Perspectives on long-acting formulations of pre-exposure prophylaxis (PrEP) among men who have sex with men who are non-adherent to daily oral PrEP in the United States.

INTRODUCTION: Pre-exposure prophylaxis (PrEP) persistence among men who have sex with men (MSM) in real world clinical settings for HIV prevention is suboptimal. New longer-acting formulations of PrEP are becoming available, including injectables, subdermal implants, and other oral medications. These longer-acting formulations have the potential to improve retention among those who have challenges remaining adherent to daily oral PrEP. METHODS: We interviewed 49 MSM who had initiated but discontinued oral PrEP at three diverse clinics across the United States. We examined participants' perspectives about long-acting PrEP formulations and how long-acting options could affect PrEP use using thematic analysis. RESULTS: Participants were not very knowledgeable about long-acting formulations of PrEP but were open to learning about them and considering use. Participants were concerned about safety and efficacy of products given that they were still newer and/or in development. Finally, participants had clear preferences for oral pills, injectables, and then subdermal implants and were most interested in options that reduced the number of visits to the clinic. CONCLUSION: Long-acting formulations of PrEP are acceptable to MSM with suboptimal PrEP persistence and have the potential to improve PrEP persistence. However, many felt they needed more information on safety, efficacy, and use to consider these options. As these long-acting formulations are implemented, public health campaigns and clinical interventions to encourage may maximize uptake particularly among those who are not currently adherent to daily oral PrEP.

Facilitators for retaining men who have sex with men in pre-exposure prophylaxis care in real world clinic settings within the United States.

BACKGROUND: Pre-exposure prophylaxis (PrEP) can significantly reduce HIV acquisition especially among communities with high HIV prevalence, including men who have sex with men (MSM). Much research has been finding suboptimal PrEP persistence; however, few studies examine factors that enhance PrEP persistence in real-world settings. METHODS: We interviewed 33 patients who identified as MSM at three different PrEP clinics in three regions of the U.S. (Northeast, South, Midwest). Participants were eligible if they took PrEP and had been retained in care for a minimum of 6 months. Interviews explored social, structural, clinic-level and behavioral factors that influencing PrEP persistence. RESULTS: Through thematic analysis we identified the following factors as promoting PrEP persistence: (1) navigation to reduce out-of-pocket costs of PrEP (structural), (2) social norms that support PrEP use (social), (3) access to LGBTQ + affirming medical providers (clinical), (4) medication as part of a daily routine (behavioral), and (5) facilitation of sexual health agency (belief). DISCUSSION: In this sample, persistence in PrEP care was associated with structural and social supports as well as a high level of perceived internal control over protecting their health by taking PrEP. Patients might benefit from increased access, LGBTQ + affirming medical providers, and communications that emphasize PrEP can promote sexual health.

Genomic sequence investigation Streptococcus pyogenes clusters in England (2010-2015).

OBJECTIVES: To analyse genomic sequence data of referred Streptococcus pyogenes isolates and those pertaining to selected elderly/nursing care or maternity clusters from 2010 to 2015 to ascertain genomic differences between epidemiologically related isolates and unrelated isolates from outbreaks of disease. METHODS: The genomic sequences of 134 S. pyogenes isolates from 21 clusters of infection in elderly care or maternity settings from 2010 to 2015 were analysed using bioinformatics to ascertain genomic phylogeny, single nucleotide polymorphism (SNP) differences and statistical outliers from epidemiologically defined outbreaks. Analysis was undertaken within clusters and compared with sporadic isolates from geographically distinct outbreaks of S. pyogenes infection. RESULTS: Genomic sequence analysis of 21 outbreaks of S. pyogenes infection ranged in size from a single patient (with colonized healthcare worker link) to 18 patient cases of group A streptococcus (GAS) infection in a single setting. Seventeen healthcare workers were identified in 8 of 21 outbreaks with the associated outbreak strain, with multiple staff in 2 of 21 outbreaks. Genomic sequences from epidemiologically linked isolates from patients, staff and healthcare environmental settings were highly conserved, differing by 0-1 SNP in some cases and mirrored geographical data. Four of 21 outbreaks had environmental contamination with the outbreak strain, indistinguishable or of limited SNP difference to the patient isolates. Genomic SNP analysis enabled exclusion of ten isolates from epidemiological outbreaks. CONCLUSIONS: Genomic discrimination can be applied to assist outbreak investigation. It enabled confirmation or exclusion of GAS cases from epidemiologically defined outbreaks. Colonization of healthcare workers and environmental contamination with the outbreak strain was demonstrated for several outbreaks.

