Eulemur mongoz) and brown lemurs (E. fulvus) at Anjamena in west Madagascar.Hybrid zones generally represent areas of secondary contact after speciation. The nature of the interaction between genes of individuals in a hybrid zone is of interest in the study of evolutionary processes. In this study, data from nuclear microsatellites and mitochondrial DNA sequences were used to genetically characterize hybridization between wild mongoose lemurs (E. fulvus) and six other individuals with putative introgressed alleles in their genotype. Principal component analysis groups both hybrids as intermediate between E. mongoz and E. fulvus and admixture analyses revealed an admixed genotype for both animals. Paternity testing proved one F1 hybrid to be fertile. Of the eight brown lemurs genotyped, all have either putative introgressed microsatellite alleles and/or the mtDNA haplotype of E. mongoz.Two segments of mtDNA have been sequenced and 12 microsatellite loci screened in 162 brown lemurs and mongoose lemurs. Among the mongoose lemur population at Anjamena, we identified two F1 hybrids (one also having the mtDNA haplotype of Introgression is bidirectional for the two species, with an indication that it is more frequent in brown lemurs than in mongoose lemurs. We conclude that this hybridization occurs because mongoose lemurs have expanded their range relatively recently. Introgressive hybridization may play an important role in the unique lemur radiation, as has already been shown in other rapidly evolving animals. Hybridization among animals has traditionally been viewed as an unusual event. However, recent genetic studies have shown that it occurs more commonly than originally believed ,2. HybriHarrison  defined The Malagasy lemurs are a spectacular example of adaptive radiation and provide an ideal model for studies of evolutionary diversification . CurrentEulemur mongoz) at Anjamena in western Madagascar an animal was observed which exhibited a pelage coloration intermediate between that of red-fronted brown lemurs (E. fulvus rufus) and mongoose lemurs [E. f. rufus individuals from the same locality for a phylogenetic study of brown lemurs [E. mongoz [Eulemur species.During a field study on mongoose lemurs  with E. mongoz but also exhibits a similar activity pattern [E. f. rufus  in the vicinity of Anjamena was much higher than that of E. mongoz  [E. mongoz and E. fulvus exhibit sexual dichromatism. The pelage coloration of brown lemur males and females is very different from that of mongoose lemur males and females  . Both E.ions see ). At Anjions see .E. mongoz and E. fulvus, to gain insights into the genetic composition of the two species, and to assess hybridization between them in Anjamena. Microsatellites show a high degree of length polymorphism, which makes it possible to obtain pedigree-level characterization of populations as well as to detect introgression of alleles between species. Analysis of mtDNA sequences allows direct determination of matrilineal relatedness, which provides information on past hybrid events.We conducted a comprehensive genetic analysis, including both nuclear microsatellites and mitochondrial DNA (mtDNA) sequences of E. mongoz were collected. Five hair samples were collected in 1994 and 1995 during a behavioural study [E. mongoz and 2 E. fulvus samples were collected from animals living near Anadabomandry across the Mahavavy river from Anjamena. Additionally, 2 mongoose lemur and 3 brown lemur samples from Ampijoroa  and brown lemurs (55) were collected from the wild and captivity  were sequenced for mtDNA [for more details see Additional file A total of 63 blood, hair or tissue samples were collected from mongoose lemurs in captivity. The origin of the captive mongoose lemur population is not entirely known, but some founder animals originated from the Comoro islands. One es Table . In addi4)2SO4, 1.5 mM MgCl2, 0.78 M DMSO, 0.025 mM for each dNTP, 1 mM for each primer, and 0.5 U Thermophilus aquaticus (Taq) polymerase. Samples were amplified for 25–35 cycles, with denaturing at 95°C for 30 s, primer annealing at 50–65°C for 60 s, and extension at 72°C for 60 s, followed by a final extension for 5 min at 72°C. The number of cycles and/or the annealing temperature was changed as necessary, to optimize the PCR conditions for each locus.DNA was extracted from hair, blood or tissue samples using a standard phenol-chloroform extraction . ApproxiE. f. rufus [L. catta [A total of 12 microsatellite loci were used for this study. Locus Efr09J was originally isolated from f. rufus . We perfL. catta , 55°C anL. catta . PCR proThr gene, the complete tRNAPro gene and the 5' end of the control region (D-loop), as well as part of the NADH-dehydrogenase subunit 4 (ND4) gene were amplified and sequenced, using the primers listed in Table The 3' end of the tRNAO) and expected (HE) heterozygosities, frequency estimates for null alleles and to test for deviations from the Hardy-Weinberg equilibrium. Additionally, non-exclusion probabilities for first and second parents on the 12 autosomal microsatellite loci and the probability for two random individuals having the same genotype were determined with CERVUS. GENEPOP 4.0.6 [is, Fst and Rst estimates in GENEPOP and by performing an analysis of molecular variance (AMOVA) in Arlequin 3.11 [For microsatellite data, CERVUS 3.0.3  was usedOP 4.0.6  was useduin 3.11 . Principuin 3.11 . STRUCTUuin 3.11  was usedLemur catta sequences were used as the outgroup.The aligned mtDNA sequences were analysed with PAUP 4.0b10  using maE. mongoz and 3.64E-14 for E. fulvus. With one exception, all individuals have unique multilocus genotypes. One adult mongoose lemur male from Anjamena sampled in 1995 has exactly the same genotype as a male from 1997. Because none of the wild animals sampled were marked permanently, it is likely that the same male was captured twice. Therefore, one of the two samples was removed from the data set.Overall, the multilocus panel employed was found to be very informative. The probability that two individuals cannot be differentiated is 3.08E-9 for is < 0.40, Table Variation among mongoose and brown lemurs for the 12 microsatellite loci is summarized in Table Eulemur species showed 23.21% between-species variability , indicating that 76.79% of the genetic variation was found within each species. Genetic differentiation between the two species is relatively high  and significant at each locus .Analysis of molecular variance applied to the two E. mongoz, the wild and captive populations are grouped apart  clearly separates brown lemurs from mongoose lemurs , all 162 samples are correctly assigned to their species Figure . Most moE. fulvus or populations/groups in mongoose lemurs (data not shown). However, while individuals might be admixed between populations or subspecies of the same species, there is no admixture of the two species (apart from JP167 and JP184). These tests confirm the clear separation of the two species and the intermediate positioning of JP167 and JP184.When K is increased from 2 up to 16, the samples are divided into more clusters, with K = 12 showing the highest probability. The different clusters mainly represent subspecies in E. mongoz and Q2 = 0.995 ± 0.004 in all E. fulvus). There are 3 mongoose lemurs that are split between the two clusters. As in the previous analyses, the two individuals JP167 and JP184 have the highest probabilities to be admixed (Q1/Q2 = 0.527/0.473 and 0.714//0.286). In addition, a third mongoose lemur (JP152) has a slightly increased proportion of membership for cluster 2 (Q2 = 0.192). When increasing K, no further subdivision at Anjamena is possible.We also ran analyses using only the 42 mongoose and brown lemurs from Anjamena. When assuming K = 2 populations, most lemurs are clearly assigned to the correct species , however, since they are very rare in mongoose lemurs , they might still be of brown lemur origin. One mongoose lemur at Anjamena has an allele (Lc8–223), not found in any other mongoose lemur and also not in the brown lemurs at Anjamena. Only one E. f. fulvus has that same allele.Five of the possibly introgressed alleles are very common (>20%) in E. f. rufus.The two hybrid mongoose lemurs (JP167 and JP184) identified from PCA/STRUCTURE analyses have 4 putative introgressed alleles each. These two animals have 7 putative introgressed alleles found only once or twice in the mongoose lemurs at Anjamena , this makes JP184 the mother of the juvenile.E. f. rufus subspecies clade, which includes the animals from Anjamena , this identification of putatively introgressed alleles is only preliminary.The 13 brown lemurs in the Eulemur species and 2 Lemur catta. The aligned nucleotide sequences span a total of 1463 base positions (bp). The analysed data set consists of 31 bp of tRNAThr, 66 bp of tRNAPro, 804 bp of D-loop and 559 bp of ND4. Details on the number of haplotypes found with D-loop data, ND4 data and the combined data set in animals at Anjamena or across the species range are given in Table EU333172–EU333247, ND4: EU333248–EU333274, see also Additional file We sequenced two fragments of the mtDNA genome for 107 mongoose lemurs and 55 brown lemurs, as well as 12 individuals from the other three E. fulvus at Anjamena with the E. mongoz haplotype MF, which is not found in any mongoose lemur at Anjamena. While the haplotypes MA-ME differ from each other by 1 or 2 bp, haplotype MF is 4–5 bp different from the other haplotypes found among mongoose lemurs in Anjamena. The 3 E. fulvus haplotypes at Anjamena differ from each other by 1 or 2 bp.There are 5 mtDNA haplotypes (MA-ME) in the mongoose lemur population at Anjamena. In addition, one mongoose lemur exhibits the mtDNA haplotype of a brown lemur. The brown lemurs at Anjamena have 3 haplotypes . One of these (FA), is the haplotype also observed in the mongoose lemur. There are 4 E. mongoz kept in various zoos. Two animals in captivity each have a unique haplotype (MJ and MK). The 3 haplotypes found in captivity differ from each other by only 1 or 2 bp. The two animals at Ampijoroa have haplotype MH and the 4 mongoose lemurs from Anadabomandry have haplotype MG.Among all 107 mongoose lemurs, 10 haplotypes (MA-MK) are found. The most common (57%) haplotype, MI, is present in 61 In the phylogenetic analyses, four distinct clades of mongoose lemurs can be recognized Figure . One claAmong brown lemurs, a total of 34 haplotypes are found. Phylogenetic analyses group them into 6 clades  the mother of the juvenile. The genotype of the juvenile contains none of the 4 putative introgressed alleles from the mother. However, admixture analyses of the lemurs at Anjamena reveal a proportion of brown lemur ancestry (Q2 = 