Convulsions associated with fever and acute onset of unknown aetiology with case fatalities have become a long observed medical condition at the Child Health Department of the Korle-Bu Teaching Hospital. Children admitted to the department with seizures of undetermined origin and fever has been a source of diagnostic confusion. Studies from the Asia Pacific region suggest a link with non-polio enteroviruses. The aim of the study was to investigate the association between non-polio enterovirus and acute encephalopathy causing neurological morbidity in children.One hundred and fifty cerebrospinal fluid (CSF), throat swab and serum samples were collected from participants at the Child Health Department of the Korle-Bu Teaching Hospital for virus isolation and characterization. Samples were cultured on cells and positive culture assayed by microneutralisation. Direct PCR as well as multiplex PCR were used to detect other viral agents present.Enterovirus isolation rate was approximately 0.67%. Intratypic differentiation by molecular characterization identified a poliovirus from vaccine origin. Further screening by real-time RT-PCR identified the virus as normal Sabin and not vaccine-derive poliovirus. No arbovirus was however detected.Non-polio enteroviruses and chikugunya virus were found not to be the etiologic agent responsible for the convulsion with neurologic morbidity observed in the Ghanaian children. Investigation for other viral agents is recommended. Convulsion with acute onset in general is a common cause of admission in paediatric emergency wards and risk for neurological, cognitive impairment and epilepsy. Early diagnosis for the etiological cause and immediate clinical management is crucial to the survival of the child –4. CliniAt the Child Health Department of the Korle-Bu Teaching Hospital (KBTH), sporadic cases of convulsion with associated fever of unknown aetiology have become a long observed medical condition with diagnostic confusion. Routine parasitological and bacteriological investigations conducted have been inconclusive and no further investigations have been able to establish the cause of the disease. Cases include children between the ages of one day and twelve years with presentations of convulsion and fever, with occasional rashes. Many of the patients without any history of neurological problem were found to have developed temporal or permanent neurological impairment. Isolation of the causative agent would help to curtail unnecessary investigations, rationalise treatment, improve reliability of prognosis and prevent overuse of antimicrobial agents with consequent antimicrobial resistance.In the Asian-Pacific regions, this manifestation of childhood convulsion associated with fever and neurological complication observed at the Child Health Department of the KBTH is usually associated with the non-polio enterovirus known as Human enterovirus 71 (HEV71) –27. CurrEpidemics of viral infections causing central nervous system effects are continuously being reported from around the world and clinicians are challenged to be abreast with local epidemiology. This study therefore aimed to investigate whether non-polio enterovirus was the etiological cause of the neurological disorders observed in the children.Study population: The study population comprised children between the ages of one day and twelve years old admitted to the hospital having clinical diagnosis of convulsion associated with fever and rash. Convulsions includes seizures lasting for at least half an hour, or convulsions followed by coma lasting two hours or more or convulsions followed by paralysis or other neurological signs not previously present and lasting 24 hours or more and convulsions that presented as encephalitis [phalitis –34. OnlyVirus isolation: Viruses in stool specimens were isolated on Hep-2C (derived from human carcinoma cells) and RD (Rhabdomyosarcoma) cell lines in accordance with standard protocols . Briefly, Hep-2C and RD cells were seeded in tissue culture tubes with growth medium (Eagle's MEM supplemented with 10% FCS) 48 hr prior to inoculation. Suspensions of faecal samples pre-treated with chloroform was inoculated on serum-free medium and incubated at 37oC and observed daily for the characteristic enterovirus cytopathic effect (CPE). The tubes with CPE up to 75% and above were harvested and kept at -20oC while those negative after 5 days of incubation were re-passaged (blind passage) on the same cell line and if it remained negative after 5 days was considered negativeMicroneutralization and real time PCR: Identification of isolates on Hep-2C and RD cells lines was carried out by microneutralisation technique using polyclonal antisera raised in horse against Coxsackie and echoviruses prepared by the National Institute of Public Health and the Environment (RIVM), Netherlands. Microneutralisation assay with HEV71 antiserum was carried out on the isolates that were untypable with the antiseraum pools. Samples that showed neutralization in the wells with polio pool antisera selected for Real-Time Reverse Transcriptase polymerase Chain Reaction (rRT-PCR).RNA extraction and PCR: Viral RNA was extracted by QIAamp Viral Mini Spin Protocol  according to manufacturer's instruction. Reverse Transcriptase Polymerase Chan Reaction (RT-PCR) with Pan-EV primers MD90 (5’- ATT GTC ACC ATA AGC AGC CA-3’) and anti-sense MD91 (5’ - CCT CCG GCC CCT GAA TGC GGC TAA T -3’) were used to amplify the VP1 region under the following conditions RT: 97°C for 3 minutes, PCR: 95°C for 45 seconds, 55°C for 45 seconds, and 70°C for 45 seconds and cooled to 4°C. The products were observed on 1% agarose gel electrophoresis.Data analysis: Data analysis was done using SPSS version 19. The analysis involves frequency distribution of responses and cross tabulation of variables. The analyzed information was presented using tables, graphs, charts and other diagrams that depicted the pattern of findings.Ethical issues: Ethical approval was obtained from the Ethical Committee of the College of Health Sciences, and Research Committee of the University of Ghana Medical School (UGMS). Informed consent was sought from parent/guardian of subjects before the commencement of the study. All ethical considerations were adhered to. Data collected from the study was handled anonymously and confidentially. Samples had only the identification numbers of the subjects to ensure anonymity. We protected the confidentiality of patients through use of codes.Demographic/clinical findings: Eighty-two subjects were recruited for the study, with 49 (59.8%) being males. Majority of the subjects (67%) were between the ages of one day old and four years old. Apart from three of the subjects in junior high, the rest were either in primary school, kindergarten or not in school. According to the study, mothers were the usual care givers of children at home and were those attending to the children at the hospital. The educational status of the guardians showed that 14.6% of them did not have formal education. Of the remaining, 32.9% of them attended primary school, 26.8% junior high, 15.8% senior high and 9.7% had tertiary education.The clinical presentations as shown in Virus isolation and characterisation: Of the 150 samples, only one (0.7%) sample showed growth on Hep-2C and RD cell lines by the second day post inoculation. The positive sample was serotyped using pools of antisera to determine the serotype of virus present. No neutralization was observed in the wells containing the non-polio enterovirus antisera apart from those containing the polio pool, an indication of poliovirus present in the sample. Conventional PCR  and oral rehydration salt (ORS) depending on the clinical presentation. The anticonvulsants were administered to manage clinically severe convulsion cases while antibiotics and antimalarial drugs were prescribed due to the febrile condition which may have either bacterial or parasitological origin.Routine bacteriological and parasitological laboratory tests requested by the physicians to isolate the aetiological agent failed to detect neither bacteria nor any parasite. The failure of the laboratory to isolate the aetiological agent compelled these emergency physicians to continue the symptomatic management. In spite of this necessity, the practise of treating patients symptomatically is not always recommended because it may mask the presence of the underlying etiology which will then be forgotten or treated with great delay. Symptomatic treatment with antimicrobial agent can lead to antimicrobial resistance , 36. TheTo many emergency room physicians, the threat of antimicrobial resistance has not sunk in yet. It has been apparent through this study that emergency department physician's fundamental and principal concern in an emergency situation is how possible he could resuscitate his patient and that, the issue of the impact of antimicrobial use on the prevalence of resistance was not a crucial consideration at that moment. Many physicians and patients do not see antimicrobial resistance as a reason to abstain from its use , 42. EmeThe findings from this study could not establish non-polio enterovirus or chikugunya association with the etiological cause of the manifestation. All molecular virological assays to determine the aetiology after RNA extraction from 150 samples yielded only one positive for poliovirus. Further characterization identified the poliovirus as a Sabin 1 poliovirus. The virus was obtained from the throat swab of a one month old baby with the presentations of fever, convulsion and fast breathing. These symptoms are not characteristic of poliovirus infection. Since Sabin 1 poliovirus is a component of the oral polio vaccine (OPV), the child might have acquired the virus from either polio vaccination during the recent national polio immunization days, contact with immunized person or from the environment. The oral polio vaccine (OPV) apart from seeding the gut of the recipient could also immunize children in contact with the faeces of the OPV recipient , 44. It Further investigation to determine whether the Sabin 1 poliovirus isolated was a normal Sabin or a VDPV have shown that the virus is a normal Sabin 1 poliovirus and not VDPV. It was very expedient to have screened for the presence of VDPV because of its latest implication in cases of acute flaccid paralysis in many parts of the world. Screening for Chikungunya virus using the multi-virus real time PCR did not also establish the aetiological agent. In West-Africa, similar clinical presentations of fever, convulsion, maculopapular rash and meningoencephalitis have been recorded to be caused by Chikungunya virus (CHIKV). Since its discovery in Tanganyika (Tanzania), Africa, in 1952, Chikungunya virus outbreaks have occurred sporadically in Africa, West Africa , South Asia, and Southeast Asia with recent outbreaks spreading the disease over a wider range.The study limitations include our inability to collect stool samples or anal swap since enteroviruses are readily present in stool. Secondly we did not investigate other DNA viruses with similar presentation. The etiological agent may be a DNA virus while the study was geared towards the detection of RNA virus. In the literature, other viruses associated with such manifestation are influenza, parainfluenza virus, herpes virus 6, Herpes virus 7 and RSV. Similar manifestations must be investigated for the presence of these viruses. Solving this puzzle will save the Government a lot of money that is spent on antibiotics, antimalarial and other drugs which in future will lead to drug resistance in the children.The findings from this study indicate that the aetiological agent for the observed convulsions in the children was neither due to non-polio enteroviruses nor chikugunya. We recommend that other viral agents capable of causing convulsion in children be investigated. The use of drugs to manage convulsions should be minimized and efforts should be made toward identifying the aetiological agent.