Whole-genome sequencing in the investigation of recurrent invasive group A streptococcus outbreaks in a maternity unit.

BACKGROUND: The clinical manifestations of group A streptococcus (GAS) (Streptococcus pyogenes) are diverse, ranging from asymptomatic colonization to devastating invasive disease. Maternity-related clusters of invasive GAS (iGAS) infection are complex to investigate and control, especially if recurrent. AIM: To investigate three episodes of emm 75 GAS/iGAS infection in maternity patients at one hospital site over a four-year period (two with monophyletic ancestry). METHODS: The episodes are described, together with whole-genome sequence (WGS) isolate analyses. Single nucleotide polymorphism differences were compared with contemporaneous emm 75 genomes. FINDINGS: Over the four-year study period, seven mothers had emm 75 GAS/iGAS and one mother had emm 3 iGAS (in year 4) (subsequently discounted as linked). Three (clinical/screening samples) of the seven babies of emm-75-positive mothers and three screened healthcare workers were positive for emm 75 GAS. WGS similarity suggested a shared ancestral lineage and a common source transmission, but directionality of transmission cannot be inferred. However, the findings indicate that persistence of a particular clone in a given setting may be long term. CONCLUSIONS: Occupational health procedures were enhanced, staff were screened, and antibiotic therapy was provided to GAS-positive staff and patients. The definitive source of infection could not be identified, although staff-patient transmission was the most likely route. The pattern of clonal GAS transmission over the four-year study period suggests that long-term persistence of GAS may have occurred.

Risk factors for relapse and recurrence of depression in adults and how they operate: A four-phase systematic review and meta-synthesis.

PURPOSE: To review and synthesise prognostic indices that predict subsequent risk, prescriptive indices that moderate treatment response, and mechanisms that underlie each with respect to relapse and recurrence of depression in adults. RESULTS AND CONCLUSIONS: Childhood maltreatment, post-treatment residual symptoms, and a history of recurrence emerged as strong prognostic indicators of risk and each could be used prescriptively to indicate who benefits most from continued or prophylactic treatment. Targeting prognostic indices or their "down-stream" consequences will be particularly beneficial because each is either a cause or a consequence of the causal mechanisms underlying risk of recurrence. The cognitive and neural mechanisms that underlie the prognostic indices are likely addressed by the effects of treatments that are moderated by the prescriptive factors. For example, psychosocial interventions that target the consequences of childhood maltreatment, extending pharmacotherapy or adapting psychological therapies to deal with residual symptoms, or using cognitive or mindfulness-based therapies for those with prior histories of recurrence. Future research that focuses on understanding causal pathways that link childhood maltreatment, or cognitive diatheses, to dysfunction in the neocortical and limbic pathways that process affective information and facilitate cognitive control, might result in more enduring effects of treatments for depression.

Vestibular rehabilitation using video gaming in adults with dizziness: a pilot study.

OBJECTIVE: To determine the effectiveness of vestibular rehabilitation using the Wii Fit balance platform, in adults with dizziness. METHODS: A single-site prospective clinical trial was conducted in a university hospital in the UK. Forty patients with dizziness, who would normally be candidates for vestibular rehabilitation, were identified and considered as potential participants. Participants were randomised into either the treatment group (the Wii Fit group) or the control group (standard customised vestibular rehabilitation protocol). Participants were assessed over a 16-week period using several balance and quality of life questionnaires. RESULTS: Both exercise regimes resulted in a reduction of dizziness and an improvement in quality of life scores over time, but no statistically significant difference between the two interventions was identified. CONCLUSION: This pilot study demonstrated that use of the Wii Fit balance platform resulted in a statistically significant improvement in balance function and quality of life. Furthermore, outcomes were comparable to a similar group of individuals following a standard customised vestibular rehabilitation protocol. The study provides useful information to inform the design and execution of a larger clinical trial.

Rapid detection and evolutionary analysis of Legionella pneumophila serogroup 1 sequence type 47.

