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The Prevention and Reactivation Care Program: intervention fidelity matters
Organization, structure and delivery of healthcare
The Prevention and Reactivation Care Program (PReCaP) entails an innovative multidisciplinary, integrated and goal oriented approach aimed at reducing hospital related functional decline among elderly patients. Despite calls for process evaluation as an essential component of clinical trials in the geriatric care field, studies assessing fidelity lag behind the number of effect studies. The threefold purpose of this study was (1) to systematically assess intervention fidelity of the hospital phase of the PReCaP in the first year of the intervention delivery; (2) to improve our understanding of the moderating factors and modifications affecting intervention fidelity; and (3) to explore the feasibility of the PReCaP fidelity assessment in view of the modifications.
Based on the PReCaP description we developed a fidelity instrument incorporating nineteen (n=19) intervention components. A combination of data collection methods was utilized, i.e. data collection from patient records and individual Goal Attainment Scaling care plans, in-depth interviews with stakeholders, and non-participant observations. Descriptive analysis was performed to obtain levels of fidelity of each of the nineteen PReCaP components. Moderating factors were identified by using the Conceptual Framework for Implementation Fidelity.
Ten of the nineteen intervention components were always or often delivered to the group of twenty elderly patients. Moderating factors, such as facilitating strategies and context were useful in explaining the non- or low-adherence of particular intervention components.
Fidelity assessment was carried out to evaluate the adherence to the PReCaP in the Vlietland Ziekenhuis in the Netherlands. Given that the fidelity was assessed in the first year of PReCaP implementation it was commendable that ten of the nineteen intervention components were performed always or often. The adequate delivery of the intervention components strongly depended on various moderating factors. Since the intervention is still developing and undergoing continuous modifications, it has been concluded that the fidelity criteria should evolve with the modified intervention. Furthermore, repeated intervention fidelity assessments will be necessary to ensure a valid and reliable fidelity assessment of the PReCaP.
Trial registration
The Netherlands National Trial Register: NTR2317
Geriatric care interventions are complex, dynamic, and patient-oriented in order to be sensitive and responsive to the unique characteristics of the elderly patient. This approach is essential to ensure clinically meaningful and relevant interventions. Yet, it is this approach, that makes it challenging to establish the reliable and competent delivery of geriatric interventions, in the literature referred to as intervention fidelity
In outcome research intervention fidelity has been described as the confirmation that the manipulation of the independent variable occurred as planned, which enables researchers to (1) determine how adequately a program model has been implemented; (2) assess conformity with prescribed components and absence of non-prescribed components; and (3) provide assurances to policy-makers that services are being implemented as intended and are reaching the target audience
Intervention fidelity has two core components: protocol adherence and competence. Adherence is the most basic and entails the extent to which the interventionists' behavior conforms to the intervention protocol in terms of content, frequency and duration
● Content
● Characteristics (e.g. complexity, resources and equipment, number of interventionists, time needed for intervention components)
● Discriminability of evaluated components
● Overlap of components (e.g. was there contamination of the independent variable)
● Process
● Description of the implementation strategy
● Implementation of components in accordance with the protocol
● Acceptance of the intervention by the interventionists and the patients
● Use of effective communication skills and behaviours
● Variation in the intervention delivery across departments
● Change of the intervention delivery across time (e.g. did the components weaken as they were delivered over the course of time?)
● Change of the intervention delivery throughout the course of the study period (e.g. did the interventionists drift as they delivered the intervention?)
● Variance in the delivery of a particular component to different types of patients (e.g. did more severe patients receive different components than less severe ones?)
● Outcomes
● Association between particular components and good (or poor) outcomes
● Association between intervention 'purity' and better outcomes
Traditional outcome research often assumed that the provision of a detailed description of the intervention (e.g. in the form of a manual) was enough to ensure the adequate implementation in the field. Consequently, it was assumed that patients received the interventions they were supposed to receive as designed
An important issue to consider in fidelity assessment is the influence of moderating factors on the implementation of the intervention. According to Carroll's Conceptual Framework for Implementation Fidelity, these factors include comprehensiveness of policy description, facilitation strategies, quality of delivery, and participant responsiveness
Despite calls for process evaluation as a necessary component of randomized clinical trials in the field of geriatric care
Case study: The Prevention and Reactivation Care Program
Hospital related functional decline in older patients is an underestimated problem. Thirty-five percent of 70-year old patients experience functional decline during hospital admission in comparison with pre-illness baseline