Varroa infestation or high level of winter mortality were also surveyed. Results from RT-qPCR showed a considerable difference of virus levels between healthy and sick colonies. In the group of healthy colonies, no virus was detected in 36% of cases, while at least one virus was found in each of the sick colonies. Virus titers varied among the samples, and multiple virus infections were common in both groups with a high prevalence of Sacbrood virus (SBV), Black queen cell virus (BQCV) and Deformed wing virus (DWV). Based on the distribution of virus titers, we established four categories of infection: samples free of virus (C = 0), samples with low virus titer (estimated number of virus copies 0 < C < 103), samples with medium virus titer (103 ≤ C < 107) and samples with high virus titer (C ≥ 107). This allowed us to statistically compare virus levels in healthy and sick colonies. Using categories to communicate virus diagnosis results to beekeepers may help them to reach an informed decision on management strategies to prevent further spread of viruses among colonies.Honey bee virus prevalence data are an essential prerequisite for managing epidemic events in a population. A survey study was carried out for seven viruses in colonies representing a healthy Danish honey bee population. In addition, colonies from apiaries with high level  Honey bee colonies provide valuable pollinators that enable secure crop productivity . The decVarroa destructor, originating from one of the Asian honey bee species (Apis cerana) is an invasive species on the European honey bee (Apis mellifera) [Varroa mites together with closely associated viruses , Kashmir bee virus (KBV), and Israeli acute paralysis virus (IAPV)) on individual bees as well as colony survival has been established in previous studies [Varroa mites, the microsporidian Nosema ceranae, another possible agent involved in collapse of honey bee colonies, also created concern in some countries [Nosema apis have been linked with infections of BQCV [The ectoparasitic mite, llifera)  and consllifera) , 12, 16.llifera) , 17, 18,llifera) , 19. The studies . In addiountries , 24. Inf of BQCV .Varroa mite. Furthermore, three viruses for which Varroa seems to play no significant role in transmission, namely Chronic bee paralysis virus (CBPV), SBV, and BQCV are frequently surveyed, too [Numerous viruses of honey bees are known and occur in different geographical regions , 27. Mosyed, too –31.Based on results of infection studies, these seven viruses can be characterized as either acute or persistent. In particular, the three closely related viruses from the family Dicistroviridae  and the yet unclassified virus (CBPV) can cause acute infection of adult bees with a high rate of viral replication leading to high mortality of workers within a short time span , 32. TheVarroa mites are being conducted in several countries [Nosema spores enables beekeepers and scientists to monitor the prevalence and quantity of parasites easily and select the best colonies for the next generations.For example, efforts to breed honey bee strains that are more tolerant towards ountries –43. In Dountries , 45. TheFor virus diagnosis, quantitative RT-PCR is an advanced and more sensitive technology than qualitative gel-based technique . The abiVarroa mites and interactions between Varroa and viruses [Varroa mites [Colony losses in Denmark reached up to 32% in the winter of 2007 to 2008 and were largely attributed to honey bee viruses,  viruses , 31, 49.oa mites . HoweverVarroa mites and were determined free of symptomatic diseases  based on thorough visual inspection. We pooled each sample of 20 bees, which is considered sufficient for a reliable quantification of virus levels in a colony [The survey was designed to determine viral presence from apparently healthy honey bee colonies all over Denmark . In totaa colony . For comBees of each sample were placed in a 15 mL plastic bottle together with 5–10 steel bearing balls. Using a technique adapted from plant virology, the samples were freeze dried, homogenized in a genogrinder and thereafter, RNA was extracted according to the manufacturer’s manual (for details see ). FollowQuantitative PCR amplifications were carried out on a vii7 apparatus (Applied Biosystems) in duplicate for each sample using SYBR Green DNA binding dye. Final volumes of 12μL with a primer concentration of 0.4μM were loaded on optical 384 well PCR plates. Primers , 51–53 uFor each virus genome, a standard curve was calculated by plotting the serial dilutions of known amounts of the amplification product against the corresponding Ct values as described previously . Slope aA total of 241 apparently healthy and 28 sick colonies were screened for SBV, BQCV, DWV, CBPV and ABPV complex viruses. No virus at all was detected in 36% (n = 86) of the healthy colonies, while all of the sick colonies had at least one virus. The frequencies of the viruses in the two groups of colonies are shown in The most prevalent viruses in the healthy colonies are SBV, BQCV and DWV, with frequencies of 39% (n = 95), 35% (n = 84) and 34% (n = 82), respectively. These three viruses are also highly prevalent and frequent  in the sick colonies. In contrast, viruses of the ABPV complex are present in only 14% of the healthy colonies, but 50% of the sick colonies. CBPV was only observed in four healthy colonies and one sick colony.Twenty-seven percent of the healthy colonies contained at least one virus, mainly SBV and DWV. Multiple virus infections are also not uncommon in healthy colonies. We observed high rates of duplicate and triplicate infections , the majority of them with DWV, SBV and BQCV . As expe7), we decided to subdivide the remaining samples into two groups: samples with low virus titer (0 < C < 103) and samples with medium virus titer (103 ≤ C < 107). For both DWV and SBV we found samples falling in all four categories, whereas high BQCV and AKI titers were never observed. Finally, CBPV was observed only in the two categories no virus or medium virus level. The distribution of samples within these four categories is displayed in P < 0.001), but not for CBPV (P = 0.3842).For each investigated virus the samples of healthy and sick colonies were categorized according to the quantified virus titer. Beyond the two obvious categories, bees free of virus  and symptomatic bees  which is in accordance with a previous study from Denmark . Sacbroark (81% , 30, 34.ark (81% , or the ark (81% . Even th studies , 56, then Europe .Deformed wing virus is the other highly prevalent virus in this study. It was detected in 34% of the healthy colonies with almost 11% of these carrying a medium or high titer which is associated with reduced life span of the adult bees [Varroa mite and strongly suspected to be one of the biological agents for honeybee colony losses [Varroa mites infestation during the preceding winter.ult bees . This reult bees , 31, 49.ult bees , 22, 30.ult bees , 59. It y losses , 59, siny losses , 60. OurBlack queen cell virus is another highly prevalent virus in the healthy colonies in this study. It has been reported as a common virus in adult European honey bees [ney bees , howeverney bees . We hypoN. apis [N. ceranae [BQCV has been detected with high titers in collapsed colonies . A recenN. apis , 29. As  ceranae .Acute bee paralysis virus, Kashmir bee virus and Israeli bee paralysis virus are three closely related viruses that were analyzed together in a single assay [Varroa mite [le assay . Almost roa mite , 21, 28.roa mite , 13, 62.roa mite , and obsroa mite , 30. TheChronic bee paralysis virus is the rarest detected virus, also amongst the sick colonies. CBPV is known as a disease of adult honey bees, mostly with outbreaks during high nectar flow [tar flow . We onlytar flow , 31.Several of the viruses we discuss above are considered serious pathogens in regard to their impact on colony survival , 12, 13.Varroa treatment, DWV seems to prevail in the colony and the infection level will build up again, when the Varroa population increases. Individual bees parasitized by Varroa mites, either in the pupal stage or as adults, exhibit extraordinary high viral titers [Varroa population in a colony increases, more and more bees carry such high infection levels and therefore the chance of including one of those bees in a random sample will also increase. Similar patterns are expected for the Acute bee paralysis virus complex, which may result in colony loss [For instance, even after successful l titers . As the ony loss .Varroa, may spread readily between colonies and is considered very contagious [On the other hand, a virus like CBPV, which is not vectored by ntagious , 66. We Varroa tolerance [Frequently, due to the non-normal distribution of virus titers over several magnitudes, problems arise when trying to statistically compare two sets of samples. Categorization of quantitative virus titers renders the results accessible to statistical evaluation beyond non-parametric tests and thus allows comparisons between different samplings. Such data enable monitoring the progression of a viral infection in a colony or apiary, or comparisons between different breeding lines in a selection program. Based on a sufficient number of observations, critical thresholds will emerge that can be used for management decisions . Especiaolerance , 67 info


The syndrome was renamed in the twentieth century after it was shown to differ from dengue. After an absence of ≈200 years, chikungunya returned to the American tropics in 2013. The virus is maintained in a complex African zoonotic cycle but escapes into an urban cycle at 40- to 50-year intervals, causing global pandemics. In 1823, classical chikungunya, a viral exanthem in humans, occurred on Zanzibar, and in 1827, it arrived in the Caribbean and spread to North and South America. In Zanzibar, the disease was known as kidenga pepo, Swahili for a sudden cramp-like seizure caused by an evil spirit; in Cuba, it was known as dengue, a Spanish homonym of denga. During the eighteenth century, dengue (present-day chikungunya) was distinguished from breakbone fever (present-day dengue), another febrile exanthem. In the twentieth century, experiments resulted in the recovery and naming of present-day dengue viruses. In 1952, chikungunya virus was recovered during an outbreak in Tanzania, but by then, the virus had lost its original name to present-day dengue viruses. The return of chikungunya virus to the Americas provides an opportunity to revisit the epidemiology of this zoonotic togavirus from Africa and to contrast it with that of dengue viruses, flaviviruses that are maintained as zoonoses in Southeast Asia. All of these viruses can be transmitted by Aedes aegypti and Ae. albopictus mosquitoes in an urban cycle. In the course of history, a remarkable name change has taken place because of the similarities between the clinical syndromes caused by dengue and chikungunya virus infections. The story of how the term dengue was originally applied to what we now call chikungunya and then subsequently applied to what we now call dengue should be well known by persons who deal with these 2 similar, but crucially different, global diseases. For more details on the name switch, the reader should consult the historical account by Carey  mosquitoes (Ae. aegypti mosquitoes, but not C. quinquefasciatus mosquitoes, are a biological vector of dengue virus (Why then did chikungunya lose and dengue gain a name? Throughout the nineteenth century, astute observers of outbreaks in the Americas and India recognized the clinical differences between dengue and breakbone fever, principally the duration of fever and the occurrence of post-illness arthritis (ue virus . During