0.192) in its genotype that is consistent with an F2.The second mongoose lemur of hybrid origin is an adult female (JP184). She is a member of a small social group, including herself, an adult male, subadult female and juvenile male. The adult male has the normal fur coloration of a mongoose lemur male. The juvenile (JP152), like JP184 exhibits a mixture of fur colours from The 6 mongoose lemurs with one or two putative introgressed alleles might be descendants of a hybrid crossing. Neither PCA nor admixture analyses were able to differentiate them from other mongoose lemurs. Consistent with this, their coat coloration was normal for mongoose lemurs and so they could not be F1s, and are unlikely to be F2s, since the confirmed offspring of an F1 hybrid and a mongoose lemur (JP152) has intermediate coat colour. If hybridization had occurred, a single event could have accounted for the allele distribution. Further data would be required to assess whether these alleles truly represent a signature of past introgression events, and to estimate the rate of introgression.We found genetic evidence for a minimum of 2 hybridization events in mongoose lemurs of Anjamena. Two hybrid crossings were very recent (F1). In addition, one of the F1 hybrids had an offspring where paternity could be genetically confirmed. Therefore, hybridization between mongoose and brown lemurs occurs in the wild and yields fertile offspring, which can successfully backcross with mongoose lemurs. A third potential hybrid crossing might have happened a few generations ago, which would again show the fertility of the hybrids.E. mongoz females and E. fulvus males as well as between E. mongoz males and E. fulvus females.One of the F1 hybrids has the mtDNA haplotype of a brown lemur and the other of a mongoose lemur. Hybrid crossings can therefore occur between E. mongoz mtDNA haplotype. As all 4 animals have the same haplotype MF, the presence of that haplotype could be the consequence of a single hybrid crossing. Interestingly, haplotype MF is not found in any of the 35 mongoose lemurs sampled at Anjamena and is the most divergent of the 5 E. mongoz haplotypes found there  and also not knowing the familial relationships among the members of each social group , no detailed information on hybrid crossings can be obtained for the brown lemurs at Anjamena. However, half of the brown lemurs sampled have an E. mongoz. The samples were collected from animals living in 7 different social groups at Anjamena. Principal component analysis failed to group the 8 animals closely together  and east (Anjamena) side of this river are higher for E. f. rufus (21–24 bp) than for E. mongoz (12–15 bp). This again indicates that E. mongoz expanded its range more recently than E. f. rufus.A possible explanation for this hybridization is that one species only recently expanded its range and there were no reproductive barriers in place between the two newly overlapping taxa. During the radiation of brown lemurs the Betsiboka river formed a barrier dividing boundary . The curE. mongoz either had a wide distribution in the past and then disappeared from large parts of its former range, or dispersed across otherwise efficient geographical barriers [E. mongoz at Ampasambazimba [E. mongoz (this study). One is, that mongoose lemurs had a large distribution in the past with regular gene flow across the range. Only once they lost their range in central Madagascar relatively recently, was gene flow no longer possible across the range because the Betsiboka and Mahavay rivers became geographical barriers. The other option is, that in the past mongoose lemurs occurred only to the east of the Betsiboka river all the way up to central highlands and only later expanded their range towards the coast on the west side of the Betsiboka and Mahavavy rivers, while disappearing from central Madagascar. In the first scenario they were sympatric with E. f. fulvus and E. f. rufus for a long time, while in the second scenario they only became sympatric with E. f. rufus relatively recently.A cladistic biogeographic analysis on the extant lemur distribution in western Madagascar suggests that barriers . In 1939mbazimba , which lmbazimba , to the mbazimba , the fosmbazimba  and the E. mongoz) and the related native species (E. fulvus). Where few reproductive barriers to gene flow exist, this frequently leads to the rapid introgression of genetic and phenotypic characters from one species into another [E. fulvus subspecies resulted in fertile offspring [Eulemur species, crossbreeding between E. macaco and E. fulvus gave rise to fertile hybrids [E. macaco macaco and E. m. flavifrons [E. f. fulvus and E. f. rufus [E. f. rufus and E. f. albocollaris [Eulemur species and subspecies indicate that reproductive barriers among Eulemur taxa are not very well developed.The influx of a new species into a novel ecosystem can result in hybridization between the new species  of the 37 animals studied show signs of introgression.Hybridization between newly overlapping species provides a unique opportunity to observe the initial stages of hybridization and its evolutionary consequences. Hybridization which results in fertile offspring, is expected to introduce sets of alleles that gradually disperse through the gene pool of the parent organisms through successive backcrosses. Such extensive introgression might have occurred among brown lemurs at Anjamena with 7 (88%) brown lemurs possibly having introgressed alleles and 4 (50%) brown lemurs having the mtDNA haplotype of Eulemur has proved to be very successful in adapting to a variety of ecological niches. Introgressive hybridization among taxa is known to quickly increase levels of variation, allowing more rapid responses to environmental changes [Eulemur taxa in the wild [[Eulemur across Madagascar.There are several possible evolutionary consequences of hybridization . In extreme cases, parental taxa may be lost in the process and/or new taxa formed ,22. A th changes ,22. Conshe wild [-18, thisE. mongoz and E. fulvus in western Madagascar. Most other hybrid zones reported so far in lemurs are also between Eulemur taxa [Varecia variegata subspecies [Eulemur may be more capable of hybridizing than other lemur taxa. However, hybrids are not easy to detect. They are much easier to identify and are generally recorded more often if the hybridizing taxa are brightly and distinctively coloured (e.g., birds, butterflies [Eulemur species and subspecies, as well as for Varecia variegata subspecies. The detection of hybridization in lemurs may follow a similar time sequence as the discovery of parent taxa, with nocturnal species lagging behind. For nocturnal lemurs, the pelage coloration of which is neither bright nor varies much between taxa, only detailed genetic studies are likely to detect hybridization events. Our study records an instance of on-going hybridization in the wild. Introgressive hybridization may hasten speciation and allow rapid ecological adaptation of taxa, hence be one of the driving forces for the adaptive radiation of lemurs in Madagascar.The genetic data presented here confirms the occurrence of hybridization between mur taxa -18. Onlybspecies . It is kbspecies  and EuleJP participated in the design of the study and the field expedition, carried out the lab work, conducted data analyses and wrote the first draft of the manuscript. AZ organized and lead the field expedition to Anjamena to collect the samples. DC participated in the design of the study, helped with the logistics of the field expedition and revised a draft version of the manuscript. CN advised and participated in the data analyses. NM critically revised the draft version of the manuscript. All authors approved the final version of the manuscript.E. mongoz haplotypes and E. fulvus samples.Locality, sample type and GenBank numbers for all  none.Click here for file


Information about Medical Emergency Teams (METs) in Australia and New Zealand (ANZ) is limited to local studies and a cluster randomised controlled trial . Thus, we sought to describe the timing of the introduction of METs into ANZ hospitals relative to relevant publications and to assess changes in the incidence and rate of intensive care unit (ICU) admissions due to a ward cardiac arrest (CA) and ICU readmissions.We used the Australian and New Zealand Intensive Care Society database to obtain the study data. We related MET introduction to publications about adverse events and MET services. We compared the incidence and rate of readmissions and admitted CAs from wards before and after the introduction of an MET. Finally, we identified hospitals without an MET system which had contributed to the database for at least two years from 2002 to 2005 and measured the incidence of adverse events from the first year of contribution to the second.P = 0.0244). The incidence of ICU readmissions and the mortality for both ICU-admitted CAs from wards and ICU readmissions did not change. Data were available to calculate the change in ICU admissions due to ward CAs for 16 of 62 (25.8%) hospitals without an MET system. In these hospitals, admissions to the ICU after a ward CA decreased from 5.0 per year in the first year of data contribution to 4.2 per year in the following year .The MET status was known for 131 of the 172 (76.2%) hospitals that did not participate in the MERIT study. Among these hospitals, 110 (64.1%) had introduced an MET service by 2005. In the 79 hospitals in which the MET commencement date was known, 75% had introduced an MET by May 2002. Of the 110 hospitals in which an MET service was introduced, 24 (21.8%) contributed continuous data in the year before and after the known commencement date. In these hospitals, the mean incidence of CAs admitted to the ICU from the wards changed from 6.33 per year before to 5.04 per year in the year after the MET service began (difference of 1.29 per year, 95% confidence interval [CI] -0.09 to 2.67; Approximately 60% of hospitals in ANZ with an ICU report having an MET service. Most introduced the MET service early and in association with literature related to adverse events. Although available in only a quarter of hospitals, temporal trends suggest an overall decrease in the incidence of ward CAs admitted to the ICU in MET as well as non-MET hospitals. Rapid Response Systems (RRSs) have been introduced into hospitals to identify and treat at-risk ward patients in an attempt to reduce unplanned intensive care unit (ICU) admissions and cardiac arrests (CAs) -3. In AuIn the United States, RRSs have been introduced into multiple hospitals in response to the '5 Million Lives campaign' promoted by the Institute of Health Improvement (IHI) . On the The Australian and