OBJECTIVES: Legionella pneumophila serogroup 1 (Lp1) sequence type 47 is the leading cause of legionellosis in north-western Europe, but, surprisingly, it is rarely isolated from environmental samples. Comparative genomics was applied to develop a PCR assay and to better understand the evolution of this strain. METHODS: Comparative analysis of 36 genomes representative of the Lp species was used to identify specific PCR targets, which were then evaluated in silico on 545 sequenced genomes and in vitro on 436 Legionella strains, 106 respiratory samples, and three environmental samples from proven ST47 sources. Phylogenetic analyses were performed to understand the evolution of ST47. RESULTS: The gene LPO_1073 was characterized as being 100% conserved in all 129 ST47 genomes analysed. A real-time PCR designed to detect LPO_1073 was positive for all 110 ST47 strains tested and agreed with culture and typing results previously obtained for 106 respiratory samples. The three environmental samples were also positive. Surprisingly, 26 of the 44 ST109 strains tested among 342 non-ST47 strains scored positive for LPO_1073. SNP-based phylogenetic analysis was undertaken to understand this result: the PCR-positive ST109 genomes were almost identical to ST47 genomes, with the exception of a recombined region probably acquired by ST47 from a ST62(-like) strain. CONCLUSION: The genomic analysis allowed the design of a highly specific PCR assay for rapid detection of ST47 strains. Furthermore, it allowed us to uncover the evolution of ST47 strains from ST109 by homologous recombination with ST62. We hypothesize that this recombination generated the leading cause of legionellosis in north-western Europe.

The ecological impacts of marine debris: unraveling the demonstrated evidence from what is perceived.

Anthropogenic debris contaminates marine habitats globally, leading to several perceived ecological impacts. Here, we critically and systematically review the literature regarding impacts of debris from several scientific fields to understand the weight of evidence regarding the ecological impacts of marine debris. We quantified perceived and demonstrated impacts across several levels of biological organization that make up the ecosystem and found 366 perceived threats of debris across all levels. Two hundred and ninety-six of these perceived threats were tested, 83% of which were demonstrated. The majority (82%) of demonstrated impacts were due to plastic, relative to other materials (e.g., metals, glass) and largely (89%) at suborganismal levels (e.g., molecular, cellular, tissue). The remaining impacts, demonstrated at higher levels of organization (i.e., death to individual organisms, changes in assemblages), were largely due to plastic marine debris (> 1 mm; e.g., rope, straws, and fragments). Thus, we show evidence of ecological impacts from marine debris, but conclude that the quantity and quality of research requires improvement to allow the risk of ecological impacts of marine debris to be determined with precision. Still, our systematic review suggests that sufficient evidence exists for decision makers to begin to mitigate problematic plastic debris now, to avoid risk of irreversible harm.

Microevolution of Monophasic Salmonella Typhimurium during Epidemic, United Kingdom, 2005-2010.

Microevolution associated with emergence and expansion of new epidemic clones of bacterial pathogens holds the key to epidemiologic success. To determine microevolution associated with monophasic Salmonella Typhimurium during an epidemic, we performed comparative whole-genome sequencing and phylogenomic analysis of isolates from the United Kingdom and Italy during 2005-2012. These isolates formed a single clade distinct from recent monophasic epidemic clones previously described from North America and Spain. The UK monophasic epidemic clones showed a novel genomic island encoding resistance to heavy metals and a composite transposon encoding antimicrobial drug resistance genes not present in other Salmonella Typhimurium isolates, which may have contributed to epidemiologic success. A remarkable amount of genotypic variation accumulated during clonal expansion that occurred during the epidemic, including multiple independent acquisitions of a novel prophage carrying the sopE gene and multiple deletion events affecting the phase II flagellin locus. This high level of microevolution may affect antigenicity, pathogenicity, and transmission.

Design and application of a core genome multilocus sequence typing scheme for investigation of Legionnaires' disease incidents.