Lates calcarifer) kept in maricultures in Southeast Asia. The most striking symptom of this emerging disease is the loss of scales. It was referred to as scale drop syndrome, but the etiology remained enigmatic. By using a next-generation virus discovery technique, VIDISCA-454, sequences of an unknown virus were detected in serum of diseased fish. The near complete genome sequence of the virus was determined, which shows a unique genome organization, and low levels of identity to known members of the Iridoviridae. Based on homology of a series of putatively encoded proteins, the virus is a novel member of the Megalocytivirus genus of the Iridoviridae family. The virus was isolated and propagated in cell culture, where it caused a cytopathogenic effect in infected Asian seabass kidney and brain cells. Electron microscopy revealed icosahedral virions of about 140 nm, characteristic for the Iridoviridae. In vitro cultured virus induced scale drop syndrome in Asian seabass in vivo and the virus could be reisolated from these infected fish. These findings show that the virus is the causative agent for the scale drop syndrome, as each of Koch’s postulates is fulfilled. We have named the virus Scale Drop Disease Virus. Vaccines prepared from BEI- and formalin inactivated virus, as well as from E. coli produced major capsid protein provide efficacious protection against scale drop disease.From 1992 onwards, outbreaks of a previously unknown illness have been reported in Asian seabass ( Lates calcarifer is a large, valuable fish kept in maricultures. Scale drop syndrome is an emerging disease in this species that currently results in significant economic losses for affected farms. Mortality rates can become as high as 50%, both in juvenile and adult fish. With the increasing mariculture of L. calcarifer, it is expected that economic losses due to the syndrome will increase as well. We provide conclusive evidence that the single causative agent for the syndrome is a newly identified member of the genus Megalocytivirus that has been designated scale drop disease virus.Asian seabass or  Lates calcarifer, Asian seabass, was first reported in 1992 in Penang, Malaysia, and since then outbreaks have been seen in Indonesia and in the Strait of Malacca. The phenotypic symptoms and pathology of the syndrome were described in detail by Gibson-Kueh et al.  / [total # control fish] * 100% = x %). The ‘percentage protected in the vaccine group’ was calculated as the number of survivors in the vaccine group, divided by the total number of vaccinated fish, multiplied by 100% . The RPS is formulated as RPS = 1-(% protection test / % protected control) or in a mathematical formulation RPS = 1-(x/y)* 100%.The relative protection percentage (RPS) in the vaccination-challenge study was calculated as follows. The ‘percentage protected control’ was calculated as the number of survivors in the control group, divided by the total number of control fish, multiplied by 100% , Institut Pasteur, 25 Rue du Docteur Roux, F-75724 Paris Cedex 15, France, under accession number CNCM I-4754)S1 Fig(PDF)Click here for additional data file.S2 FigThe deduced amino acid sequences of the major capsid protein encoding gene was aligned with the corresponding gene of all viruses of which this gene has been sequenced. The neighbor-joining method with pairwise deletion within the MEGA-5 package was used, bootstrap values (for 500 replications) are provided at the root of the clusters and the scale bar is a measure of the proportion of divergence. A • indicates SDDV.(PDF)Click here for additional data file.S3 Fig10log TCID50 was 6.5.Confluent monolayer at day 10 post inoculation of controls cells (A) and SDDV infected cells (B). Note round-up cells, which can clearly be distinguished from normal, attached cells on the monolayer. The culture was harvested and the (PDF)Click here for additional data file.S4 Fig10Log TCID50/mL) was incubated with 10% or 50% chloroform. A 100-fold decrease in infectivity was observed due to incubation with chloroform. The remaining infectivity of 5,5 10Log TCID50 /mL indicates that a lipid envelope is not essential for replication of the SDDV.Virus (7.5 (PDF)Click here for additional data file.S5 FigLane 1, Protein size marker, Lane 2 to 4: concentration range of BSA used for quantification 25-50-100 μg/lane; Lane 5: SDDV-MCP undiluted; Lane 6: SDDV-MCP 2x diluted.(PDF)Click here for additional data file.S1 TableN/A: Below the threshold, so no virus detected; POS: positive; NEG: Negative.(PDF)Click here for additional data file.S2 TableaSDDV: scale drop disease virus; ISKNV: infectious spleen and kidney necrosis virus; OSGIV: orange-spotted grouper iridovirus; RBIV: rock bream iridovirus; TRBIV: turbot reddish body virus; RSIV: red sea bream iridovirus; LCDV-1: lymphocystis disease virus 1; LCDV-C: lymphocystis disease virus China; SGIV: Singapore grouper iridovirus; GIV: grouper iridovirus; FV3: Frog virus 3; STIV: soft-shelled turtle iridovirus; TFV: tiger frog virus; ATV: Ambystoma tigrinum virus; EHNV: epizootic haematopoietic necrosis virus; IIV-6: invertebrate iridovirus type 6;IIV-3: invertebrate iridescent virus 3. bGene name according to Eaton et al. (reference [eference  in main (PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.


Early life respiratory viral infections and atopic characteristics are significant risk factors for the development of childhood asthma. It is hypothesized that repeated respiratory viral infections might induce structural remodeling by interfering with the normal process of lung maturation; however, the specific molecular processes that underlie these pathological changes are not understood. To investigate the molecular basis for these changes, we used an established Sendai virus infection model in weanling rats to compare the post-infection transcriptomes of an atopic asthma susceptible strain, Brown Norway, and a non-atopic asthma resistant strain, Fischer 344. Specific to this weanling infection model and not described in adult infection models, Sendai virus in the susceptible, but not the resistant strain, results in morphological abnormalities in distal airways that persist into adulthood. Gene expression data from infected and control lungs across five time points indicated that specific features of the immune response following viral infection were heightened and prolonged in lungs from Brown Norway rats compared with Fischer 344 rats. These features included an increase in macrophage cell number and related gene expression, which then transitioned to an increase in mast cell number and related gene expression. In contrast, infected Fischer F344 lungs exhibited more efficient restoration of the airway epithelial morphology, with transient appearance of basal cell pods near distal airways. Together, these findings indicate that the pronounced macrophage and mast cell responses and abnormal re-epithelialization precede the structural defects that developed and persisted in Brown Norway, but not Fischer 344 lungs. The inception of childhood asthma depends on interactions between environmental factors and development of the lung and immune system. For example, the relationship between infection with virus  and subsequent asthma onset is influenced by immune and lung factors A number of animal models have been developed to further explore the relationships among viral respiratory infection, innate immune dysfunction, and the development of chronic lower respiratory tract diseases. For example, the effects of respiratory syncytial virus have been extensively studied in neonatal and adult mice and rats Using a weanling rat model of virus-induced airway dysfunction, we have found that the development of a chronic asthma phenotype occurs in an “atopic” asthma susceptible strain  (Th2-like), but not in a non-atopic asthma non-susceptible strain  The rats were housed and all experimental procedures were performed in an American Association for Accreditation of Laboratory Animal Care-accredited laboratory animal facility at the University of Wisconsin School of Medicine and Public Health. The study was approved by the University of Wisconsin School of Medicine and Public Health Animal Care and Use Committee (assurance number A3368-01) and conformed to the Guide for the Care and Use of Laboratory Animals.Male BN rats  and F344 rats (Charles River) were purchased and housed within HEPA-filtered isolation cubicles . All invasive procedures were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.8 plaque-forming units (pfu) virus/ml, 2.5 ml of which was delivered into the chamber of a Glas-Col Aerosol Exposure Apparatus  over 20 minutes. The apparatus can accommodate up to 50 weanling rats per inoculation batch. Noninfected controls were sham-inoculated in the same manner, using an aerosol of sterile vehicle (PBS). In previous inoculations using the same virus in this weanling rat model, it was shown that the viral titer peaks at 3–5 days post-inoculation, and was largely cleared by day 7, the earliest time point assayed here Weanling (3–4 week old) BN and F344 rats were inoculated with aerosolized Sendai virus strain P3193 or vehicle (PBS) as previously described Lung fixation, processing, and embedding were performed as previously described Immunofluorescent staining was carried out using standard protocols and citric acid antigen retrieval. The primary antibodies used were: mouse anti-CD68 ED1 at 1∶100 (Abcam), mouse anti-mast cell tryptase at 1∶500 (Abcam), mouse anti-P63 4A4 (Santa-Cruz Biotechnology), and rabbit anti-KRT5 (Covance). The secondary antibodies used were goat anti-mouse CY3 1∶200 and goat anti-rabbit FITC (Jackson Immunoresearch).Total lung RNA was isolated using RNeasy Mini Kit (Qiagen). Two micrograms of total RNA from each of the 15 rats in each group was pooled  All analyses were carried out in R (R Development Core Team 2013).The Affymetrix probe level data was processed using Robust Multi-Array Analysis (RMA) to obtain normalized summary scores of expression for each probe set on each array EB-HMM was used to identify genes differentially expressed across multiple biological conditions over time. The approach has been previously described in detail EB-HMM extends EBarrays, an empirical Bayesian method for identifying differentially expressed genes at a single time point across two or more conditions EB-HMM uses a hidden Markov model to accommodate dependence in expression patterns over time and like EBarrays, for every gene, provides a gene-specific distribution that quantifies the posterior probability of that gene being in each particular expression pattern. The posterior probabilities may be used to generate false discovery rate (FDR) controlled lists of genes. Here, a gene is assigned into a particular expression pattern if its