New Zealand Intensive Care Society (ANZICS) Research Centre for Critical Care Resources (ARCCCR) maintains a database recording information on critical care resources, including the timing of introduction of METs. In addition, ANZICS maintains an Adult Patient Database (ANZICS-APD), which currently contains information on the demographics, admissions source, and outcomes of more than 450,000 ICU admissions. The development and details of the ANZICS-APD have been described in detail elsewhere .The aims of this study were (a) to describe the timing and extent of the introduction of MET services into ANZ hospitals in relation to relevant publications, (b) to assess the association between MET service introduction and the incidence and rate of ICU admissions due to ward CAs, (c) to assess the association between MET service introduction and the incidence and rate of ICU readmissions, and (d) to assess changes in the same adverse events in hospitals that had not introduced an MET service.The collection, analysis, and reporting of de-identified data by the ANZICS-APD comply with Australian Commonwealth legislation (1994) enabling national quality assurance activities. They also comply with the quality assurance amendment of the Australian Health Insurance Act (1973) . This enWe obtained information from a database maintained by the ANZICS ARCCCR and derived from surveys of ICU resources and activity. The information related to the timing of commencement of an MET into hospitals in ANZ which were not involved in the MERIT study . HospitaAn electronic search was conducted to identify literature related to serious adverse events and METs to assess the timing of MET introduction in relation to such literature. Studies were selected from a Medline search from 1990 to 2006 using the key words 'adverse event', 'medical emergency team', 'cardiac arrest', and 'rapid response team'. The date of these publications was then related to the timing of commencement of MET services.The ANZICS-APD was interrogated to obtain data on the incidence of ICU admissions secondary to CAs in ward patients and on the incidence of readmissions to the ICU.Hospitals were eligible for analysis if they had an MET service with a known commencement date and had contributed to the ANZICS-APD for the two continuous years spanning the introduction of the MET service (12 months before and 12 months after). Hospitals were excluded from analysis if they were participants in the MERIT study , if the The APD was interrogated using commercially available software  for data in the 12 months before and 12 months after commencement of the MET service. In the case of ward CAs, the patient cohort was constructed by restricting the 'ICU admission source' field to 'patients admitted from the ward' and restricting the 'admission diagnostic codes' field to the APACHE  III 'non-operative diagnostic code 114 – post cardiac arrest'. The cohort of patients experiencing ICU readmission was constructed by including all patients admitted to the ICU on two or more occasions in the same hospital admission, regardless of admissions source. We also obtained information on the overall number of ICU admissions and the hospital mortality of patients admitted after a ward CA or readmission.We assessed similar changes in hospitals that had contributed at least 24 months of data to the APD during the same period (2000 to 2005) but had not introduced an MET service and had not participated in the MERIT study by comparing the first year of data submission to the second. Finally, in an additional sensitivity assessment, we extended our analysis to hospitals involved in the MERIT study which had submitted information to the APD before participation in the MERIT study and which had continued to submit data thereafter.Descriptive data are presented as raw numbers and as a percentage of overall cases or events. Data on adverse events (ICU admission due to ward CA and readmission to ICU) are presented as means ± standard deviation for absolute values and rates of events  in the 12-month periods before and after the commencement of the MET service.P value of less than 0.05 was considered statistically significant.The difference in the incidence and hospital mortality for adverse events before and after commencement of the MET was tested for with the Wilcoxon signed rank test. A similar comparison was performed for hospitals that had not introduced an MET service using the first year of data as baseline and the second year as comparator. Finally, an additional and similar analysis was performed for hospitals that had participated in the MERIT study. A The MET status was known for 131 of the 172 (76.2%) ANZ hospitals that did not participate in the MERIT study  in the incidence of ward CAs admitted to the ICU in the year after the introduction of an MET service. A similar decrease was seen in their rate   Table .P = 0.779)  Table . There w9) Table .P = 0.3). Similar to MET hospitals, there was no change in other outcome measures . When hospitals were analysed in their aggregate, this temporal trend was statistically significant (P = 0.023).Twenty-three hospitals participated in the MERIT study. Of those randomly assigned to an MET service (n = 12), all continued to have an MET system in 2007. Of those randomly assigned to the control arm (n = 11), five had introduced an MET service by 2005. Twelve hospitals could be identified which participated in MERIT, had an MET system, contributed to the database, and had contributed data for at least one year before the introduction of the MET and one year thereafter. Six hospitals could be identified which participated in MERIT, did not have an MET system, contributed to the database, and had contributed data for at least two consecutive years during our study period. These hospitals showed no temporal trends in readmission rates. However, both control hospitals and MET hospitals showed a trend toward a decreased percentage of ICU admissions being secondary to CAs  without an MET which contributed 24 months of consecutive data during the same time frame, however, similar changes in outcome were seen. Finally, we also obtained information on those hospitals that had participated in the MERIT study and had contributed sufficient data for analysis. We found that 5 of 11 MERIT control hospitals had introduced an MET system and that the temporal trends toward reduced CA admission to the ICU seen in the main cohort were confirmed in MERIT hospitals.To our knowledge, this is the only study to assess the implementation of METs in two countries and the timing of such implementation. It is also the first to seek to relate the introduction of METs to available evidence. It is also the first multi-centre before-and-after comparison in a broad cohort of hospitals for relevant outcomes in a 'real life' setting outside of trial-modified situations. As such, it provides some insights into the triggers and consequences of the process of translating research into practice. We believe that the information we obtained may provide a perspective on the possible applicability and generalisability of clinical research in general and of research on the MET/RRS in particular.Despite the above features, the study is retrospective and observational, with all of the associated limitations. We are able to comment on the uptake of MET services until April 2005 only and cannot assess the effect of the publication of the MERIT study  publish. Thus, oThe assessment of the possible effect of the MET service on ICU admissions due to ward CAs and unplanned ICU admissions is also from a retrospective and before-and-after design, not from a randomised controlled trial. As such, it describes association and not causality. The reduction in ward CAs could be demonstrated only for patients subsequently admitted to the ICU. Therefore, we are unable to comment on the number of CAs that occurred in hospital wards but did not lead to ICU admission. These CAs may have increased or more people may have died from them, thus artificially reducing the number of patients admitted to the ICU. Furthermore, we observed that the introduction of the MET service was not associated with a reduction in the incidence of ICU readmissions or the mortality from admissions due to either ward CAs or ICU readmission. In addition, our study does not report on the incidence of other outcomes such as unexpected deaths or unplanned ICU admissions, which were used as the major outcomes for the MERIT study . The ANZApproximately 60% of ICU-equipped hospitals in ANZ report having introduced an MET service. In most of these hospitals, the service commenced prior to the publication of literature demonstrating the possible effectiveness of the MET on patient outcomes. In the 24 hospitals for which before-and-after data were available, introduction of an MET service was associated with no effect on the incidence of ICU readmissions, their mortality, or the mortality of patients admitted to the ICU after a ward CA. However, it was associated with a significant reduction in the incidence and rate of ICU admissions due to ward CAs. A similar reduction was also seen over a similar period of time among hospitals that had not introduced an MET service and in a cohort of hospitals that had participated in the MERIT study.• A majority of hospitals in Australia and New Zealand (ANZ) appear to have introduced a Medical Emergency Team (MET) system.• The introduction of such systems in ANZ occurred mostly before any publications reporting the possible effectiveness of such systems.• The introduction of MET systems in ANZ appeared to be a response to publications highlighting the incidence of adverse events in hospitals.• The introduction of MET systems in ANZ was associated  with a temporal trend toward reduced intensive care unit admissions secondary to a ward cardiac arrest. However, a similar trend was seen in hospitals that did not have an MET system.ANZ = Australia and New Zealand; ANZICS = Australian and New Zealand Intensive Care Society; ANZICS-APD = Australian and New Zealand Intensive Care Society Adult Patient Database; APD = Adult Patient Database; ARCCCR = Australian and New Zealand Intensive Care Society Research Centre for Critical Care Resources; CA = cardiac arrest; ICU = intensive care unit; IHI = Institute of Health Improvement; MERIT = Medical Emergency Response and Intervention Trial; MET = Medical Emergency Team; RRS = Rapid Response System.The authors declare that they have no competing interests.DJ and RB designed, executed, and wrote up the study. CG, GKH, and JM obtained the study data and assisted with study development and execution. All authors read and approved the final manuscript.