Sequence-based typing (SBT) for Legionella pneumophila (Lp) has dramatically improved Legionnaires' disease (LD) cluster investigation. Microbial whole genome sequencing (WGS) is a promising modality for investigation but sequence analysis methods are neither standardised, nor agreed. We sought to develop a WGS-based typing scheme for Lp using de novo assembly and a genome-wide gene-by-gene approach (core genome multilocus sequence typing, cgMLST). We analysed 17 publicly available Lp genomes covering the whole species variation to define a core genome (1,521 gene targets) which was validated using 21 additional published genomes. The genomes of 12 Lp strains implicated in three independent cases of paediatric humidifier-associated LD were subject to cgMLST together with three 'outgroup' strains. cgMLST was able to resolve clustered strains and clearly identify related and unrelated strains. Thus, a cgMLST scheme was readily achievable and provided high-resolution analysis of Lp strains. cgMLST appears to have satisfactory discriminatory power for LD cluster analysis and is advantageous over mapping followed by single nucleotide polymorphism (SNP) calling as it is portable and easier to standardise. cgMLST thus has the potential for becoming a gold standard tool for LD investigation. Humidifiers pose an ongoing risk as vehicles for LD and should be considered in cluster investigation and control efforts.

Linking effects of anthropogenic debris to ecological impacts.

Accelerated contamination of habitats with debris has caused increased effort to determine ecological impacts. Strikingly, most work on organisms focuses on sublethal responses to plastic debris. This is controversial because (i) researchers have ignored medical insights about the mechanisms that link effects of debris across lower levels of biological organization to disease and mortality, and (ii) debris is considered non-hazardous by policy-makers, possibly because individuals can be injured or removed from populations and assemblages without ecological impacts. We reviewed the mechanisms that link effects of debris across lower levels of biological organization to assemblages and populations. Using plastic, we show microplastics reduce the 'health', feeding, growth and survival of ecosystem engineers. Larger debris alters assemblages because fishing-gear and tyres kill animals and damage habitat-forming plants, and because floating bottles facilitate recruitment and survival of novel taxa. Where ecological linkages are not known, we show how to establish hypothetical links by synthesizing studies to assess the likelihood of impacts. We also consider how population models examine ecological linkages and guide management of ecological impacts. We show that by focusing on linkages to ecological impacts rather than the presence of debris and its sublethal impacts, we could reduce threats posed by debris.

Rapid identification of major Escherichia coli sequence types causing urinary tract and bloodstream infections.

Escherichia coli sequence types (STs) 69, 73, 95, and 131 are collectively responsible for a large proportion of E. coli urinary tract and bloodstream infections, and they differ markedly in their antibiotic susceptibilities. Here, we describe a novel PCR method to rapidly detect and distinguish these lineages. Three hundred eighteen published E. coli genomes were compared in order to identify signature sequences unique to each of the four major STs. The specificities of these sequences were assessed in silico by seeking them in an additional 98 genomes. A PCR assay was designed to amplify size-distinguishable fragments unique to the four lineages and was validated using 515 E. coli isolates of known STs. Genome comparisons identified 22 regions ranging in size from 335 bp to 26.5 kb that are unique to one or more of the four predominant E. coli STs, with two to 10 specific regions per ST. These regions predominantly harbor genes encoding hypothetical proteins and are within or adjacent to prophage sequences. Most (13/22) were highly conserved (>96.5% identity) in the genomes of their respective ST. The new assay correctly identified all 142 representatives of the four major STs in the validation set (n = 515), with only two ST12 isolates misidentified as ST95. Compared with MLST, the assay has 100% sensitivity and 99.5% specificity. The rapid identification of major extraintestinal E. coli STs will benefit future epidemiological studies and could be developed to tailor antibiotic therapy to the different susceptibilities of these dominant lineages.

Extension of the Legionella pneumophila sequence-based typing scheme to include strains carrying a variant of the N-acylneuraminate cytidylyltransferase gene.

Sequence-based typing (SBT) combined with monoclonal antibody subgrouping of Legionella pneumophila isolates is at present considered to be the reference standard during epidemiological investigation of Legionnaires' disease outbreaks. In some isolates of L. pneumophila, the seventh allele of the standard SBT scheme, neuA, is not amplified, because a homologue that is refractory to amplification with the standard neuA primers is present. Consequently, a complete seven-allele profile, and hence a sequence type, cannot be obtained. Subsequently, primers were designed to amplify both neuA and the homologue, but these yielded suboptimal sequencing results. In this study, novel primers specific for the neuA homologue were designed and internationally validated by members of the ESCMID Study Group for Legionella Infections at national and regional Legionella reference laboratories with a modified version of the online L. pneumophila sequence quality tool. To date, the addition of the neuAh target to the SBT protocol has allowed full typing data to be obtained for 108 isolates of 11 different serogroups, namely 1, 2, 3, 4, 5, 6, 7, 8, 10, 13, and 14, which could not previously be typed with the standard SBT neuA primers. Further studies are necessary to determine why it is still not possible to obtain either a neuA or a neuAh allele from three serogroup 11 isolates.