posterior probability of being in that pattern exceeds 0.95. This threshold was chosen as it controls the FDR at 5%.An empirical Bayes hierarchical modeling approach implemented in the R package PCA was performed using R function prcomp (R Development Core Team 2013). Prior to applying the PCA, the normalized gene expressions were rescaled to mean 0 and standard deviation 1 for each gene.allez. Gene-sets with Z-scores above 5 were considered enriched as suggested in the approach The random-set scoring method was used to measure the enrichment of gene sets defined by GO functional categories Il-1b, A2m, C3, Cfi, Tgfb3, and Gp2 were designed and purchased from Integrated DNA Technologies . Primers and probes were: β-actin F: GCCGTCTTCCCCTCCAT, β-actin R: AGGAGTCCTTCTGACCCATACC, β-actin probe: CCATCACACCCTGGTGCC: Il-1β F: CTGCAGCTGGAGAGTGTG, Il-1β R: CTCCACTTTGGTCTTGACTTCT, Il-1β probe: TCCCAAACAATACCCAAAGAAGAAGATGGA; A2m F: GAAGTGAGAGTGACAGTTCCAG, A2m R: GCTTCCCATAGGTGTATATTCCA, A2m probe: ACACGGACACATTCATCTCTTCCTCC; C3 F: GTGTAGGCACGCTGGTG, C3 R: CCTCGATCCTTAGTGTCGTTTG, C3 probe: TGACCCAAGAGATAACCGACAGCC; Cfi F: AGTTTGGCAGAGTGTACCTTTA, Cfi R: GACTTGTTGGGAAATCTGCATC, Cfi probe: TAGCACACTACACCTGCCAAGCC. All values were normalized to β-actin. Standard curves were generated by serial dilutions of cDNA from the lungs of a Sendai virus-inoculated rat and used to determine relative expression levels.A set of rats independent of those used for the microarray experiments was inoculated, harvested, and total RNA was prepared. RNA was reverse transcribed into cDNA using SuperScript III reverse transcriptase following the manufacturer's instructions . Quantitative real-time PCR was performed using an ABI 7500 Real Time PCR System . PrimeTime primer and probe sets for rat th to 5th day (D4 to D5) after exposure to the aerosolized virus. Both exhibited reduced body weight and delayed growth during the acute illness, with similar differences relative to the respective non-infected controls for the two strains (P>0.9 for strain-by-inoculation interaction), and mean body weight at D7 being 61% of the sham-inoculated controls (P<0.0001). To understand the morphological origins of BN airway obstruction, histological analyses were carried out on lung sections from infected and control rats of each strain  and harvested at 7, 10, 14, 21, and 35 days post-infection .BN rats, but not F344 rats, exhibit long-term airway obstruction following respiratory Sendai virus infection during the weanling period Histological analyses of infected and control tissue revealed clear temporal and strain-related differences in the appearance and resolution of specific histological features. D7 was characterized by a significant inflammatory response in both strains. BN-virus and F344-virus lungs exhibited terminal airway inflammation, necrosis, macrophage and neutrophil alveolar exudates, and perivascular edema -D. FocusEB-HMM analysis identified genes that were differentially expressed between BN-virus and F344-virus . Principal component analysis (PCA) of the data show that the transcriptomes of BN-virus lungs at D7 and D10, and F344-virus lungs at D7 and D10 are most different from the transcriptomes of lungs at other conditions and time points . e points . After Dallez. The number of differentially expressed genes between BN-virus and F344-virus increased from 200 at D7 to 408 at D35, while the number of significant GO terms decreased from 137 at D7 to 6 at D35 (Gene ontology (GO) analysis was then performed on differentially expressed genes using 6 at D35 . These oThe GO terms fell into two major categories: immune response-related genes and lung structure-related genes . We plotFor lung structure-related GO terms, “Collagen Catabolic Process” was most enriched at D7 and by D10 it was no longer significant . During Strain-related differences in virus-induced gene expression could arise from differences in baseline expression or from the magnitude of induction. To determine the relative contribution of each mechanism, directional differences (increase vs. decrease) were identified through inter-strain  and intra-strain  comparisons. We grouped genes into sixteen categories based on possible inter- and intra- strain expression differences . Here weEB-HMM), 172 genes were expressed at a higher level in BN-virus compared to F344-virus. It is interesting that when all 200 genes were subdivided into the sixteen categories, some categories contained no genes . This allowed us to visualize the dynamic gene expression changes related to infection and rat strain over time. Three overall temporal expression patterns emerged and the genes were manually sorted into these groups: an early D7 peak, a middle D10/14/21 peak, and a late D35 peak    ; and 5 Irf family members   associated with epidermal growth factor (EGF)/receptor tyrosine kinase (RTK) signaling, which has been implicated in virus-induced airway inflammation  ; 10 CCL ; Ccrl2) ; 6 Stat 5,7,8,9) . Similarammation  [35]. MaKrt4,5,14,19), as well as Mmp7, and Sprr1al (and A2m), a known inhibitor of inflammatory molecules , which are key genes in allergic responses Tslp which encodes a pro-inflammatory cytokine-like factor with polymorphisms linked to asthma susceptibility Cp, which encodes an antioxidant and free radical scavenger produced by the airway under stressful conditions such as endotoxin administration    [49]. ThCfi, which encodes a serine protease that can degrade activated complement components such as aforementioned C3  to assess gene expression levels at D7 and D21 in an independent set of BN-virus, F344-virus, BN-sal, and F344-sal rats. We assessed expression of Cd68, Cd14, and Cd163 were found in the D7 BN-virus peak group . Consistent with this pattern, multiple effecter molecules produced by CAMΦs, including Il6, Il1b, Ccl3/4, Tnfa, and Cxcl10, also displayed increased expression in BN-virus lungs, us at D7  [55], [5rus lungs. BN-virump3/9/13 . SimilarBN-virus  [57]. Hond Il4ra . In addi44-virus . Furtherus lungs . TogetheMcpt1/3/8/9/10, Cma1, Cpa3, Tpsab1, and Tpsb2  lungs and the incomplete repair, aberrant remodeling, and persistent chronic airway physiologic dysfunction in susceptible (BN) lungs. Our data reveal two major mechanisms: first, differences in the magnitude and composition of the immune response, and second, differences in the resolution and/or repair of the tissue damage, which together may cause the persistent airway morphological and physiological defects observed in infected BN lungs.It should be noted that the acute viral infection in this rat model is similar in time course and intensity for the two strains, with peak lung viral titers occurring at days 3–5 post inoculation, followed by a rapid clearance on days 6–8, with no virus detectable on plaque assay after day 8 CCL type chemokines/receptors, Stat, and Irf family members exhibited enhanced BN-virus expression peaks at D7 compared to F344-virus lungs. The D7 BN-virus peak gene expression demonstrated a significant macrophage signature early, which then transitioned to a mast cell signature at later time points (D10-21).Beginning from D7, the BN lungs mount an enhanced immune response compared to F344 lungs. Multiple interleukins, Il12 and Ifng in BN-virus lungs compared to F344-virus lungs at D7, previous work using the same weanling model has shown the opposite protein level change at D1-3 post-infection Macrophages have been implicated in a number of respiratory illnesses. For example, in mice, macrophages are a major player in mediating neonatal carbon monoxide-induced lung injury leading to bronchopulmonary dysplasia Mmp9, a key protease involved in cleaving TGFb pro-proteins, thereby activating its ability to signal Fn, Col1a1, Col5a1, Col5a3, Fap and Cdh11) Ctgf and Il6) Il1b, Tnfa, and Ptgs1) were also increased in BN-virus lungs Previous studies have shown that macrophages in BN-virus lungs exhibit higher TGFb1 expression relative to F344-virus lungs The mast cell gene expression and immunostaining data presented here is consistent with prior results of greater mast cell numbers in BN-virus compared to F344-virus lungs Mmp7expression, which has been shown to stimulate epithelial cell migration in the repair process following denudation in both the mouse and human trachea Upk3a, the lower airway progenitor marker, our results suggest that the increase in basal and UPK3A-positive progenitor cells may underlie the more effective and uniform repair in F344-virus airways compared to BN-virus airways.There is strong evidence that viral infection can cause epithelial barrier dysfunction A unique and persistent feature of the BN airway that may contribute to airflow obstruction and reduced lung function is the convolution and irregularity of the terminal airways The virus-induced rat model of airway dysfunction reported herein provided the impetus for the design of the Childhood Origins of ASThma (COAST) study, an ongoing high risk prospective birth cohort study that has explored both environmental (respiratory tract infections) and genetic (immune response) factors that contribute to the inception of childhood asthma Due to lack of samples, it has not been shown whether persistent lung or airway remodeling occur in human infants following rhinovirus or respiratory syncytial virus infections. However, both rhinovirus and respiratory syncytial virus have been shown to induce similar factors as observed in the rat model here. These include chemokines such as CXCL10 Figure S1Histological lung sections comparing global morphology of FS, BS, FV, and BV at representative time points. (A-L) Less magnified views of H&E stained lung sections of control F344-sal (FS) and BN-sal (BS) at D7  and F344-virus (FV) and BN-virus (BV) at D7 , D10 , D14 , D21  and D35 . Shown scale bars in micrometers (µm).(TIF)Click here for additional data file.Figure S2Principle component analysis (PCA). PCA was performed using transcriptome-wide gene expression profile. The x axis and the y axis show the first 2 principle components respectively. Four strains are shown in different colors.(TIF)Click here for additional data file.Figure S3Additional D7 BN-virus peak gene plots. Gene names indicated. F344-sal (FS) (green), BN-sal (BS) (black), F344-virus (FV) (blue), or BN-virus lungs (BV) (red).(TIF)Click here for additional data file.Figure S4Additional D7 BN-virus peak gene plots. Gene names indicated. F344-sal (FS) (green), BN-sal (BS) (black), F344-virus (FV) (blue), or BN-virus lungs (BV) (red).(TIF)Click here for additional data file.TIF Figure S5Additional D7 BN-virus peak gene plots. Gene names indicated. F344-sal (FS) (green), BN-sal (BS) (black), F344-virus (FV) (blue), or BN-virus lungs (BV) (red).(TIF)Click here for additional data file.Figure S6Additional D7 BN-virus peak gene plots. Gene names indicated. F344-sal (FS) (green), BN-sal (BS) (black), F344-virus (FV) (blue), or BN-virus lungs (BV) (red).(TIF)Click here for additional data file.Figure S7D7 BN-virus peak gene plots. Gene names indicated. F344-sal (FS) (green), BN-sal (BS) (black), F344-virus (FV) (blue), or BN-virus lungs (BV) (red).(TIF)Click here for additional data file.Figure S8Additional D7 F344-virus peak gene plots. Gene names indicated. F344-sal (FS) (green), BN-sal (BS) (black), F344-virus (FV) (blue), or BN-virus lungs (BV) (red).