1. To systematically assess intervention fidelity of the hospital phase of the Prevention and Reactivation Care Program;
2. To improve our understanding of the moderating factors and modifications affecting intervention fidelity;
3. To explore the feasibility of the PReCaP fidelity assessment in view of the modifications.
It is anticipated that the results of the intervention fidelity study will assist in strengthening the impact of the evaluation study
A literature review was conducted to determine procedures for fidelity assessment utilized by other researchers, which resulted in a synthesis of methodologies across studies
Step 1: identification of the essential intervention components
The various components of the PReCaP were outlined in the description of the intervention, based on a me-thod proposed by Perera, Heneghan & Yudkin
Step 2: construction of scale items
Fidelity criteria often include specification of the four adherence components, i.e. content, frequency, duration, coverage
Step 3: measuring fidelity
Fidelity data were collected by means of document analysis in the Vlietland Ziekenhuis, Schiedam, The Netherlands. Taking a pragmatic approach
Step 4: assessing reliability and validity of the fidelity criteria
The components of the intervention fidelity instrument were established to ensure an accurate reflection of the PReCaP. The PReCaP Recovery Team, consisting of the program director/psycho geriatrician, program leader, and casemanagers with geriatric expertise critically reviewed the intervention fidelity instrument to ensure construct validity
Descriptive analysis in IBM SPSS Statistics 19 was performed to obtain levels of fidelity for each of the 19 PReCaP components. Fidelity levels were determined as follows: never (0%), seldom (1-33%), sometimes (34-66%), often (67-99%), and always (100%).
The interviews were transcribed and analyzed using framework analysis
Patient records and individual GAS care plans were assessed for evidence of delivered PReCaP components. The patients' demographics are presented in Table
The PReCaP encompassed 19 components during the hospital phase (Table
Moderating factors
Although the components 'identification of patient at risk within 48 hours after admission' and 'assessment of risk factors for functional decline' were performed according to the intervention description (Table
Facilitation strategies
The provision of guidelines and manuals, monitoring and feedback are known strategies to optimize and standardize implementation fidelity, i.e. to ensure that all interventionists receive the same training and support, aimed at a uniform delivery of the intervention
Since December 2011, the casemanagers use issue reports to explore unplanned implementation issues (i.e. incidents or deviations from the PReCaP description), and to prevent these issues from happening in the future. These reports incorporate a description of the issue and the situation, the impact on the patient, the level of priority, and the planned action. The PReCaP Recovery Team, including program director/geriatrician, program leader, and casemanagers convenes four-weekly to discuss the quality of the intervention and to address implementation issues if necessary. Below is an outline of an issue report.
● Issue: GAS care plans are not designed during MTM
● Description: According to PReCaP description GAS care plans should be designed during MTM
● Attending team members refuse to cooperate due to time constraints and lack of motivation
● Impact: Goals and advice for reactivation treatment (aimed at functional preservation) are not formulated. Reactivation treatment does not start within the set time frame of 48 hours after hospital admission
● Priority: High
● Action:
1. During the next MTM the casemanagers emphasize the importance of the timely design of the PReCaP care plan to team members to enable timely start of the reactivation treatment
2. During the MTM the team members draft the GAS care plan on paper. Casemanagers enter the GAS care plan draft in the GAS data base on the same day
● Deadline: four weeks
Contextual factors had an immediate effect on the intended implementation of the PReCaP. Despite earlier agreements, the management of the Vlietland Ziekenhuis allowed only three of the ten hospital units to participate in the intervention. This decision undoubtedly affected the recruitment and the case mix of the participants in the PReCaP, which shows the complex interrelationship between the context and recruitment
According to the description of the PReCaP, the research nurse performs the components 1 and 2 (i.e. 'Identification of patient at risk within 48 hours after admission' and 'Assessment of risk factors for functional decline'). Therefore, permission was obtained from the Vlietland Ziekenhuis to access the admissions department database, enabling identification of the patients at risk (65 years or older and admitted for at least two days) within 48 hours after admission.
Given that polypharmacy is a known risk factor for elderly patients
The PReCaP prescribes the casemanager to conduct the exit interview with the patient and the informal caregiver. However, it is the Vlietland Ziekenhuis policy that the transfer nurses perform all exit interviews to ensure coordination and continuity of the discharge procedures. Therefore, this component was modified to comply with hospital policy.
Quality of delivery
The quality of delivery concerns the extent to which an intervention is delivered in a way appropriate to achieving what was intended
The Vlietland Ziekenhuis and Argos Zorggroep have collaboratively developed a geriatric training program for registered nurses and nurse practitioners. Prior to the commencement of the PReCaP in 2010, fifty Registered Nurses in the Vlietland Ziekenhuis have undertaken the particular training program. In addition, three clinical geriatricians were employed, nursing home physicians have undertaken gerontology-rehabilitation training, and psychologists have undertaken training in system therapy.
Participant responsiveness
Participant responsiveness refers to how well participants respond to, or are engaged by, an intervention. It also involves judgments by the interventionists about the outcomes and relevance of an intervention
Since mid-2011 the Geriatrics unit team leader and the PRC nursing home physician participated in the MTM. In particular, the extra-mural vision of the nursing home physician was considered to be of added value, in particular with regard to the post-discharge period.