Benedikt Huttner and Stephan Harbarth discuss the implications of a new study that examined the impact of a national campaign in France to reduce antibiotic overuse. PLoS Medicine:This Perspective discusses the following new study published in doi:10.1371/journal.pmed.1000084Sabuncu E, David J, Bernède-Bauduin C, Pépin S, Leroy M, Boëlle P-Y, Watier L, Guillemot D (2009) Significant reduction of antibiotic use in the community after a nationwide campaign in France, 2002–2007. PLoS Med 6(6): e1000084. Didier Guillemot and colleagues describe the evaluation of a nationwide programme in France aimed at decreasing unnecessary outpatient prescriptions for antibiotics. The campaign was successful, particularly in reducing prescriptions for children.Antibiotic resistance is an important public health concern Streptococcus pneumoniae in Iceland There has been great interest in the public health community in avoiding unnecessary prescriptions, not only by providing treatment guidelines and decision support to physicians, but also by educating the public about appropriate antibiotic use. But the most effective strategy to achieve this goal remains unknown PLoS Medicine, Didier Guillemot and colleagues analyze the impact of a similar campaign in France, which used to be known for the highest rates of antibiotic use and pneumococcal resistance in Europe In a new study published in this issue of Analyzing prescribing data provided by the national health insurance (covering over 90% of the population) for two winters before and five winters after the launch of the first campaign, Guillemot and colleagues observed a decline by 26.5% in the number of antibiotic prescriptions, surpassing even the national target of a 25% reduction over five years. The decrease was seen in all French regions and age groups, with the highest decrease observed in children. Despite this decline in antibiotic use, a relative increase in fluoroquinolone use by 12.8% was observed, indicating the possibility that the choice of antibiotic may have become less appropriate for certain treatment indications.This study provides the largest and most sophisticated analysis published thus far correlating a nationwide public campaign to decreased antibiotic use over an extended period of time. The researchers address many potential sources of bias and confounding without major campaigns. The paper does not provide any information about surrogate markers that might explain the campaign's success, such as the public's awareness of the campaign or changes in knowledge and attitudes among physicians and the public. A limitation inherent to any multifaceted, complex intervention is that no clue is provided to identify which of the numerous interventions were most effective and could be recommended to other countries. Finally, although the impact on antibiotic prescriptions is impressive, the effect on antimicrobial resistance is still unclear and difficult to separate from the effect of the conjugate pneumococcal vaccine Nevertheless, some issues warrant further comment. Although it is likely that the reduction in antibiotic prescribing was causally related to the campaign, the ecological before-and-after study design cannot provide definitive proof of causality. As mentioned by the authors, in a few European countries a decrease in antibiotic use was also seen Guillemot and colleagues report that developing and conducting the campaign cost 500 million euros over six years, which seems extremely costly considering that the Belgian campaign cost only about 400,000 euros per year The amount of money spent by the pharmaceutical industry to promote antibiotics should also be considered when assessing cost issues. Unfortunately, we do not have access to these figures for Europe. However, in the United States in 1998, it was estimated that pharmaceutical companies spent about US$1.6 billion to promote antibiotics, an amount that vastly exceeds any budget allocated to antibiotic campaigns failed to show a major impact on antibiotic prescriptions, although few of these data have been published Overall, the results of the French public campaign are very encouraging and confirm those of the Belgian campaign: in countries with high baseline antibiotic use, sustained and multifaceted campaigns using mass media as well as targeting physicians can decrease antibiotic use substantially. Nonetheless, given the variations in antibiotic usage patterns, sociocultural determinants, and health care systems, different approaches are warranted for different countries Since the ultimate goal of any campaign to reduce antibiotic use is to curb antibiotic resistance, more longitudinal and modeling studies are needed. Realistically, these studies may require individual patient data from small-scale surveillance networks, including monitoring of potential adverse effects of reduced prescribing.All in all, the French have to be congratulated for their success story demonstrating that, if policy makers and opinion leaders are serious about reducing antibiotic misuse, this goal can be achieved by well-coordinated national efforts. Throughout the world, we should no longer accept antibiotic misuse and resistance as “automatic”.


Against a backdrop of increasing antibiotic resistance, and the emergence of new and evolving pathogens, clinicians are increasingly forced to consider alternative therapies - probiotics are one such alternative. With life-cycles measured in minutes as opposed to years, bacteria have an extraordinary ability to evolve and adapt rapidly to changes in their environment . Thus, ivia a specific mode of action; for example, penicillin interferes with the cross-linking of two linear polymers by inhibiting the transpeptidase reaction, bacteriocins on the other hand have quite diverse activities. Nisin and many other structurally related lantibiotics for example, use the cell wall precursor lipid II bound to the membrane as a docking molecule for pore formation and combine at least two modes of action, i.e., pore formation and inhibition of cell wall biosynthesis, for antibacterial activity at nanomolar concentrations [Faced with an emerging pandemic of antibiotic resistance, clinicians and scientists alike are now struggling to find viable therapeutic alternatives to our failing antibiotic wonder drugs . One suctrations . These mtrations .However, despite their potent anti-pathogenic effect, a significant limitation of this approach is that probiotic bacteria tend to be physiologically fragile; often not surviving to sufficiently high numbers during prolonged storage in delivery matrices such as foods (yogurt and probiotic drinks) or tablet formulations . FurtherListeria monocytogenes in the probiotic strain Bifidobacterium breve, not only improves gastrointestinal colonisation and persistence, but also significantly bolsters the clinical efficacy of the probiotic strain [One approach to improving the physiological robustness and stress tolerance of probiotic strains is patho-biotechnology ,14. Essec strain .In addition to improving their physiological stress tolerance, resulting in improved delivery and persistence within the gut, recent studies have led to the development of 'designer probiotics' which specifically target enteric infections by blocking crucial ligand-receptor interactions between the pathogen and its target host cell ,18,19. Min vivo sensitivity to gastric acidity associated with oral application of therapeutic or prophylactic compounds, they can also be produced cheaply; grown to high levels, dried and stored for years at ambient temperatures.In addition to infection control Fig. , probiotIn conclusion then, "designer probiotics" can be engineered to kill pathogens, neutralise toxins, and facilitate re-colonisation of the resident beneficial microflora while at the same time priming both the innate and adaptive immune system; strengthening the host against subsequent infection - an approach far beyond the reach of conventional antibiotic therapies. Thus, the war against the antibiotic resistant "super bugs" may eventually be won by recruiting engineered "good bugs" as our allies.Bioengineered Bugs http://www.landesbioscience.com/journals/biobugs.Sleator is Editor-in-Chief of the peer reviewed scientific journal


Individuals living with HIV/AIDS in sub-Saharan Africa generally take more than 90% of prescribed doses of antiretroviral therapy (ART). This number exceeds the levels of adherence observed in North America and dispels early scale-up concerns that adherence would be inadequate in settings of extreme poverty. This paper offers an explanation and theoretical model of ART adherence success based on the results of an ethnographic study in three sub-Saharan African countries.Determinants of ART adherence for HIV-infected persons in sub-Saharan Africa were examined with ethnographic research methods. 414 in-person interviews were carried out with 252 persons taking ART, their treatment partners, and health care professionals at HIV treatment sites in Jos, Nigeria; Dar es Salaam, Tanzania; and Mbarara, Uganda. 136 field observations of clinic activities were also conducted. Data were examined using category construction and interpretive approaches to analysis. Findings indicate that individuals taking ART routinely overcome economic obstacles to ART adherence through a number of deliberate strategies aimed at prioritizing adherence: borrowing and “begging” transport funds, making “impossible choices” to allocate resources in favor of treatment, and “doing without.” Prioritization of adherence is accomplished through resources and help made available by treatment partners, other family members and friends, and health care providers. Helpers expect adherence and make their expectations known, creating a responsibility on the part of patients to adhere. Patients adhere to promote good will on the part of helpers, thereby ensuring help will be available when future needs arise.Adherence success in sub-Saharan Africa can be explained as a means of fulfilling social responsibilities and thus preserving social capital in essential relationships. Using ethnographic data from Nigeria, Tanzania, and Uganda, Norma Ware and colleagues examine why levels of adherence to HIV/AIDS drugs are so much higher in sub-Saharan Africa than in North America. The acquired immunodeficiency syndrome (AIDS) epidemic has killed more than 25 million people since 1981, and about 30 million people  are currently infected with the human immunodeficiency virus (HIV), which causes AIDS. HIV destroys immune system cells, leaving infected individuals susceptible to other infections. Early in the AIDS epidemic, most HIV-infected individuals died within ten years but in 1996, combination antiretroviral therapy (ART)—a mixture of powerful drugs—was developed. For HIV-infected people living in affluent, developed countries, HIV/AIDS became a chronic disease, but for the millions of infected people living in low- and middle-income countries, HIV/AIDS remained a death sentence—ART was simply too expensive. In 2003, this situation was declared a global health emergency. Today, through the concerted efforts of governments, international organizations, and funding bodies, nearly one-third of the people in developing and transitional countries who are in immediate need of life-saving ART receive free, reliable supplies of the drugs they need.For ART to work, it must be taken regularly. If drug doses are missed, the virus can rebound and resistance to ART is more likely to develop. In poor countries, even though free antiretroviral drugs are increasingly available, many obstacles to good adherence to ART remain. These include economic obstacles , and social, cultural, and behavioral barriers. Some patients fear disclosure, for example. Others receive conflicting messages about the benefits of ART. However, despite worries that the scale-up of ART provision in developing countries would be dogged by inadequate adherence, people living with HIV/AIDS in sub-Saharan Africa generally take more than 90% of their prescribed doses of ART, a better level of adherence than in North America. In this study, the researchers investigate why ART adherence is so high in sub-Saharan Africa by analyzing qualitative data from an ethnographic study done in Nigeria, Tanzania, and Uganda. Qualitative data are often used to address “how” and “why” research questions: ethnography is a comprehensive qualitative approach to describing and explaining human behavior and culture.For their study, the researchers interviewed 158 patients, 45 treatment partners (lay-people who help HIV-positive people keep to their treatment), and 49 health care workers. Patients were asked about their experiences of ART and about the help they received from their treatment partners; partners were asked about the type of help they gave and about their feelings about this help; health care workers were asked to describe a typical clinic visit and to indicate how adherence was discussed. From these interviews and observations of clinic sessions, the researchers identified several strategies used by patients and their treatment partners to overcome economic obstacles to ART adherence. These included borrowing and “begging” funds to pay for travel to clinics and making “impossible choices” to prioritize adherence, and “doing without.” The researchers' analysis also indicates that the prioritization of adherence to ART reflects the importance of relationships as a resource for managing economic hardship. So, for example, they found that treatment partners and health care workers expected patients to adhere to ART  and made their expectations known, thereby creating a responsibility among patients to adhere. Patients, in turn, adhered to their treatment to promote good will from their helpers and thus ensure their continuing help.The findings offer a possible explanation of adherence success in sub-Saharan Africa. The high level of adherence to ART can be explained as a means of fulfilling social responsibilities. Adherence, the researchers suggest, not only improves personal health (the main driver for ART adherence in resource-rich environments) but also preserves “social capital” in essential relationships. In other words, in sub-Saharan Africa, adherence to treatment may protect the relationships that individuals living in extreme poverty rely on to help them survive.http://dx.doi.org/10.1371/journal.pmed.1000011.Please access these Web sites via the online version of this summary at PLoS MedicinePerspective by Agnes Binagwaho and Niloo RatnayakeThis study is further discussed in a HIV infection and AIDSInformation is available from the US National Institute of Allergy and Infectious Diseases on HIV InSite has comprehensive information on all aspects of HIV/AIDS, including an article about to antiretroviral therapyHIV and AIDS in Africa (including detailed information on HIV/AIDS in Nigeria and Uganda) and on providing AIDS drug treatment for millionsInformation is available from Avert, an international AIDS charity, on universal access to HIV treatment The World Health Organization provides information about global efforts to deal with the HIV/AIDS pandemicThe US Centers for Disease Control and Prevention also provides information on  People living with HIV/AIDS in sub-Saharan Africa generally take more than 90% of prescribed doses of antiretroviral therapy (ART) –12. ThisThese near-perfect levels of adherence are being achieved despite formidable obstacles in the poorest regions of the world. Prohibitive costs of self-pay medications and drug stock-outs create insurmountable access barriers and lead to missed doses ,12,17–20Social