(TIF)Click here for additional data file.Figure S9Additional D10/14/21 BN-virus peak gene plots. Gene names indicated. F344-sal (FS) (green), BN-sal (BS) (black), F344-virus (FV) (blue), or BN-virus lungs (BV) (red).(TIF)Click here for additional data file.Figure S10Additional D10/14/21 BN-virus peak gene plots. Gene names indicated. F344-sal (FS) (green), BN-sal (BS) (black), F344-virus (FV) (blue), or BN-virus lungs (BV) (red).(TIF)Click here for additional data file.Figure S11Additional D10/14/21 F344-virus peak gene plots. Gene names indicated. F344-sal (FS) (green), BN-sal (BS) (black), F344-virus (FV) (blue), or BN-virus lungs (BV) (red).(TIF)Click here for additional data file.Figure S12Additional classically activated macrophage marker gene plots. Gene names indicated. F344-sal (FS) (green), BN-sal (BS) (black), F344-virus (FV) (blue), or BN-virus lungs (BV) (red).(TIF)Click here for additional data file.Figure S13Additional classically activated macrophage effector molecule gene plots. Gene names indicated. F344-sal (FS) (green), BN-sal (BS) (black), F344-virus (FV) (blue), or BN-virus lungs (BV) (red).(TIF)Click here for additional data file.Figure S14Additional alternatively activated macrophage-related gene plots. Gene names indicated. F344-sal (FS) (green), BN-sal (BS) (black), F344-virus (FV) (blue), or BN-virus lungs (BV) (red).(TIF)Click here for additional data file.Figure S15Additional mast cell-related gene plots. Gene names indicated. F344-sal (FS) (green), BN-sal (BS) (black), F344-virus (FV) (blue), or BN-virus lungs (BV) (red).(TIF)Click here for additional data file.Figure S16Additional ciliated cell marker gene plot. F344-sal (FS) (green), BN-sal (BS) (black), F344-virus (FV) (blue), or BN-virus lungs (BV) (red).(TIF)Click here for additional data file.Table S1List of all differentially expressed genes at each time point.(XLSX)Click here for additional data file.Table S2List of all significant GO groups at each time point.(XLSX)Click here for additional data file.Table S3List of D7 differentially expressed genes by category.(XLSX)Click here for additional data file.Table S4GO terms for D7 Category 1 and Category 2 genes.(XLSX)Click here for additional data file.Table S5List of genes in the shape plot categories.(XLSX)Click here for additional data file.


Integrated community case management (iCCM) can be an effective strategy for expanding the provision of diarrhea, pneumonia, and malaria services to children under 5 years old but there are concerns in some countries about the corresponding cost and impact. This paper presents and compares findings from a multi–country analysis of iCCM program costs.Data on coverage, utilization, and costs were collected as part of two sets of studies conducted between 2011 and 2013 for iCCM programs in seven sub–Saharan African countries: Cameroon, the Democratic Republic of the Congo, Malawi, Senegal, Sierra Leone, South Sudan and Zambia. The data were used to compare some elements of program performance as well as costs per capita and costs per service .Among the seven countries, iCCM utilization ranged from a total of 0.26 to 3.05 contacts per capita (children 2–59 months) per year for the diseases treated, representing a range of 2.7% to 36.7% of the expected numbers of cases. The total recurrent cost per treatment ranged from US$ 2.44 to US$ 13.71 for diarrhea; from US$ 2.17 to US$ 17.54 for malaria (excluding rapid diagnostic testing); and from US$ 1.70 to US$ 12.94 for pneumonia. In some of the country programs, the utilization of iCCM services was quite low and this, together with significant fixed costs, particularly for management and supervision, resulted in services being quite costly. Given the differences across the countries and programs, however, these results should be treated as indicative and not definitive.A comprehensive understanding of iCCM program costs and results can help countries obtain resources and use them efficiently. To be cost–effective and affordable, iCCM programs must be well–utilized while program management and supervision should be organized to minimize costs and ensure quality of care. iCCM programs will not always be low–cost, however, particularly in small, remote villages where supervision and supply challenges are greater. Further research is needed to determine the cost–effectiveness of iCCM programs and corresponding patient and service delivery costs. Due to limited access to effective treatment, diarrhea, malaria and pneumonia remain the leading causes of child mortality in sub–Saharan Africa and result in nearly 41% of global deaths in children under five years old [To be effective, iCCM services must be available from a single provider (“one–stop shopping”) within 24 hours of the onset of symptoms. For example, if a child has a fever, the parent should be able to see a CHW in his or her community within 24 hours and the CHW should be able to provide diagnosis and treatment if the case is simple and refer the case if it is not. The integration of these services is important – there is growing evidence that this increases the utilization of malaria and pneumonia treatment [Despite the reported success of iCCM in several low– and middle–income countries, it has yet to be implemented as a national strategy in some other countries. This is partly due to concerns about the costs and financing of iCCM programs and the justification of the extra investment in terms of the related health outcomes. A comprehensive understanding of the costs and results will help countries who are considering implementing or expanding iCCM programs to advocate for funding and to plan and budget appropriately. It will also allow for costs to be better monitored and controlled, thus contributing to the efficient use of scarce resources.This paper describes and compares the results of iCCM cost analyses conducted under two separate sets of studies in seven sub–Saharan African countries.The cost analyses were conducted between 2011 and 2013. The first two studies were conducted of national programs in Malawi and Senegal in 2011 and 2012 as part of the testing of an iCCM costing and financing tool under the United States Agency for International Development (USAID) Translating Research into Action Project, and these countries were selected because they have mature iCCM programs and sufficient data. A third study conducted in Rwanda was excluded because data were not comparable. The second set of five studies was conducted of sub–national programs in Cameroon, Democratic Republic of Congo (DRC), Sierra Leone, South Sudan, and Zambia in 2013 with funding from the Bill and Melinda Gates Foundation (BMGF). These five countries were selected by BMGF to estimate the costs of five iCCM projects funded by another international donor. The areas where these five studies were conducted were, reportedly, based on need and feasibility, and all included areas with hard–to–reach populations.Data were obtained from records and through interviews with the CHWs who provided iCCM services, their supervisors, and program managers. A standard questionnaire was used for the interviews. The samples of districts, health centers, and communities were selected purposefully in terms of access and availability of health facility staff and CHWs. Time limitations and access constraints, such as poor road conditions, meant that some samples may not have included health facilities and CHWs from very remote areas. The samples were relatively small but were sufficient to validate service delivery protocols and to collect data on the work and supervision of the CHWs. An average of 12 health centers were visited in each country, and interviews were conducted with an average of three CHWs per health center, totaling approximately 36 CHWs in each country.www.msh.org/iccm and is described in detail in the individual country studies). At the service delivery level, this is a bottom–up, activity–based costing tool, in which standard costs are used to estimate total direct costs per service. Indirect costs, such as supervision and training, are then allocated based on CHW time estimates using a top–down methodology. The resulting figures are a mixture of standard and actual costs, obtained from accounting and budget records and through interviews, in what is sometimes known as an “ingredients” approach [The costs were analyzed using the USAID iCCM Costing and Financing Tool , supervision, supply chain, CHW remuneration, user fees, and other aspects. These many differences limit the usefulness of direct comparisons of the findings, but add richness to the analysis. More information on the country studies can be found in the individual country reports –13 – briMalawi’s national iCCM activities began in 10 districts in 2008 and, with support from donors, were scaled–up throughout the country by 2010. CHWs were remunerated by the government and were expected to spend two days per week on iCCM activities at village clinics and to participate in active case finding through household visits. iCCM services comprised the treatment of malaria, diarrhea, pneumonia, and red eye as well as the identification and referral of anemia and malnutrition. The costing study was based on a sample of iCCM services in the 2328 hard–to–reach communities covered by iCCM.Senegal’s iCCM program started in 2003. Services were provided through USAID’s Community Health Program which covered the whole country in collaboration with the Ministry of Public Health (MoPH). Services were provided at health huts in the communities and were meant to cover rural, remote areas that did not have health posts (the lowest level of facility). The iCCM service package included rapid diagnostic tests (RDTs) and malaria treatment, and diarrhea and pneumonia treatment. User fees were charged and patients were supposed to purchase the medicines; the prices included a mark–up of 5% to 25%. The funds were intended to be used to replenish stocks and to cover other costs.Cameroon, a local NGO, in collaboration with the MoPH, began implementing an iCCM project in 2009 in two remote districts – Nguelemendouka and Doumé. Through this project, volunteer CHWs provided free treatment to children between the ages of 2 to 59 months for cases of malaria and diarrhea. Treatment for pneumonia was added in Nguelemendouka District in 2013.In Democratic Republic of the Congo (DRC), implementation of a national iCCM program began in 2005 under the leadership of the MoPH. In 2010, iCCM services were expanded to include family planning services, and the MoPH mandated that malaria treatment be integrated at community sites, along with pneumonia and diarrhea treatment services. The focus of the costing study was the iCCM component of a project in 9 of the 16 health zones of the Sud–Ubangi District in Equateur Province. The project started in 2010 and was implemented by a local NGO in coordination with the MoPH.In the Sierra Leone, an international NGO led an iCCM project which began in Kono district in May 2006. Unpaid Community Health Volunteers provided free treatment to children ages 2 to 59 months for malaria, diarrhea, and pneumonia. Starting in September 2013, the plan was to expand the role of the CHW to