. Expectations, however, are not as fixated and are formed specific to a response or situation. According to our participants, expectations are greatly shaped through 1) Personal beliefs 2) Training and briefing supports and 3) Organizational and Media influence. Thus, expectations are derived both intrinsically  and extrinsically . Not surprisingly, the data shows that previous experience with disaster response plays a strong role in developing personal beliefs and expectations regarding future responses and availability of supports. Delegates who had previously deployed to Haiti, or had completed similar disaster relief missions, typically held a better understanding of their roles and responsibilities than novice volunteers. Thus, having previous experiences with disaster, shapes a piece of the lens. If organizations consistently employ support strategies that provide a delegate with positive experiences, then, over time, the delegate’s lens will be shaped positively, thus affecting their outlook regarding the disaster response. These effects can be reversed if support strategies are not provided and the delegates experience negative deployments, thus deterring them from participating in future responses.Our data indicates that, in regards to extrinsic factors, training plays a large role in shaping expectations. In fact, 40% of our participants would have liked to receive better pre-departure or early on-site briefing to better prepare them for their deployment. Briefs regarding the political and economic climate of the country, roles and team dynamics, and medical concerns specific to the disaster response were requested by the participants. The participants also indicated that the media  plays a role in shaping participant experiences and expectations. Inaccurate media coverage causes delegates to enter a context under false expectations, which may impact their ability to respond once on-site. Furthermore, negative or pessimistic media may discourage delegates who just returned from a large-scale disaster, such as Haiti, and lead to feeling under-appreciated. Holding briefing sessions and following up with delegates post-disaster can mitigate some of these negative outcomes. The biggest difference between the lens and expectations in relation to the available supports is that expectations are formed on a short-term basis, and can be easily swayed with information through training, briefing and media, while the lens is more integrated and takes time to develop. Therefore, the lens is not easily changed or affected by information or training. However, the lens can be swayed based on an individual’s experiences, which can be shaped by the types of supports available during deployment. In essence, the supports will ultimately determine whether or not the individual continues to respond to disasters, and if they do, what their attitude towards the response will be. This point is especially pertinent to health and social service providers, who are typically volunteers, and not mandated by their professional organization to respond to disasters.(3) Supports  should be used effectively to shape the lens and expectations and ensure an overall positive responseAccording to the data, it is evident that overall experiences were greatly dependent on the types of supports that were provided by the responder’s affiliated organization. Those who deployed with these larger, more established organizations typically felt well-supported and understood they were part of a ‘bigger picture’, while those who deployed with smaller organizations felt the response to be disorganized. As stated by one of the participants,Everybody [organizations] wants to show that they are there and they are the ones doing the work. They are well intentioned, there’s no question. But because it’s not coordinated, sometimes they can harm each other.” “All of the aforementioned supports are key to ensuring overall positive outcomes. As seen in the data, those who reported overall positive experiences in Haiti typically had formal and communicative and leadership supports available to them (regardless of whether or not they chose to use them or found them effective). Those who were not provided with these supports either reported a negative experience or cited these supports as lacking. In addition to training, participants highlighted the importance of safety and psychological/emotional supports for an effective response. In a comparison of two quotes reporting the effectiveness of training supports, one can see how similar supports can be perceived differently, based on an individual’s lens and experience. As stated by the first participant,“I know volunteers...some of them were a little shocked at the level of security and wondered ‘why do we have so much security, is there something we need to be worried about here?’ And that got them more upset. And then some of them were really relieved to see all of the security.” (Faith-based organization)This statement is reflected in a quote by a second participant, who highlights seemingly comprehensive  supports, yet does not believe these to be adequate for ensuring feelings of safety and security.“That’s where I thought things were really lacking [in regards to safety supports]. It’s kind of crazy when you think about it, you’re just kind of given a truck and a driver and you go out and there’s a radio...and there’s a security officer that briefs you on security and you have orders to be indoors by nightfall. But other than that, you’re really kind of on your own there.” (Canadian government)These types of examples are found throughout our data. It seems that when protocols were explained and supports were organized , volunteers typically felt safe. However, excessive protocols sometimes led to feelings of worry or confusion, as did a lack of briefing. Ultimately, however, the individual’s lens and experiences shaped the perceived effectiveness of supports. A model outlining the theoretical relationships between factors that influence the overall experiences and outcomes in the disaster response.According to Strauss and Corbin, grounded theory is developed through an identification of emergent themes within the Context, Causes, Strategies and Consequences. In order to understand the implications of this theory, it is important to consider the context in which this theory takes place. The context is especially important in Haiti, as it was challenging for both inexperienced delegates and organizations. According to a 2010 report created by the Pan American Health Organization (PAHO-WHO), there are four characteristics that created disadvantages for the response, which are as follows: “1) Haiti is a small country, among the poorest of the world, and had limited response capacity; 2) Haiti had weak institutions with little control over thousands of donor-supported NGOs; 3)Haiti had a lack of governance and a high level of corruption; and 4) Haiti had the absence of armed forces.”  facilitate a feedback loop with the supports. As seen in Figure 2, when organizations effectively target the extrinsic factors that affect these causes, they are more likely to have their volunteers perceive their supports positively. Furthermore, as the efficiency and effectiveness of supports are improved, the lens and expectations of the participants will also change. It is an interaction between the expectations and lens, in addition to the available supports, that determine the overall experience of a delegate. Therefore, there is an interaction between extrinsic and intrinsic factors. Support strategies are extrinsic interventions aimed at assisting the delegates, while the lens and expectations are intrinsic support strategies, unique to each responder. Organizations must target both components to ensure an overall positive experience for their responders.While a sample size of N=21 interviews is considered sufficient for the purpose of a grounded theory qualitative study, further research should be conducted to validate these findings within a larger population. Furthermore, it is important to remember that Haiti was a unique and extensive disaster, and data regarding the country context and conditions of a disaster should always be considered before developing support strategies. Regardless, all of the identified supports can be tailored to suit the particular context of the disaster-stricken country as well as the needs of responders.The authors have declared that no competing interests exist.