and cultural obstacles also threaten adherence success. Stigma and the fear of disclosure cause patients to skip doses if privacy is unavailable at a scheduled dosing time ,21. ConfAdherence facilitators in resource-scarce settings have received less attention than adherence barriers. Currently, home-based adherence “help” is emerging as an important resource to support adherence. One form of home-based help centers on community health workers. Community health workers are laypersons trained and paid through treatment programs to provide adherence support and other services at home. They deliver medications, observe dosing, refer individuals for HIV-testing, offer encouragement, provide nutritional and housing support, help with transportation, and in general represent important links between clinics and communities ,28–32.A second form of home-based help is treatment supporters . TreatmeMore structured support in the form of clinic-based modified directly observed therapy (MDOT) has shown promising results in Kenya . The facTo better understand exceptional adherence in sub-Saharan Africa, we analyzed qualitative data from an ethnographic study carried out in three countries: Nigeria, Tanzania, and Uganda.This patient-centered ethnographic study took place in public HIV-treatment settings in Nigeria, Tanzania, and Uganda see . The HIVWe used a purposeful sampling design. The goal of purposeful sampling is to systematically represent a variety of perspectives on the topic under study . Three dTreatment partners who participated in the study were referred by patient participants. Patients were asked to name “someone who assisted them in their efforts to take antiretroviral medications.” Health care providers who participated were volunteers who responded to a letter of invitation introducing the study.In Nigeria and Tanzania, patient participants were recruited at the clinic during routine follow-up visits. At a free moment , staff approached eligible individuals to describe the study and extend an invitation to participate. In Uganda, permission to contact prospective patient participants for recruitment was first obtained by UARTO research staff. Once permission was obtained, a research assistant for this ethnographic study approached the participant for consent.Treatment partners were visited or telephoned by staff after giving permission for contact. Health care providers who indicated interest in response to the letter of invitation were invited to participate.The research was approved by the institutional review boards at Harvard Medical School ; Jos University Teaching Hospital ; Muhimbili University of Health and Allied Sciences ; Mbarara University of Science and Technology ; and the Uganda National Council for Science and Technology . Written informed consent was obtained from all participants.Data were collected through in-person qualitative interviews and observations of clinic activities. Information on adherence was collected through the 3-d self-report instrument from the AIDS Clinical Trials Group edited and adapted for cultural context . All datMultiple interviews were conducted with patient and treatment partner participants to allow for elaboration of areas whose significance emerged as analysis proceeded. The interviews were semistructured, meaning that predesignated core topics, but not specific questions, were covered in each session. This approach ensured that specific areas were covered in each interview, but also allowed unanticipated themes to emerge. Core topics for patient interviews included: (1) specific experiences of taking ART ; (2) clinic visits; (3) help received from treatment partners. Core topics for treatment partner interviews included: (1) types of help provided; (2) feelings about being a treatment partner; (3) perceptions of the impact of help. Core topics for health care provider interviews included: (1) description of typical clinic visit; (2) the ways adherence comes up in clinical visits; (3) perceptions of barriers to adherence for patients.The goal of the interviews was to understand ART adherence from the interviewees' points of view. Patient participant and treatment partner interviews were conducted in homes or at other locations of the interviewee's choosing outside the clinic. Health provider interviews were conducted at the clinic. Privacy was protected by conducting the interviews in locations where the conversation could not be overheard. Interviewees had the option of conducting the interview in the local language  or in English. Interviews were audio-recorded with permission and averaged about an hour in length. Patients received compensation in the form of reimbursement for transportation (where applicable) or a small stipend.Observations are a hallmark of ethnographic data collection. They involve the presence of a researcher in a naturalistic setting and the witnessing of events and activities of interest to a given research inquiry . ObservaShortly after the completion of each interview, the interviewer produced a detailed write-up in English on the interview content, using the audio-recording and notes taken during the interview to ensure accuracy and completeness. The interview write-ups took the form of “stream-lined” transcripts. A “stream-lined” transcript is produced in English  and consists only of interview questions asked and their responses. This approach to data preparation has several advantages. First and foremost, it captures the desired level of detail often lost in a summary and preserves the exact words of the interviewee. It can be completed in a reasonable amount of time by the interviewer, eliminating the expense of separate transcription. Accuracy is also improved using this approach. The write-ups contain a section in which the interviewer adds relevant contextual details and impressions not captured in the transcript. Descriptions of activities observed were written up following each session as field notes.Analysis aimed at explaining adherence to ART using an inductive approach to category construction and interpretation of data ,42. Desc158 patients, 45 treatment partners, and 49 health care providers participated in this study. 414 interviews and 136 observation sessions were conducted across the three sites.Approximately two-thirds (65%) of patients who participated in this study were women. The mean age of the patient group was 38 y. Less than half (44%) were married or living with a partner. Approximately three-quarters (71%) of the patients were Christian; the remainder were Muslim. Primary school was the highest level of education completed for slightly more than half (57%) of the patient sample. 65% of patients met criteria for Stage III or Stage IV disease according to World Health Organization (WHO) criteria at initiation of HIV care.More than half (56%) of treatment partners were female. The mean age of the group was 39 y. Three-quarters (73%) were married or living with a partner. 60% were Christian; the remainder were Muslim. Primary school was the highest level of education completed for about a third (35%) of the group.Approximately two-thirds (64%) of health care provider participants were women, and the mean age of the group was 38 y. About a quarter (27%) of the sample were physicians; a third (35%) were nurses. Nineteen percent were counselors and 16% were pharmacists or medication dispensers. Two full-time “tracking” officers, responsible for locating patients lost to follow-up, were included in the sample (2%), as was another expert patient fulfilling an administrative function (1%).On the basis of category construction and interpretation of the data, we organized the findings from ethnographic data to demonstrate: (1) prioritization of adherence to overcome economic obstacles, and (2) responsibility in social relationships as an explanation of prioritization.Participants reported that most adherence obstacles were related to resource scarcity. Resource scarcity means that basic commodities required for daily living are not easily available. For example, obtaining money for transportation to clinic appointments (where prescriptions are refilled) is rarely a simple matter of dipping into a ready supply of cash. Concerted effort and specific strategies were required to raise needed funds to secure medications.Interviewer: “How do you re-pay the debts?”) “I work and look for money digging and weeding in people's plantations.” And: “When you do not have money you think of how you will come to clinic for medicine. [Last time] I borrowed from a friend but now I have paid it back.” (Interviewer: “How did you feel about borrowing?”) “I felt raw and depressed because I got worried. Actually I lost kilograms. When I came to the clinic and got weighed I had lost 2 kgs.”Patient participants spoke of raising transport funds through loans and handouts, what they termed borrowing and “begging.” Loans—from treatment partners and other family and friends—were a reliable source of cash but were expected to be repaid, often with interest. Some interviewees reported selling possessions or earning income through work to repay loans. Others borrowed repeatedly but saw no way to resolve the debts they had incurred. Chronic indebtedness was a source of considerable emotional distress for borrowers, as these interview excerpts indicate: “Most times I borrow money [to get to clinic] and worry about re-paying the debt.”  to look for transport to send me back to the village. Some people, if they see you in terminal illness, they start to value their money. They think, ‘this illness is getting worse. It'll really cost me to transport a dead body, whereas to arrange to transport a living body is different….'”Patient participants in this study received adherence assistance from several sources. In addition to money for transport, treatment partners, other family and friends, and fellow patients provided regular reminders of dosing times. They offered encouragement by emphasizing the benefits of treatment and continually reinforcing the importance of taking medications as prescribed. Treatment supporters also worked to destigmatize HIV/AIDS. They socialized in public with infected persons; likened HIV/AIDS to common, but less stigmatized conditions ; and challenged myths by deliberately sharing food and eating utensils. “…when they [a set of friends of the patient] started understanding the problem [HIV/AIDS], everybody started withdrawing from eating in the same container with him. …I saw everyone was running away from him so I called him and lied to him. I did not want to create division and break him the more. So I told him, ‘look, I met one of your doctors and she told me that you have to be very careful because of infection. You don't eat anything anybody else eats, you don't use the same cup anybody else uses, because you could get another infection and it could be even worse.' And then I ate with him, so the others would see.”Health care providers were also important sources of help. They supported adherence through education, reinforcement, even the organization of care. At one site, providers maintained a flexible schedule to accommodate patients, even though it made their days longer. They instituted a policy of accepting “latecomers”—individuals who arrived after the deadline for being seen on a given day had passed.Not infrequently, providers stepped outside their professional roles to make resources available to those in need. They provided money and food at their own expense, and organized larger efforts to generate support. In the following quote, a provider describes her practice of helping indigent women patients toward financial independence by securing small loans and then indicating how they might use the funds to start small businesses: “I say, ‘go around in your area, your community. Look at what people like that is not available there. Go and get some of these things and begin to sell them.' That is how I do it.”Interviewer: And would you really?) If she comes back with the same story, I am serious. We will discontinue her. We will.”Providers expected adherence and did not hesitate to make their expectations known. In clinic, staff communicated expectations of adherence by stating repeatedly that antiretroviral therapy must be taken as prescribed. In extreme cases, they even threatened those who repeatedly missed clinic appointments with withdrawal of access, as we see from the following report: “I told [the patient] this morning, ‘You had better decide if you are going to take these drugs. Because the way you are doing, you are creating resistance. I am giving you my last warning. If you come next time with the same story, even if you beg from heaven, we will discontinue you.' That is what I told her.  social structure, e.g., patterns of inequality; (2) infrastructure, e.g. weaknesses in health systems; (3) culture, e.g., values, religious beliefs; and (4) individual experience and behavior, e.g., side effects, lack of information, psychological distress. We offer here a social relational theory to explain adherence differences between resource-rich and resource-poor settings, while acknowledging other kinds of determinants that merit further study.. Verification of such hypotheses will guide interventions to sustain adherence and improve treatment effectiveness.Qualitative analyses contribute to quantitative research in medicine and public health by delineating useful constructs and testable hypotheses. In detailing the explanatory power of social capital, we point to its likely predictive value in examining hypotheses such as: Greater social capital will be associated with better ART access and adherenceA question that immediately arises is whether the benefits of social capital are sustainable? How long can patients realistically expect to receive resources from others who are struggling with resource scarcity themselves? Social capital, unlike many other forms of capital, increases with use . HoweverWe are not the first to address the social dimensions of adherence to ART in the context of international scale-up. A call for a biosocial framing laid out general principles of a socially grounded analysis and proposed relevant analytic concepts—social capital among them . The impThere are several limitations to our findings. First, African societies are highly heterogeneous. We collected data in urban and rural settings in East and West Africa. However, the extent to which these concepts explain adherence behavior in other African contexts will be an important area of future study. Second, we have developed a social explanation but left other domains of influence unexplored. As we develop theoretical models of adherence for resource-scarce locations, these types of influences should be more comprehensively represented. Third, while ART scale-up has been highly successful, the dynamics of adherence will likely evolve as patients confront long-term treatment. Finally, we draw upon social capital as an explanatory construct, but stop short of elaborating underlying cross-cultural differences in definitions of personhood .Like persons living with HIV/AIDS all over the world, sub-Saharan Africans adhere to ART because they want to be healthy. But the desire for health alone does not adequately explain adherence success. A more complete explanation highlights the role of social capital in relationships as a resource for prioritizing adherence and overcoming economic obstacles to care. Adherence preserves social capital by protecting relationships required for survival in settings of poverty. This may be what patients are referring to when they tell us they have “no choice” but to adhere.