include the delivery of community–based maternal and newborn health care interventions.In South Sudan, an international NGO began implementation of an iCCM program in 2009 in hard–to–reach areas in five states and ten counties. These include Kapoeta North County which was selected for the costing study. Unpaid Community–Based Distributors provided free treatment to children ages 2 to 59 months for cases of malaria, diarrhea, and pneumonia.In Zambia, volunteer CHWs began conducting iCCM activities in four districts of Luapula Province in late 2010, then scaled up to all seven districts in 2012, serving a population of 741 373 in remote communities. The iCCM package included RDTs and treatment of pneumonia, diarrhea and malaria, and was implemented in areas where access to health facilities and services was limited. The program had a demand generation element, including having CHWs conduct behavior change communication activities. The project was managed by an international NGO working closely with the Ministry of Health (MoH) as part of a national iCCM program being implemented by the MoH across the country, although this project may not have been completely representative of the overall MoH program.In A summary of key elements of the iCCM programs studied are shown in The package of iCCM services varied across the programs, with only six of the seven covering the three illnesses in an integrated way . These comparisons should be treated as indicative, as estimating the catchment populations in the hard–to–reach areas was difficult. A major difference was the treatment of malaria (diagnosed or presumptive) which accounted for higher numbers of treatments in Zambia, Sierra Leone and, to some degree, in the DRC. Numbers of referrals were only available in DRC, South Sudan and Zambia and amounted to 0.11, 0.01, and 0.38 per child per year, respectively. In South Sudan and Zambia these figures translate to about 1% and 14% of total cases, respectively. A rule of thumb used by some providers is that around 10% of cases need to be referred – a referral rate that is too low may indicate that the provider is treating too many severe cases, whereas one that is too high may indicate a lack of medicines or supplies or a lack of confidence.The costs of starting an iCCM program can include the development of plans, policies, guidelines and training materials– most of which are generally financed by the national government and/or partners. For this study, we only took into account the training and equipping of CHWs . All costs for these activities were included irrespective of who incurred or funded them. If the training included more health topics than iCCM, we only included the proportion related to iCCM. The start–up costs for training and equipment were mostly in the range from US$ 202 to US$ 352 per CHW, with Malawi and Zambia being outliers at US$ 1058 and US$ 897, respectively . In Malawi, costs were higher because they included a portion of general CHW training and an incentive payment. In Zambia costs were higher because they included training–of–trainers and supervisors, the training was longer than in the other countries, and per diem rates were high relative to those in other countries.The reported annual CHW attrition rates varied significantly across the study sites, ranging from 2% in Malawi (where they are remunerated) to an estimated 10% in South Sudan . The costs of training and equipping replacement CHWs can be significant, as shown above, with most costs in the range of US$ 202 to US$ 352 per provider.While it is possible to amortize non–recurrent costs over the expected period of use and include them with recurrent costs, this was not done here as it would have been difficult to estimate certain aspects, such as the length of the use of equipment or how long a CHW will work after the initial training.A comparison of recurrent costs can provide meaningful perspectives on the resourcing, equity, and efficiency of service provision and support, and can provide input into cost–effectiveness analyses, involving comparisons of costs per output or outcome. Recurrent costs are those repeated on an ongoing basis and, in this study, include medicines and supplies, management, supervision, and refresher training. These costs are expressed in two ways: per capita and per service. The costs of the training and equipping of replacement CHWs were not included in recurrent costs in these studies although it would be reasonable to do so.Per capita recurrent costs are calculated here by dividing the total recurrent cost by the number of children in the catchment population. With the exception of the costs of medicines and supplies (which represent estimates of the quantities consumed), these figures represent the iCCM resources made available to the catchment populations. Per service recurrent costs, on the other hand, are calculated by dividing the total recurrent cost by the number of services provided. These figures represent the iCCM resources that should have been used in providing a single service. The ratio between per–capita and per–service costs is the same as the rate at which services are used per capita.Medicines and supplies are variable costs which change based on the numbers of services provided. Provider remuneration, management, supervision, refresher trainings and other similar costs are generally fixed and do not vary with the number of services provided. It is important to note that the average total costs of medicines and supplies vary with the mix of services provided as well as with the unit costs of the various medicines. So if a greater proportion of services with higher–cost medicines is used, the average costs across all services will be higher.The average total recurrent costs per capita (children aged 2–59 months) were much lower for the two national programs , which were US$ 2.16 and US$ 2.07, respectively, than for the four sub–national programs with the complete iCCM package , which ranged from US$ 5.50 to US$ 10.20 . These costs depended mainly on the frequency of meetings, per diem rates, and amounts reimbursed to CHWs for transport. Refresher training was sometimes provided as part of the routine supervision system or through meetings – in others it was provided as a separate dedicated training activity. In the programs where it was a separate activity, the average cost per capita ranged from US$ 0.09 to US$ 0.75.Recurrent costs can be more meaningfully compared by type of service  since the average total costs across all services are affected significantly by variations in service mix. Diarrhea treatment was the only service provided in all the studies and the recurrent cost ranged from US$ 2.44 per service in Malawi to US$ 7.80 in South Sudan  and the indirect costs  across the service types. The estimates of time used in the studies were obtained through CHW interviews and ranged widely, for example, from 26 minutes to 91 minutes for pneumonia diagnosis and treatment in Malawi and Sierra Leone, respectively. Such figures should be treated with caution as in some cases the CHWs had difficulty in understanding the concept of estimating the times spent in diagnosing and treating patients. They can also be affected by the way the time is estimated, for example, if a CHW includes the time taken to travel to the house of the patient. It is important to note that some CHWs reported making follow–up visits to patient’s homes and this was not generally taken into account.The reported unit cost per episode for tests and medicines also varied across the study sites, for example ranging from US$ 0.04 to US$ 0.56 for pneumonia treatment in Senegal and Zambia, respectively  and many also provide other voluntary health services. Factors influencing the demand for services include the size of the catchment population, the incidence of the illnesses, the distance a person’s home to the place where the CHW is based, perceptions of quality of care, and the availability of medicines.The average catchment population of children (aged 2–59 months) per CHW differed considerably, with CHWs in Sierra Leone covering an average of 38 children and CHWs in Malawi covering an average of 454 children , as shown in the same table, and the higher rates are a concern since experienced, skilled providers may be lost and the cost of training and equipping replacements can be high.The CHWs were remunerated in the two national programs but not in the five sub–national programs and it is notable that the attrition levels were lower in the national programs. There does not, however, seem to be a relationship between CHW remuneration and the numbers of iCCM cases seen, and a deeper analysis would be needed to explore this due to the contextual factors.User fees were charged to patients in one of the national programs  and the average numbers of iCCM cases seen were low. Again, a deeper analysis would be needed to try to determine if there was a relationship between user fees and utilization levels.Other studies have been conducted which add value to the discussion of CHW costs. In particular, an overview of community health workers by Perry et al  which exStudies have also examined how patient costs, such as transport and user fees, can restrict or delay access to health services and can negatively impact on a poor family’s financial situation, as well as indicating how iCCM can alleviate this economic burden by bringing services closer to the family. A study in Uganda showed, for example, that community treatment of malaria and pneumonia resulted in significant cost–saving for rural, poor communities, who would otherwise lose productive time travelling to health facilities . And a sTo have maximum impact on child health and mortality, iCCM services should be available within 24 hours of the onset of illness and from a single provider (“one–stop shopping”). This is especially important if there are co–morbidities. If the case is complex or severe, the CHW should be able to refer the case to the nearest health facility and help arrange transport, if necessary.It is clear that effective iCCM can reduce morbidity and save lives but for the services to be widely accepted and implemented by governments, they must also be affordable and cost–effective. Based on this analysis, there are two main factors that affect this: utilization of services, and management and supervision costs.The analysis shows that low utilization of iCCM services contributes significantly to high unit costs per service, as fixed supervision and management costs have to be absorbed by fewer cases. The results indicate that iCCM services may have been under–utilized in several of the programs, with less than 20% of the expected number of episodes of illness seen by CHWs and, in some cases, CHWs seeing less than one case per week. Low utilization can also be an issue in terms of quality of care because a provider should see sufficient cases per month to build and retain the necessary experience and skills.Utilization depends partly on the number and types of service included in the iCCM package. In Cameroon, for example, where pneumonia was not treated as part of iCCM in the year of the study, overall utilization was low and this contributed to a higher average unit cost per service. On