The PReCaP description prescribed that the casemanagers handed out information flyers to the eligible patients for the Prevention and Reactivation Centre. Yet, this appeared to be ineffective, and an alternative option to inform the patient and the informal care giver was chosen.
The Prevention and Reactivation Care Program (PReCaP) was developed as a means to reduce hospital related functional decline among elderly patients by offering interventions that are multidisciplinary, integrated and goal-oriented at the physical, social, and psychological domains of functional decline. Evaluation of the PReCaP, currently underway, is expected to determine the extent to which the program leads to improved geriatric care as well as cost reduction in comparison to current geriatric care in The Netherlands
Fidelity assessment is considered essential in order to maintain internal validity and to ensure a fair comparison of the results between the intervention and control settings. Results without a fidelity check may be due to an effective intervention or contamination from other interventions
Studies assessing fidelity of geriatric care interventions are rare. Thus far, most fidelity studies were performed in the field of psychiatric rehabilitation (Carroll et al. 2000, Bond et al. 2000, Song, Happ & Sandelowski 2010) and prevention programs in schools (Dane, Schneider 1998). Despite calls for process evaluation as a required component of randomized clinical trials in the field of geriatric care (Buurman 2011), fidelity studies are lacking in the field of geriatric care interventions.
This paper presents the results of a fidelity assessment, carried out to evaluate the adherence to the PReCaP (hospital phase) in the Vlietland Ziekenhuis in The Netherlands and to improve our understanding of the moderating factors affecting intervention fidelity. The results showed that ten of the nineteen intervention components were always or often delivered to the group of twenty elderly patients. In particular, the identification of the patient at risk and the assessment of risk factors were performed in accordance with the PReCaP. This was in contrast with a study performed in the Emergency Department, where participant recruitment was problematic due to time constraints in conducting the geriatric assessment, and difficulties finding individuals that fulfilled the inclusion criteria
Multi-disciplinary team meetings (MTMs), inter-disciplinary consultations and treatments were largely conducted according to the PReCaP. Yet, it is not clear whether the latter two were the result of the PReCaP intervention (or more specifically the MTM component) or the specialist's orders, an issue known as the 'essential but not unique' components of an intervention
The absence of a comprehensive protocol may have caused reduced fidelity rates due to interventionists' (e.g. nurses in the ward) unfamiliarity with the PReCaP components. A well written comprehensive protocol outlines the intervention and presents the theory, the goals, and the strategies for achieving these goals. Moreover, a protocol provides objective means for comparing interventions and facilitating transfer from research to practice. Therefore, utilizing a comprehensive protocol will lead to greater consistency and precision in the intervention delivery and enhanced internal validity
The interview results demonstrated that the PReCaP is still developing and undergoing continuous modifications. For example, in contrast with the initial PReCaP description, the casemanagers do not visit the patient until Day 2 to prevent mental overload, and since mid-2011 additional professionals (the Geriatrics unit team leader and the PRC nursing home physician) participate in the MTM to enhance the multidisciplinary input. Evidence suggests that programs are usually not static and often undergo substantial changes over time
There has been a long-standing controversy between schools of thought that advocate for exact replications of effective program models versus the need to adapt models to local conditions to maximize efficiency as well as local ownership, also referred to in the literature as reinvention
Although the PReCaP fidelity assessment was demonstrated to be a feasible measurement instrument, it also showed the need for further articulation of the criteria for inclusion of the conditional intervention components (e.g. 'Support and provide treatment to informal caregiver by social worker or psychologist). Other components (e.g. 'Consultation with patients and relatives to discuss vulnerability and risk factors'), though considered essential, did not apply to some patients. In addition, a component might be deemed 'essential', but its frequency or dose might be open to modification. Therefore, refining and further articulation of the criteria will be necessary to ensure ecological validity of the instrument.
The current PReCaP instrument includes two components which are - at least implicitly – double-barreled, i.e. 'Identification of patient at risk within 48 hours after admission' and 'Assessment of risk factors for functional decline' by specifying both activity and timing in its label. In rating the component, it was broken apart to allow credit for adherence independent of time followed by categorization of the timing of delivery. Although it did not present a problem at this preliminary stage, feasible application, including quantification of assessment results, may require expanding the measure to put adherence and timing in separate items.
In this measurement, we have based the assumption of quality of intervention delivery on the pre- and in-service training. Yet, a study evaluating fidelity of community-based treatments for people with serious mental illness suggested that introductory training, supplemented by the use of written materials and phone-based consultation is not an adequate proxy for the assumption of quality of intervention delivery
As part of the larger evaluation study
Methodological limitations
The design of the intervention fidelity instrument was critically reviewed by the program director/psycho geriatrician, program leader, and casemanagers to ensure the construct validity of the instrument. Although expert panel consensus has been used in various phases of the development and use of fidelity instruments
The results suggest that intervention fidelity assessment is associated with potential under-reporting, a view also expressed by Carroll et al.