Lysmata is characterized by simultaneous hermaphroditism, a very rare sexual system among Decapoda. Specialized cleaning behavior is reported in a few pair-living species; these life history traits vary within the genus. Unfortunately, the systematics of Lysmata and the Hippolytidae itself are in contention, making it difficult to examine these taxa for trends in life history traits. A phylogeny of Lysmata and related taxa is needed, to clarify their evolutionary relationships and the origin of their unique sexual pattern. In this study, we present a molecular phylogenetic analysis among species of Lysmata, related genera, and several putative hippolytids. The analysis is based upon DNA sequences of two genes, 16S mtDNA and nuclear 28S rRNA. Phylogenetic trees were estimated using Bayesian Inference, Maximum Likelihood, and Maximum Parsimony.The hippolytid genus Lysmata, eight genera of Hippolytidae and two genera of Barbouriidae based on a single  and combined gene approach (16S+28S) indicates that three groups of Lysmata differentiate according to antennular morphology: (1) Lysmata, having a multi-segmented accessory branch, (2) Hippolysmata (prior to Chace 1972), with a one-segmented accessory branch, and (3) a third group of Lysmata outliers, with one-segmented unguiform accessory branch, and close affinity to the genera Exhippolysmata and Lysmatella. The monophyly of the clade bearing a multi-segmented accessory branch is robust. Within the short accessory branch clade, species with specialized cleaning behaviors form a monophyletic clade, however, the integrity of the clade was sensitive to alignment criteria. Other hippolytid and barbouriid genera used in the analysis are basal to these three groups, including one displaying simultaneous hermaphroditism (Parhippolyte). The two barbouriid species occur in a separate clade, but among hippolytid taxa.Phylogenetic analysis of 29 species of Lysmata into clades based on accessory branch morphology. The position of the "cleaner" shrimps, indicates that specialized cleaning behavior is a derived trait. The topologies of the cladograms support the monophyly of the barbouriids, but do not support their elevation to familial status. Taxa ancestral to the genus Lysmata display simultaneous hermaphroditism, suggesting that this life history trait evolved outside the genus Lysmata.The data support the historical morphological division of  Lysmata , has attracted the attention of biologists for several decades. Members of this genus are small caridean shrimp and occur in tropical to warm temperate marine coastal waters worldwide. They are popular marine aquarium pets, with some species engaging in cleaning behavior of fishes. One species (L. seticaudata) was used as a model organism for ground breaking studies on sexual differentiation in Crustacea [Lysmata species was a form of simultaneous hermaphroditism, or euhermaphroditism [Lysmata species examined . Among  in life -7. This  in life ) to deschrodites .Lysmata was related to social systems and/or behavioral characteristics among members of the genus. He divided Lysmata into two informal, non-taxonomic ecological groupings: 1) low density, pair living, specialized "cleaner shrimps", with bright and contrasting coloration, including yellow and red colors and long white antenna, and famous for their ability to actively "clean" fish ; 2) high density, group living, "peppermint shrimps", with color patterns consisting of semi-translucent bodies with longitudinal and lateral red bands . Bauer [Lysmata and its socioethological patterns. Furthermore, there are indications that PSH may have evolved outside the genus. Recent studies have shown that PSH occurs in the hippolytid genera Exhippolysmata Stebbing, 1916 [Lysmatella Borradaile, 1915 (Rhyne unpub.), and one barbouriid genus (previously a hippolytid) Parhippolyte Borradaile, 1899 . The presence of PSH within these few taxa suggests an opportunity to examine the evolution of this unique system via a molecular phylogenetic approach.Bauer ,13 postu). Bauer ,13 hypotng, 1916 ,15, LysmLysmata and the family Hippolytidae are still unsettled. Recent revisions in the caridean genus Lysmata have increased the number of species to nearly 40, an expansion of 33% over the last 10 years, and this has not abated [Lysmata were originally split into two genera: Hippolysmata Stimpson, 1860 and Lysmata. These two genera were previously differentiated by the presence of a multi-segmented accessory antennal branch in Lysmata species, and the lack thereof in Hippolysmata species . FurtheLysmata with the closely related Lysmatella and Exhippolysmata in its own family, the Lysmatidae Dana, 1852. Chace [Barbouria Rathbun, 1912, Janicea Manning and Hart, 1984, and Parhippolyte from the Hippolytidae to a new family, Barbouriidae Christoffersen, 1987. Martin and Davis [Lysmata formed a distinct clade, well separated from the other hippolytids [The family Hippolytidae has also been under recent scrutiny. Christoffersen  conclude2. Chace  did not 2. Chace . He conc2. Chace  suggestind Davis  recogniznd Davis , and usend Davis  kept thend Davis  other nepolytids , supportLysmata species and eight genera of related hippolytids and two barbouriids, based upon sequences from both mitochondrial and nuclear ribosomal gene sequences. Our use of two genes from independently evolving genomes, a thorough taxonomic coverage of the Lysmata and related genera, and a robust analysis in terms of alignment strategies improves upon a very recent preliminary phylogeny of the genus Lysmata [Lysmata, as it is present in at least one ancestral taxon. Our phylogenetic analyses support the past division of Lysmata and Hippolysmata species based on the morphology of the antennular accessory flagellum, and the need for revision of both past and present Hippolytidae.In this paper, we present a phylogeny of 29  Lysmata . We demoLysmata and other hippolytid genera, from all over the world . Most of the Indo-Pacific specimens were collected by the first author in Hawaii, Japan, and other Pacific locations; the majority of West Atlantic specimens were provided by AR. Other specimens were kindly provided by individuals from a variety of locations, including Indonesia, the Mediterranean, and Brazil. Many specimens were photographed prior to fixation, as color information is critical in the ultimate determination of species identity [Lysmata wurdemanni was sampled from two locations in Florida and one location in Texas.We obtained specimens from ld Table . Hereaftprior to ) based oidentity . Speciesidentity ), the moidentity ,22), andidentity  SpecimenAlope, Exhippolysmata, Heptacarpus, Tozeuma, and Thor, as well as two barbouriids  to explore their phylogenetic relationship with Lysmata. The snapping shrimp Synalpheus brevicarpus from the closely related Alpheidae [We have also included representatives of the hippolytid genera lpheidae  was selelpheidae , Baeza elpheidae , and Rhylpheidae . SamplesDNA was isolated from individual specimens using one (or more) of three techniques, dependent upon sample condition, fixation method, and laboratory location. Total DNA extractions from EtOH-fixed specimens were performed in one of two ways: a) using the PureGene DNA isolation kit (Gentra) for fixed-tissue or b) via SDS & phenol/chloroform extraction ,30. WhenThe 16S region was amplified with the 1471-1472 primers . The 28SP) and bootstrap support (bp) values are used to indicate clade support.We analyzed the phylogenetic relationships of the sequences by using MCMC-based Bayesian inference (BI) as implemented in MrBayes v. 3.2  and maxiLysmata , in addition to 16 species belonging to 8 other hippolytid genera and two genera of Barbouriidae, from all over the world (Table Lysmata and 9 species (eight genera) of other hippolytids and barbouriids . The low clade values (pP < 50) supporting the basal position of L. olavoi with respect to species possessing a short accessory branch reflect the uncertainty of its placement in the phylogenetic tree . The three species  that are ornamented with an unguiform branch are recovered outside Lysmata and clustered with Exhippolysmata and Lysmatella . The placement of L. californica, which is considered a peppermint shrimp, is contingent to the alignment strategy of the 16S data. Different alignments placed this species ancestral to cleaners or ancestral to non-cleaners  but the placement is either weakly supported  vs. peppermint shrimps (pP and bp = 100). These data also support the historical division  and Hippolysmata , though neither L californica, L. olavoi or L. nayaritensis) are included. Lysmata cf. anchisteus and Lysmata lipkei are clustered outside Lysmata and Hippolysmata forming a clade with Exhippolysmata and Lysmatella, an association observed in the 16S analysis. Similar to the 16S tree, Merguia, Parhippolyte, and Barbouria are basal to Lysmata.The resulting phylogeny from the 28S data alone Figure  and the prior to ) betweenLysmata based on accessory flagellum morphology. It also partly supports Rhyne's [Lysmata according to morphology and/or color pattern: (1) Lysmata, having a long accessory branch, (2) Hippolysmata , renders the genus Lysmata paraphyletic. The BI recovers the different groups more consistently than both ML and MP, especially in the combined dataset  in a basal position in the group with short accessory branch , havingpattern:  Lysmata, branch,  Hippolyst Figure . Howevera et al.  analysispattern:  Lysmata,n Azores . LysmataL. amboinensis, L. debelius, and L. grabhami) may form a monophyletic group [L. californica within the cleaner clade. The placement of L. californica is unresolved, because it is strongly influenced by the alignment strategy. Lysmata californica has "peppermint shrimp" characteristics, lacking white legs and antenna, having translucent body with red stripes and living in groups. The monophyly of the remaining peppermint shrimps  was strongly supported  and insensitive to the alignment conditions and the dataset used.The "cleaners"  are robustly placed outside the Lysmata + Hippolysmata clade in the 16S phylogeny. Similarly, the 16S/28S concatenated phylogeny places L. cf. anchisteus and L. lipkei in the same clade with Lysmatella and Exhippolysmata outside of their traditional taxonomic boundaries  may help clarify the occurrence of this clade. These "outlier" Lysmata also present a challenge to any revision of the nomenclature of the genus. If one proposes to resurrect Hippolysmata and Lysmata to their previous status based upon phylogenetic data, the outliers could not be placed into either genus. A new genus