Effects of zinc on microalgal biofilms in intertidal and subtidal habitats.

Microalgal biofilms are sensitive to environmental conditions. Impacts of contaminants on assemblages of marine biofilm are often investigated in laboratories or in mesocosms. Such experiments are rarely representative of the effects of contaminants on biofilms under natural conditions. Studies in field situations, with enough power to detect impacts, are necessary to develop a better understanding of the effects of contaminants on ecological processes. Metals are a common contaminant of marine systems and can cause disturbances to assemblages. Using a new technique to experimentally deliver contaminants to microalgal assemblages, hypotheses were tested regarding the effects of zinc on microalgal biofilms growing on settlement panels in subtidal and intertidal habitats. PAM fluorometry was used to assess the amount and physiological state of biofilms on panels. Control panels deployed for 1 month in each habitat had significantly greater amounts of biofilm than those exposed to zinc. After deployment for 3 months, the results varied with location. The observed effects on the biofilm did not, however, cause significant changes in the macro-invertebrate assemblages that developed on the panels.

Urban structures provide new insights into interactions among grazers and habitat.

A major challenge in ecology is to understand and predict consequences of environmental changes to biological assemblages. Urbanization and associated alteration and destruction of habitat cause profound changes in local biodiversity and the ecology of and interactions among organisms. This study tested hypotheses about interactions among intertidal species on urbanized shores to examine predictions from their known ecology. On natural shores, grazing limpets, Cellana tramoserica, outcompete the smaller limpets, Patelloida latistrigata. The latter shelter among barnacles. On seawalls in Sydney Harbour (Australia), P. latistrigata were found among and on oysters. C. tramoserica were found directly on walls where there were no oysters. To explain these observations, several models were proposed and tested by manipulative experiments in the field: (1) oysters provide habitat for P. latistrigata; (2) negative interactions with C. tramoserica cause P. latistrigata to move on and among oysters; and (3) in areas with oysters, space available for grazing is insufficient for C. tramoserica to survive well. The results showed that C. tramoserica had a negative impact on the survival of P. latistrigata, although not causing the latter to move onto oysters. The oysters directly and indirectly affected P. latistrigata. First, oysters provided habitat and increased survival of P. latistrigata. Second, oysters influenced the interaction between C. tramoserica and P. latistrigata. This "interaction modification" diminished the negative impact of C. tramoserica. The material used to construct a wall did not directly influence the distribution of the limpets. Maintaining oysters on seawalls is, however, important because of their direct and indirect effects on P. latistrigata. It was possible to predict some processes on urbanized shores from known ecology. Other processes could not be predicted, making it necessary to do experiments to understand how built structures influence biological assemblages. This research contributes to understanding how to conserve biodiversity in urban areas.

Independent effects of patch size and structural complexity on diversity of benthic macroinvertebrates.

Despite a long history of work on relationships between area and number of species, the details of mechanisms causing patterns have eluded ecologists. The general principle that the number of species increases with the area sampled is often attributed to a sampling artifact due to larger areas containing greater numbers of individuals. We manipulated the patch size and surface area of experimental mimics of macro-algae to test several models that can explain the relationship between abundance and species richness of assemblages colonizing different habitats. Our results show that patch size and structural complexity have independent effects on assemblages of macroinvertebrates. Regardless of their structural complexity, larger habitats were colonized by more species. Patch size did not have a significant effect on numbers of individuals, so the increased number of species in larger habitats was not simply a result of random placement associated with sampling increased number of individuals. Similarly, random placement alone could not explain differences in numbers of species among habitats with different structural complexity, contrary to suggestions that the relationship between number of species and surface area might also be a sampling artifact due to more complex habitats having larger areas and therefore sampling more individuals. Future progress would benefit from manipulating properties of habitat in conjunction with experimental manipulations of area.

Pandemic (H1N1) 2009 influenza in the UK: clinical and epidemiological findings from the first few hundred (FF100) cases.