the other hand, including other services, such treatment of red eye in Malawi, increased utilization and contributed to the lower average unit cost per service. The degree to which other services can or should be added is, however, an important topic that is beyond the scope of this paper.Utilization is affected by several other factors such as the size of the catchment population, incidence of illness, CHW access and availability, perceptions of medicine supply and quality of care and perhaps, in some cases, user fees as well. In some of the programs, utilization was low because the catchment populations were small, for example in Sierra Leone where each CHW only covered an average of 38 children. Also, incidence rates were lower in some program areas, such as in Malawi, with 5.3 expected episodes per child per year, compared with 9.6 in Senegal.The availability of the CHWs does not appear to have been a reason for low utilization, since less than 20% of the reported available time was used for iCCM in 6 of the 7 programs. However, it appears likely that medicine stock–outs have been a factor since this was reported as a problem in several of the studies. The studies did not seek to determine if user fees had an impact on utilization in Senegal, which was the only program that had them, and the results did not indicate any obvious relationship.The lack of maturity of programs was a possible factor in the low utilization levels seen in three of the sub–national programs that had been running for three years or less. At the 2014 iCCM Symposium in Accra, Ghana, it was noted that it can take at least 3 years before an iCCM program reaches maturity in terms of utilization of services and it may take at least 12 months of implementation at scale (with greater than 80% of CHWs trained) to have higher utilization [As noted above the other key factor in terms of iCCM program costs is management and supervision. In the five sub–national programs, this was over 70% of the total recurrent costs and even though it was much lower in the Malawi national program it was 79% in Senegal, where the national program was run by an international organization. It is understandable that the costs of setting up and running pilot projects can be relatively high and even more so if they are run by local or international organizations. These costs should become much lower in relative terms if the programs are scaled–up and taken over by the government. Nevertheless all programs should aim to minimize these costs while maintaining good support for the CHWs so that the availability and quality of iCCM is optimal. Costs can be minimized, for example, by integrating supervision , by combining supervision with outreach visits where additional curative services are provided by the supervisor during the visit, and by using local peer supervisors to supplement professional supervisors.Another key program cost relates to replacing CHWs who stop working. The cost of training and equipping new CHWs can be significant and is often not budgeted. Moreover, the loss of experienced, knowledgeable CHWs can affect the performance of the program. Attrition rates were 5% or more in all of the programs where CHWs were not remunerated, compared with 2% in Malawi where they were remunerated.In terms of the impact of remunerating CHWs, there was not enough information to assess whether the additional costs were outweighed by increased utilization and reduced attrition, but that is a possibility that is worth exploring in other studies.The additional costs of iCCM may be offset to some degree by savings elsewhere in the health system. As mentioned previously, there is some evidence that iCCM can reduce workload at primary health centers, and cost savings should also be achieved by treating cases before they become severe. In addition, there is evidence that iCCM results in savings to families with sick children. Unfortunately, it was not possible to investigate these possibilities in the costing studies.Finally, it is important to note that iCCM programs were sometimes established as a transition strategy to save lives because primary care facility services were weak. Where this is the case, it should be accepted that iCCM services will be costly and may be unsustainable until primary health facilities are fully functional, taking into account that they need to provide the supervision and support  and serve as reliable referral units for severe cases. In small, hard–to–reach communities, however, iCCM will probably be the most cost–effective way to provide services in the long term, even if they are costly.There were a number of limitations to the studies that could have affected the results and which necessitate the need for caution in interpreting and comparing them. The most significant overall limitation is that the studies were carried out at different time periods in seven very different countries which were selected purposively for reasons other than cross–country comparisons. Other limitations include the following. Some of the sub–national programs only started in 2010, and the use of data from 2011 and 2012 to measure costs and efficiency may be premature as it can sometimes take 3 years before programs reach maturity in terms of utilization of services. The samples of facilities and communities were relatively small and were limited in terms of remote communities. Recurrent costs may be underestimated because of lack of complete information on services provided, such as follow–up visits, numbers of referrals, and treatment of fever which is not diagnosed as malaria or pneumonia. Costs do not include the removal of bottlenecks and other health system strengthening activities or economic costs, such as the value of a voluntary CHW’s time, family out–of–pocket costs or income losses due to treatment seeking. Finally, the measurement of costs and efficiency depend significantly on CHWs’ estimates of time available and time needed for services, and some inaccuracy in these estimates is likely.While there are a growing number of studies on iCCM costs, additional analyses are needed to assess the cost–effectiveness of iCCM. Such analyses are important in making a stronger case that iCCM is a worthwhile investment, while simultaneously helping to determine the most affordable ways to provide quality services. There is a need to look at the role of iCCM within the primary health care system, not as an alternative to facility–based or other community services, but as an effective way of providing treatment for key childhood illnesses in hard–to–reach communities. It is important to take into account patient financial and economic costs as well as service provision costs, and to include factors such as timeliness, quality and appropriateness of treatment. There is also a need to look at the costs of removing bottlenecks, including the costs of improving medicine supply and demand generation, as well as the impact of CHW remuneration and the impact of charging patients for services. Supervision and management can be costly element of iCCM, and the cost–effectiveness of strategies to minimize these costs should be explored. Analysis of financing and financial sustainability is also needed, including the use of medicine sales to patients as a way of financing supplies. Finally, system improvements are generally needed to ensure the availability of routine iCCM and CHW service data, which is necessary for in–depth analysis and performance monitoring.The results of this analysis show that in order to be cost–effective and affordable, iCCM programs must be well–utilized and management and supervision must be organized in a way that minimizes cost while ensuring quality of care. This requires the removal of bottlenecks, such as medicine stock–outs, and of any barriers to access. It also requires activities that encourage the utilization of iCCM services such as the promotion of those services and the involvement of community leaders. To minimize the costs of iCCM management and supervision it is important they are an integral part of the routine systems under which, for example, a supervision visit to a community covers multiple health services, not just iCCM.In some cases, however, it must be accepted that iCCM will not be low–cost even if the CHWs are volunteers. For example, a sub–national iCCM program that is established by an NGO to save lives because facility–based services are weak is likely to be relatively costly until the health system is strengthened. And in the case of small, remote villages, while iCCM is likely to be the most cost–effective way to provide services, it may never be low–cost because of the supervision and supply challenges.


We report a new approach to constructing a peptide–polysaccharide hybrid hydrogel via the calcium-ion-triggered co-assembly of fluorenylmethyloxycarbonyl-diphenylalanine (Fmoc-FF) peptide and alginate. Calcium ions triggered the self-assembly of Fmoc-FF peptide into nanofibers with diameter of about 30 nm. Meanwhile, alginate was rapidly crosslinked by the calcium ions, leading to the formation of stable hybrid hydrogel beads. Compared to alginate or Fmoc-FF hydrogel alone, the hybrid Fmoc-FF/alginate hydrogel had much better stability in both water and a phosphate-buffered solution (PBS), probably because of the synergistic effect of noncovalent and ionic interactions. Furthermore, docetaxel was chosen as a drug model, and it was encapsulated by hydrogel beads to study the in vitro release behavior. The sustained and controlled docetaxel release was obtained by varying the concentration ratio between Fmoc-FF peptide and alginate. Hydrogels are a class of soft materials with attractive applications in the field of biomedicine because of their high water content, biocompatibility, and tissue-like elastic properties –4. PartiOne potential solution is to introduce other functional components into the self-assembled peptide systems to form complex hydrogels. Nanoparticles , graphenπ–π stacking, hydrogen bonding) and ionic interactions when compared with peptide or alginate hydrogel alone.The polysaccharide–protein complex widely exists in biology and contributes to many unique functions of biological organisms, such as chondroitin sulfate proteoglycan. This has greatly inspired us to develop a new peptide–polysaccharide hybrid hydrogel with a specific set of the desired properties. Herein, alginate, a kind of natural polysaccharide, is proposed as a hybrid component in view of its good hydrophilicity, high water-absorption ability, and ease of gelation through ionic crosslinking , 31. How2 solution to trigger co-assembly of Fmoc-FF peptide and alginate, and the morphological structures of the resulting hybrid hydrogel beads were also characterized. Furthermore, the release kinetics of Fmoc-FF–alginate hydrogel beads was evaluated, using docetaxel as a drug model, for its potential application in drug delivery. We also investigated the effect of concentration ratio of Fmoc-FF and alginate on the release of docetaxel.In this study, we created a new peptide–polysaccharide hybrid hydrogel via calcium-ion-triggered co-assembly of Fmoc-FF peptide and alginate. The physicochemical properties including morphology, stability, and supramolecular structures were investigated. For comparison, hydrochloric acid (HCl) was introduced into a CaClMw: 32–250 kDa) was obtained from Aladdin Industrial Corp. . Docetaxel (purity >98 %) was purchased from Cenway Pharmaceutical Co. . All other chemicals, including calcium chloride, sodium hydroxide, Tween 80 (polysorbate 80), and acetonitrile, were of analytical grade and were obtained from commercial sources.The lyophilized form of Fmoc-FF peptide was purchased from Bachem . Alginate (2 (2 wt.