may have to be erected, once their relationship with Exhippolysmata and Lysmatella is clarified. Alernatively, the presence of Exhippolysmata and Lysmatella in the putative third clade of Lysmata, may indicate that the generic definitions of these two genera based on raised basal crest (Exhippolysmata) and lack of epipods (Lysmatella) may be insufficient to raise these species to the genus level. Unlike the Lysmata species of the third clade that are ornamented with an unguiform antennal flagella, Exhippolysmata and Lysmatella have a short blunt flagella.Three s Figure ; this grm shape [,20,26,49Exhippolysmata within Lysmata, rendering the genus Lysmata paraphyletic. We have shown that the position of Exhippolysmata depends on the alignment strategy for 16S and the taxon sampling. Additionally, single gene approaches of closely related species should be interpreted cautiously as they often represent the phylogeny of the genes [Lysmata and other closely related genera. When the morphology of the two genera is taken in to consideration  it seems highly improbable that species with vastly different morphological characters would be nested within a clade of Lysmata. Even though we present a phylogeny based on a denser taxon sampling and an additional gene from the independently evolved nuclear genome, our approach is still limited. We have proceeded by concatenating the two gene sequences prior to the phylogenetic analysis , because it has been demonstrated empirically that concatenation of multiple genes often results in a single well-supported topology [One of the surprising findings in  is the phe genes -58 or thhe genes  and not topology . Theorettopology , concatetopology .Lysmata californica, a peppermint shrimp that commonly associates with moray eels is placed within the clade that includes species living in pairs and bright coloration indicating strong specialized behavior  and Parhippolyte , a genus placed ancestrally to Lysmata, regardless of the alignment strategy. Clearly, studies attempting to determine the origins of PSH must focus on related genera that are ancestral to Lysmata, a point that has been highlighted also in [Parhippolyte and Lysmata in different families  would further support that PSH evolved well outside of Lysmata and could be far more common than previously considered. The cave dwelling, group-living shrimp genus Parhippolyte possesses PSH and all phylogenetic analyses support the ancestral position of this group relative to all Lysmata and Exhippolysmata. When Bauer [Lysmata and why there are two distinct ecological clades, he was unaware that PSH is secondary to the divisions within the genera. The evolution of PSH likely predates the diversification of Lysmata and may have little or no bearing on the evolution of different ecological groups within the genus. For Lysmata, the question is not how PSH is related to socio-ecological systems, but rather why pair living and specialized fish cleaning behavior evolved from a group living ancestor.PSH has been recently been reported in olysmata , Lysmate also in . Christo also in  subdividen Bauer ,13,47 poLysmata, Calliasmata Holthuis 1973, Exhippolysmata, and Mimocaris Vereshchaka 1997 were assigned to the family Lysmatidae, while Barbouria, Parhippolyte, among other genera were assigned to the Barbouriidae. These two families were included with the Processidae Ortmann, 1890 and Crangonidae, and the genera Merguia and Glyphocrangon Milne-Edwards 1881, in the superfamily Crangonoidea. The rest of the hippolytids are assigned by Christoffersen to various families within the superfamily Alpheoidea. Chace [Barbouria + Parhippolyte in all trees, therefore we cannot reject Christoffersen's suggestion for separating the Barbouriidae. However, it is not clear whether or not the level of differentiation from Lysmata, Exhippolysmata, Lysmatella, and Merguia is sufficient to propose a separate family. Although the relative placement of Merguia and the two barbouriids is susceptible to alignment strategies, they are clearly more closely related to Lysmata than the other hippolytid genera in our phylogenies. The ancestral relationship of Merguia to Lysmata and its basal position to the barbouriids could invalidate the Crangonoidea sensu Christoffersen. A denser sampling of taxa from Christoffersen's proposed groups may help to clarify the level of differentiation present among these taxa and others that have traditionally been a part of the Hippolytidae. Until more convincing conclusions can be drawn, the current delineations proposed by Martin and Davis [Based on cladistic analysis, Christoffersen  split tha. Chace  rejecteda. Chace . We consnd Davis  should bLysmata based on accessory branch morphology. Shrimps within the short accessory branch clade differentiate according to behavior and color pattern. The monophyly of the Lysmata group which is bearing a multi-segmented accessory branch is strongly supported, underlying the taxonomic importance of this character. Lysmata with an unguiform accessory branch are part of a third clade which includes Lysmatella and Exhippolysmata. The third clade does not conform to the historic division between Lysmata and Hippolysmata. PSH is ubiquitous within Lysmata and occurs in Barbouriidae, suggesting that this rare reproductive system that evolved ancestrally to the genera Lysmata, Exhippolysmata and Lysmatella. The two representative species of barbouriids form a monophyletic group and are consistently placed within the Hyppolytidae, therefore not providing support for the family Barbouriidae. The ribosomal data provides a unique view of the phylogeny of Lysmata and life history traits, however, the position of some taxa is sensitive to alignment strategies.Our mitochondrial and nuclear ribosomal data generally support the historic morphological division of GCF and AR conceived the project and collected the specimens. RS, TA and NVS provided monetary support, facilities and contributed to the manuscript. GCF, RS and NVS generated the molecular data. GCF, AR and NVS carried out the analyses and wrote the manuscript. All authors read and approved the final manuscript.Table S1: Sequence alignment data for the phylogenies presented in the paper. Includes 16S, 28S and 16S/28S concatenated data sets as a single MSWord file.Click here for fileLysmata and other related genera based on alternative alignment strategies of mitochondrial 16S sequencesFigure S1: Bayesian phylogenies of . Tree A was constructed after the removal of highly variable alignment regions via GBlocks using the most stringent criteria. Tree B was constructed using the alignment resulting from the default settings in ClustalX. Clade support values are shown along the corresponding branches (Bayesian Inference/Maximum Likelihood/Maximum Parsimony). Numbers before sample locations represent the number of specimens sequenced. Superscript numbers indicate which sequences/taxa are represented on the tree (see Tree Identifier in Table Click here for file


The question of whether to initiate ART at higher CD4+ cell counts than currently recommended by World Health Organization (WHO) treatment guidelines received much attention at the XVII International AIDS Conference (AIDS 2008). If studies presented at the conference ultimately lead to a revision of WHO treatment guidance, the estimated number of people who will need ART globally will increase substantially. Task-shifting is emerging as an important strategy for dealing with the acute shortage of health care workers in many high-burden countries, and several studies presented at AIDS 2008 demonstrated the impressive health system efficiencies garnered by using nurses or other health care providers to deliver HIV care and treatment. Other key presentations and discussion at the conference focused on the optimal time to start TB treatment in HIV-infected patients, the growing risk of resistance in high-burden countries, including its impact on future treatment options, and several large cohort trials testing optimal drug regimens in resource-limited settings.Biomedical prevention research continues to confirm the long-term, protective benefits of circumcision. Several studies involving HIV serodiscordant heterosexual couples have produced data suggesting a strong protective effect of ART for HIV-negative partners. Disappointing results from recent vaccine and non-ARV based microbicides trials are nevertheless providing important data to this field, and the expanding number of pre-exposure prophylaxis (PrEP) trials and ARV-based microbicides appear to provide the best hope for a new, efficacious biomedical prevention intervention. AIDS 2008, of course, took place two years before the deadline for universal access, and significant attention was devoted to the theme of the conference: Universal Action Now!, particularly in the context of both increases in the pace of scaling up treatment and care interventions and the growing realization that few countries are on target to meet universal access goals. Strategies for improving access to care in resource-limited countries, the risk of antiretroviral resistance in these countries, and evolving antiretroviral tactics dominated the AIDS 2008 agenda on clinical research and treatment of HIV infection.3 and to include people with active hepatitis B or C infection, cardiovascular disease risk, or compromised kidney function [As AIDS 2008 began, an international panel of treatment experts convened by the IAS-USA updated antiretroviral treatment guidelines for adults. The panel recommended broadening options for starting ART at a CD4 count above 350 cells/mmfunction  The pane3 when first measured independently raised the risk of new cardiovascular disease more than 75% [3, while suggesting clinicians should "consider treatment" for people with 200 to 350 cells/mm3 and defer treatment for people with more than 350 cells/mm3 [A cohort study at AIDS 2008 added to accumulating evidence favouring earlier ART. This 1,679-person analysis of the US HIV Outpatient Study cohort found that a CD4 count under 350 cells/mmDuring AIDS 2008 incoming IAS President Julio Montaner predicted that revamped treatment guidelines for high-income countries could "revolutionize the treatment of HIV" by recognizing HIV infection as a chronic inflammatory disease that "affects the heart, liver, kidneys, and in due course we are going to learn the rest of the assorted organs in the body" ,8. MontaRelated to the growing debate regarding optimal start and switch times is the issue of how and what information to use in clinical decision-making regarding switching drug