The UK was one of few European countries to document a substantial wave of pandemic (H1N1) 2009 influenza in summer 2009. The First Few Hundred (FF100) project ran from April-June 2009 gathering information on early laboratory-confirmed cases across the UK. In total, 392 confirmed cases were followed up. Children were predominantly affected (median age 15 years, IQR 10-27). Symptoms were mild and similar to seasonal influenza, with the exception of diarrhoea, which was reported by 27%. Eleven per cent of all cases had an underlying medical condition, similar to the general population. The majority (92%) were treated with antiviral drugs with 12% reporting adverse effects, mainly nausea and other gastrointestinal complaints. Duration of illness was significantly shorter when antivirals were given within 48 h of onset (median 5 vs. 9 days, P=0.01). No patients died, although 14 were hospitalized, of whom three required mechanical ventilation. The FF100 identified key clinical and epidemiological characteristics of infection with this novel virus in near real-time.

Do modified habitats have direct or indirect effects on epifauna?

Replacing natural habitats with artificial structures such as pier-pilings, jetties, and seawalls has important consequences to abundances of biota. It is, however, not often known whether these are direct (the novel habitat alters abundances of some species) or indirect (the novel habitat directly alters some aspect of the behavior or ecology of some species, which, in turn, alter abundances of other species). Marine animals in some modified habitats in Sydney Harbour provide experimental opportunities to test hypotheses to distinguish between direct and indirect processes. Covers of bryozoans and hydroids were greater on kelp growing on pilings than on kelp growing on natural reefs. The epifauna may be affected directly by the pilings or indirectly, i.e., the structure affects characteristics of the kelp which, in turn, influence covers of epifauna. Thus, differences in covers of epifauna on kelp can be due to: (1) factors associated with the primary habitats (pilings vs. reefs), (2) differences between characteristics of the kelp found in each habitat, or (3) an interaction between these factors (habitat and/or type of kelp). Kelp were experimentally transplanted between pilings and reefs, demonstrating that properties of the habitat directly affected covers of epifauna, which were not influenced by the type of kelp that grows on pilings or rocky reefs. Manipulative experiments to unconfound multiple components of habitats influencing disturbances to biota are needed to understand human impacts on natural systems.

Evaluating accuracy and precision of species-area relationships for multiple estimators and different marine assemblages.

This paper evaluates the accuracy and precision of six nonparametric estimators and six regression models for predicting the number of species in an area using random subsamples of that area. Such extrapolations are important for increased understanding of species-area relationships in different assemblages of species to inform ecological theories, and because they have direct implications in many conservation issues. Often, such extrapolations are evaluated by considering how well they fit existing data, rather than the actual number of species in the larger area. Also, many studies have focused on larger species in terrestrial habitats, where numbers of species are quite well known. Assemblages of invertebrates and smaller plants, especially in marine habitats, are less well known, and their distributions are dominated by small-scale variability. We examined species-area relations for two assemblages on boulders from six locations, for rocky shore assemblages in three locations, and for gastropods colonizing artificial habitat. Changing the focus of subsampling relative to the extent of the study and changing the size of the subsamples was also evaluated for subsets of data. Most estimators fitted the number of species poorly, with the second-order Jackknife consistently the best of the nonparametric measures, and the Negative Exponential the best of the regression models. Increasing the number of replicates improved most models, but some only slightly, and others considerably. Changing focus had little effect, probably because marine assemblages such as these vary at small scales as much as at many larger scales. These extensive analyses provide clear evidence about which estimators should not be used for measuring species-area relationships in assemblages such as these, and which will consistently over- or underestimate the number of species. The findings are applicable to many assemblages dominated by small organisms with strong stochastic variation.

Evidence necessary for taxa to be reliable indicators of environmental conditions or impacts.

Using taxa as indicators of environmental impacts is widespread. Indicators are chosen because they are considered to be easy to measure, sensitive to stresses and respond to stresses in predictable ways. Here, we review these criteria by addressing the nature of the relationships between some characteristic of taxa and the environmental variables they are supposed to indicate. It is crucial that variables measured as indicators be strongly and consistently correlated (through space and time) with levels of the environmental variables. Appropriate experiments must be done to establish that an observed correlation is causal, or the correlation cannot be considered sufficient to identify a useful indicator. Finally, it is necessary to establish that the taxa directly respond to changes in the environmental variables they are supposed to indicate. Appropriate methodologies to establish these criteria are considered and we evaluate studies in which these criteria have or have not been met.