%) solution was slowly added to 1 mL of the fresh Fmoc-FF stock solution, and the mixture was aged at room temperature for 2 h without disturbance, leading to formation of transparent hydrogels.In a typical experiment, 3 mg of Fmoc-FF was added to 1 mL of deionized water and homogeneously dispersed by ultrasound for 15 min, after which, it was solubilized by dropwise addition of 0.5 M NaOH. Next, 2 mL of a CaCl2 (2.0 wt.%), using a syringe needle with an inner diameter of 0.7 mm. Hydrogel beads could be formed in a few seconds when the Fmoc-FF–alginate liquid droplets were brought into contact with the CaCl2 solution. In addition, we used an aqueous solution of CaCl2-HCl (pH = 6) as a control to study the effect of HCl on the structure of the hydrogel beads.First, fresh Fmoc-FF stock solution (3 mg/mL) was prepared by the above method. Next, 3 mg of alginate powder was slowly added to 1 mL of the fresh Fmoc-FF stock solution and stirred (1000 rpm) at room temperature for 30 min, resulting in a uniform and translucent mixed solution. The mixture was then centrifuged at 2000 rpm for 2 min to remove bubbles. Finally, the Fmoc-FF–alginate solution was added drop by drop to an abundant volume of an aqueous solution of CaClw/v) Tween 80 and 0.02 % (w/v) NaN3. The vials were then incubated in a rotary shaker at 100 rpm and 37 °C to carry out the stability test. After 24 h, the unbroken hydrogel beads were collected by filtering and then freeze-dried. The retention of hydrogels was calculated based on following equation:Fmoc-FF or Fmoc-FF–alginate hydrogel beads were placed inside small vials of water or buffer solution , each containing 0.1 % , and 1.5 mg of alginate was dissolved in 15 mL of ddH2O. The pH values of the resulting solutions were adjusted with 0.5 M NaOH or 0.1 M HCl. The pH values of all the solutions were measured using a pH meter.A Zetasizer Nano ZS  was used to measure zeta potential of the Fmoc-FF peptide solutions and alginate solutions. To prepare fresh solutions, 1.5 mg of Fmoc-FF peptide was dissolved in 15 mL of double-distilled water (ddH2 (2.0 wt.%) using a syringe needle with an inner diameter of 0.7 mm to form drug-loaded hydrogel beads. The docetaxel-loaded hydrogel beads were then placed inside small vials containing 8 mL of PBS solution , 0.1 % (w/v) Tween 80, and 0.02 % (w/v) NaN3. The vials were incubated in a rotary shaker at 100 rpm and 37 °C to perform the drug release experiments. An aliquot of 0.5 mL of the supernatant solution was removed from each vial at the designated sampling intervals and replaced with the same amount of fresh buffer. The docetaxel concentration in the supernatant solution was analyzed by high-performance liquid chromatography (HPLC). All the samples were centrifuged at 10 000 rpm for 5 min before they were tested by the chromatograph. The HPLC measurements were performed on an Agilent 1200 HPLC system  operating with an Aglient Eclipse XDB-C18 column. The parameters used in the HPLC analysis were as follows: the mobile phase consisted of acetonitrile water ; the wavelength of UV detector was set at 229 nm; flow rate was set at of 1.0 mL min–1; the injection volume was 20 μL.The release of docetaxel from different hydrogel beads was carried out in the PBS buffer at 37 °C. The docetaxel-loaded hydrogel beads were prepared according to the following protocol: Docetaxel was first dissolved in the solvent dimethyl sulfoxide (DMSO) with a concentration of 45 mg/mL. Next, 20 μL of the docetaxel-loaded DMSO solution was added to 3 mL of the fresh Fmoc-FF or Fmoc-FF–alginate stock solution, and the resulting mixture was added to 10 mL of an aqueous solution of CaCl–1 with 20 scans and a resolution of 4 cm–1.The morphologies of the hydrogels were characterized using a S-4800 field-emission scanning electron microscope  at an acceleration voltage of 3 kV. All samples were freeze-dried and sputter-coated with platinum using an E1045 Pt-coater  before characterization. Fourier-transform infrared (FTIR) spectra of Fmoc-FF, alginate, and Fmoc-FF–alginate hydrogels were recorded on a Nicolet-560 FTIR spectrometer . The hydrogels were freeze-dried and deposited on the surface of the KBr plate, and the FTIR spectra were recorded across the range of 400–4000 cm2, the alginate could be rapidly crosslinked by calcium ions. On the other hand, the Fmoc-FF molecules could self-assemble into nanofibers via noncovalent interactions because their charge was decreased by the calcium ions. The crosslinked alginate molecules were interwoven with the Fmoc-FF fibrous network, resulting in the formation of hybrid hydrogel beads.In this study, we tried to fabricate a new peptide–polysaccharide hybrid hydrogel with a set of desired properties. Figure 2+ ions. As shown in Fig. We first studied the self-assembly behavior of Fmoc-FF peptide triggered by Ca2 to trigger co-assembly. The formation of hydrogel beads was observed within a few seconds when the Fmoc-FF–alginate liquid droplets made contact with the CaCl2 solution. These hydrogel beads had a diameter of approximately 1 mm and showed good uniformity , a porous fibrous network, in which Fmoc-FF nanofibers with different diameters were interwoven with the lightly crosslinked alginate, was observed on its surface  occurred among the hybrid hydrogel beads. For the Fmoc-FF hydrogel beads, the bead retention increased from 44.0 to 94.5 % as the alginate amount was varied from 0 to 3 mg/mL in water, demonstrating that the introduction of alginate could enhance the stability of the Fmoc-FF hydrogel. For the alginate hydrogel, the retention increased from 28.2 to 78.9 % as the Fmoc-FF concentration was varied from 0 to 3 mg/mL in PBS. In addition, self-assembled Fmoc-FF nanofibers improved the salt tolerance of the alginate hydrogel. Combining the findings of enhanced stability in water and salt tolerance in the PBS buffer, we could infer that the synergistic effect of noncovalent  and ionic interactions contributed to the high stability of the Fmoc-FF–alginate hybrid hydrogels.The stability of hydrogel beads was evaluated by immersing them in water and a phosphate buffer solution (PBS) with pH of 7.4, both at a temperature of 37 °C. As shown in Fig. −1 in the amide I region for the Fmoc-FF hydrogel, suggesting an anti-parallel β-sheet arrangement of Fmoc-FF peptide [−1, which is attributed to the large amount of the carboxylic acid group (–COO−). For the hybrid hydrogel, one obvious peak appears at 1692 cm−1 and a relatively broad peak appears at 1625 cm−1, indicating that alginate did not change the molecular arrangement of Fmoc-FF peptide in the co-assembly process.The zeta potentials of Fmoc-FF peptide and alginate were measured at different pH values to analyze the deprotonated degree of carboxylic acid and hydroxyl Fig. . Because peptide , 32. AlgThe high stability of these hydrogel beads may allow them to deliver good performance in drug delivery. Docetaxel was chosen as a drug model, and docetaxel-loaded hydrogel beads were prepared to investigate how the drug would be released. As shown in Fig. The in vitro release behavior of the docetaxel from the Fmoc-FF–alginate hydrogel beads was then studied using Fmoc-FF hydrogel beads as a control. As shown in Fig. In order to better understand the release mechanism of docetaxel from the hydrogel beads, the following Korsmeyer–Peppas equation was employed to fit the release profiles: , 352\docMt/M∞ represents the fraction of drug released from hydrogel beads at time t, k represents the kinetic constant characteristic of the drug/hydrogel matrix, and n is the diffusional exponent characteristic of the release mechanism. When n = 0.5, the drug release is purely controlled by Fickian diffusion; when 0.5 < n < 1, more than one mechanism is at work; when n = 1, the drug release is controlled by hydrogel swelling (case II transport).where n), rate constant (k), and squared correlation coefficient (R2) is presented in Table n = 0.3184 for Fmoc-FF hydrogel beads indicates that docetaxel was mainly released in a Fickian diffusion-mediated mode. The value of n < 0.5 might be attributable to the hydrophobic interaction between Fmoc-FF nanofibers and docetaxel. For the hybrid hydrogel beads, the n values are close to 0.5, indicating a Fickian diffusion-controlled process for docetaxel release. The value of exponent n increases with the increasing alginate concentration, which might be due to the fact that the hydrophilic alginate could weaken the hydrophobic interaction between Fmoc-FF nanofibers and docetaxel, and thus lead to faster drug release. In the late stage of drug release (1.25–24 h), n = 0.0885 for Fmoc-FF hydrogel beads, and n = 0.0890 for the hybrid hydrogel beads . There are two reasons for these values: the encapsulation of docetaxel in a deeper part of the hydrogel beads; hydrophobic interaction between Fmoc-FF nanofibers and docetaxel may have been responsible for the slow release behavior. Thus, the Korsmeyer–Peppas empirical model is no longer suitable for docetaxel release from these hydrogel beads in this stage. However, the value of n was 0.3359 for hybrid hydrogel beads with an alginate concentration of 3 mg/mL, indicating that the docetaxel release continued to exhibit Fickian diffusion-mediated behavior. It also indicates that the release of docetaxel from the hydrogel beads took longer when the amount of alginate in these hydrogel beads was increased. Moreover, the highly stable structure of hydrogel caused by co-assembly resulted in release behavior that was close to the empirical model.Figure n suggests that docetaxel was mainly released from the hybrid beads in a Fickian diffusion-mediated mode. We thus conclude that our method provides a way to fabricate new supramolecular materials with fascinating properties.A new peptide–polysaccharide hybrid hydrogel bead was prepared by calcium ion-triggered co-assembly of Fmoc-FF peptide and alginate. The calcium ions triggered self-assembly of Fmoc-FF peptide into nanofibers with a diameter of about 30 nm by decreasing their charge; at the same time, they allowed alginate to be crosslinked, leading to the formation of hybrid hydrogel beads. SEM results verified that the hybrid hydrogel beads had a dense surface structure on the outside and a structure of stacked sheets with a porous nanofibrous network in its interior. Compared to the alginate or Fmoc-FF hydrogel beads, the hybrid hydrogel beads had higher stability in both water and PBS, probably because of the synergistic effect of noncovalent and ionic interactions. Furthermore, we embedded docetaxel in the hydrogel beads during the hydrogel formation process and studied the drug release behavior. By varying the concentration ratio of Fmoc-FF to alginate, controlled release of docetaxel could be obtained. The release exponent