regimens. A Haitian study raised concerns about relying on clinical or immunological criteria to detect ART failure, based on WHO treatment guidelines, in the absence of VL and CD4+ laboratory monitoring. In this study almost half (47%) the participants had HIV RNA levels below the limit of detection after being assessed as failing ART using clinical criteria, suggesting that the lack of VL PCR and other laboratory diagnostics in clinical decision-making could lead to premature switching .In many parts of the world, tuberculosis is the first AIDS diagnosis and a leading cause of death among people living with HIV. Yet the best time to start ART in HIV/TB-co-infected individuals remains controversial. Two studies presented at AIDS 2008 - one in Brazil and one in Argentina - addressed this question but did not reach the same conclusion.at any point after beginning anti-TB therapy independently halved the risk of death, while completing the course of anti-TB drugs independently lowered the risk of death more than 85% [Valéria Saraceni's 632-person analysis of THRio, a Brazilian observational cohort study, found that starting ART than 85%  , regardless of treatment modality, between men and women. There were also no significant differences in adverse events  when stratified and analyzed by gender, and the safety and efficacy profile for women in the GRACE study was comparable to other studies comparing PI-boosted regimens.When comparing treatment efficacy and the incidence and frequency of adverse events among different ethnic groups, the Artemis study produced comparable results regardless of race. Ethnicity data from the CASTLE study indicated that while both treatment modalities in this study provided promising virologic and immunological results across ethnic sub-groups, there was some variation in the magnitude between different ethnic/racial groups, with ranges of those reaching undetectable levels of viremia ranging from 71% to 83% in the atazanari/r arm to 73% to 90% in the lopvinavir/r arm, with the lowest proportion of those achieving undetectable viremia among African Americans and the highest proportion among Asians or Caucasians. Similar immunological results were reported with respect to CD4+ count increases. There were modest differences in adverse events noted, with diarrhea higher among Caucasians (14%) than among African Americans (5%).Research into new HIV prevention technologies in the years leading up to AIDS 2008 has been discouraging. Between 2006 and 2008, five advanced stage trials (four for broad-spectrum microbicides and one for a vaccine) announced nil or negative results, and there are currently no vaccine candidates ready for field-testing. The long-term circumcision results reported at AIDS 2008 indicated that prevention interventions do have the potential to substantially reduce new infections. Other presentations mapped out prevention strategies using antiretroviral agents, which could be introduced in the field relatively quickly.Most optimistically, Robert Bailey  reported results from extended follow-up of participants in their randomized controlled trial of male circumcision in Kisumu, Kenya at a late-breaker session. Previously, participants in the three trials of circumcision had data up to only 21 - 24 months post-randomization. Now, with the inclusion of 42 months of follow-up, Bailey and his colleagues reported a 65% protective effect of circumcision against HIV acquisition in young men in Kisumu  presented the results of a study subsequent to his group's circumcision trial in Orange Farm, South Africa. After counselling 1,207 men on safe sex and treating them for sexually transmitted infections, the researchers offered free circumcisions in a medical clinic. Among the uncircumcised men , 65% eventually accepted the offer .Auvert's group and the Kisumu trial also attempted to gauge the effect of circumcision on sexual pleasure and performance. They found that there was no appreciable difference between circumcised and uncircumcised men, according to reports from both males and females .Fred Sawe  in addition reported that HIV prevention and reproductive health training was very well received during traditional male rites of passage in the Great Rift Valley . These cAuvert presented the results of the first circumcision randomized clinical trial three years ago at the 3rd IAS Conference on HIV Pathogenesis and Treatment in Rio de Janeiro . StudiesWhile the implementation of circumcision as a viable prevention strategy lags, a debate has emerged about using antiretroviral therapy as a prevention tool. In January 2008, Switzerland's Federal Commission on AIDS-Related Issues (EKAF) released a statement indicating that people living with HIV who were taking an effective  antiretroviral regimen could not transmit the virus ,39. TherNonetheless, arguments ensued over the Swiss statement's implications at an EKAF-sponsored satellite symposium that took place just before the official opening of AIDS 2008. EKAF president Pietro Vernazza presented his Commission's point of view, which places the position in a very carefully-defined context . He saidSuzanna Attia  presented the results of a meta-analysis of studies investigating transmission risk under ART . There hA randomised controlled trial is currently under way to quantify the relationship between the level of treatment-suppressed viral load and HIV transmission. The trial, sponsored by the US National Institutes of Health, will follow 1,750 HIV sero-discordant couples assigned to immediate or deferred treatment. Results will not be available before 2016, a problematic timeline given the research suggesting that persons with treatment-suppressed HIV are already reducing their condom use .In addition to viewing ART as a potentially effective prevention modality involving those already infected, at the conference there was also discussion of administering ART as pre-exposure prophylaxis (PrEP) for HIV-negative persons who are at high risk for infection, including members of HIV-discordant couples . AlthougThere are a number of on-going and planned PrEP trials testing daily regimens Figure . AlthougThere are obvious safety and economic concerns about supplying antiretroviral drugs to large HIV-negative populations. If it proves effective, topical application to genital and anal areas might be more feasible. As conference attendees heard, tenofovir gel applied locally yields vaginal drug concentrations 100- to 1,000-fold higher than systemic oral administration . In contTopical microbicides have not performed well in human HIV prevention studies, with 10 trials of surfactant and polyanionic compounds yielding negative results. These non-specific, broad-spectrum compounds inactivate bacteria, viruses and even sperm by emulsifying (surfactants) or coating (polyanions) their outer layers.The field is clearly moving forward. AIDS 2008 marked the shift from broad-spectrum to antiretroviral microbicides ,48. Besiin vivo results at AIDS 2008 came from a small macaque trial of a vaginal gel, combining two antiretrovirals, tenofovir and emtricitabine. The trial applied this combination gel 30 minutes before exposure to HIV. Six out of six macaques were protected from 20 vaginal challenges over ten weeks. In contrast, seven of eight control macaques became infected after a median of 3.5 challenges [But the results of many of these trials are not expected until 2010-12. The only encouraging microbicide allenges .Recent setbacks in both the vaccine and microbicides field have required a refocus of research efforts in both fields. At the conference, Seth Berkley  reviewed the International AIDS Vaccine Initiative's (IAVI) efforts to screen human subjects for broadly neutralizing antibodies against HIV . These aCurrent HIV vaccine vectors are all nonreplicating. Among these is the adenovirus vector used in the Merck vaccine that yielded negative results in the well-known STEP trial terminated last year. A US National Institutes of Health study comparing chimpanzee responses to replicating and nonreplicating adenovirus-based HIV vaccines was one of the early warnings about the Merck vaccine design . The repCombination prevention strategies was a popular topic of discussion at the conference . CircumcAs the Kisumu trial results show, circumcision alone will not eradicate HIV incidence in men. There is some optimism that adding antiretroviral agents in one of the forms described above may have a major impact in reducing incidence. Research is moving forward slowly despite this potential, and mass implementation will likely also be slow, if the circumcision experience is any example. The prevention research legacy of AIDS 2008 is an increasing recognition that fulfilling the promise of emerging prevention technologies requires a renewed sense of urgency.AIDS 2008 became known for the "marriage" of treatment and prevention, with many speakers underscoring the need to integrate prevention and treatment interventions to ensure a more effective and sustainable response to HIV/AIDS. The term 'combination prevention' - the need to integrate a full range of biomedical, socio-behavioural and structural interventions is covered in greater detail in Section 5 of this supplement. New advances in the clinical management of HIV in resource limited settings and the success of approaches such as task-shifting could serve as important models for ART expansion as the HIV field enters what is increasingly known as the 'second wave' of ART access. However, cautions regarding the potential emergence of resistance strains and pressures on health care systems are highlighting the need for operations research to ensure the optimal management in treatment delivery, including ensuring low-cost second line regimens are available.The vaccine field is returning to the laboratory to assess different options, such as harnessing the body's innate immunity using non-replicating viral vectors. But the only two recently proven or promising biomedical interventions at this point are circumcision and ART-based prevention, either as PrEP or as part of increased ART scale-up that - it is hypothesized - will have a significant impact on community load and the course of the epidemic.Mark Mascolini, Rodney Kort and David Gilden are independent consultants contracted by the International AIDS Society for the purpose of preparing and editing the AIDS 2008 Impact Report for publication.Mark Mascolini and David Gilden drafted the initial text for this section and Rodney Kort provided editorial input and advice. All authors have approved the manuscript for publication.