Another issue to consider is the potential effect of the fidelity assessment on the intervention, also known as the police car effect
Although registered nurses, nurse practitioners, nursing home physicians, and psychologists have undertaken specialist training prior to the commencement of the PReCaP, the competence of the core interventionists was not specifically measured. Clearly, the provision of training has a moderating effect on the quality of the intervention delivery, which, in turn may affect the intervention fidelity. Yet, this relationship is more complex than may be captured in a simple correlation of training and fidelity numbers through the moderating role of the intervention complexity
Finally, we acknowledge the narrow selection of the patient sample and setting. Therefore, we emphasize that the intervention fidelity results, although indicative for the PReCaP, are not necessarily generalizable to other settings and populations. Yet, the considerations with regard to the moderating factors and modifications affecting intervention fidelity can be viewed in the broader context.
As part of a large evaluation study of a geriatric care intervention (PReCaP), a fidelity study was carried out in the first year of the program delivery to determine the extent to which each intervention component in the hospital phase was delivered as intended. A number of useful messages emerged from this study. Ten of the nineteen components were always or often delivered as set in the PReCaP description. The intervention adherence varied substantially per component due to a range of moderating factors, e.g. the lack of a comprehensiveness protocol resulted in interventionists not being familiar with the PReCaP intervention components. Furthermore, a number of intervention components were modified or added, which leaves the question to what extent the intervention objectives will be achieved by means of the modified intervention. Hence, it would be recommended to determine the essential intervention components and conduct repeated assessments of intervention fidelity over time in order to ensure a relevant and meaningful intervention.
MTM: Multi-disciplinary team meeting; PReCaP: Prevention and Reactivation Care Program; PRC: Prevention and Reactivation Centre; GAS: Goal Attainment Scaling.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
AJBMdV prepared the manuscript and revised it for important intellectual content. TJEMB designed the intervention protocol, participated in the design of the evaluation study and revised the manuscript for important intellectual content. PLdV revised the manuscript for important intellectual content. JDHvW participated in the design of the evaluation study and revised the manuscript for important intellectual content. EWS participated in the design of the evaluation study and revised the manuscript for important intellectual content. JPM participated in the design of the evaluation study and revised the manuscript for important intellectual content. APN participated in the design of the evaluation study and revised the manuscript for important intellectual content. All authors read and approved the final manuscript.
The authors acknowledge the patients and informal caregivers who participate in the PReCaP; the Vlietland Ziekenhuis, the Sint Franciscus Gasthuis, and the Ruwaard van Putten Ziekenhuis for supporting the program; the Argos Zorggroep for initiating and developing the intervention; Judith van den Bosch for contributing expertise to the grant application; the Geriatric Network Rotterdam for their invaluable contribution to the program development.
This work was supported by a grant from the Netherlands Organisation for Health Research and Development (ZonMw) [grant number 60-61900-98-130] as part of the National Care for the Elderly Program, which aims to improve the quality of care for elderly by means of developing integrated care that is adjusted to the individual needs of the elderly.
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Pre-publication history
The pre-publication history for this paper can be accessed here:


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Sugarcane cultivation plays an important role in Brazilian economy, and it is expanding fast, mainly due to the increasing demand for ethanol production. In order to understand the impact of sugarcane cultivation and management, we studied sugarcane under different management regimes (pre-harvest burn and mechanical, unburnt harvest, or green cane), next to a control treatment with native vegetation. The soil bacterial community structure (including an evaluation of the diversity of the ammonia oxidizing (amoA) and denitrifying (nirK) genes), greenhouse gas flow and several soil physicochemical properties were evaluated.
Our results indicate that sugarcane cultivation in this region resulted in changes in several soil properties. Moreover, such changes are reflected in the soil microbiota. No significant influence of soil management on greenhouse gas fluxes was found. However, we did find a relationship between the biological changes and the dynamics of soil nutrients. In particular, the burnt cane and green cane treatments had distinct modifications. There were significant differences in the structure of the total bacterial, the ammonia oxidizing and the denitrifying bacterial communities, being that these groups responded differently to the changes in the soil. A combination of physical and chemical factors was correlated to the changes in the structures of the total bacterial communities of the soil. The changes in the structures of the functional groups follow a different pattern than the physicochemical variables. The latter might indicate a strong influence of interactions among different bacterial groups in the N cycle, emphasizing the importance of biological factors in the structuring of these communities.
Sugarcane land use significantly impacted the structure of total selected soil bacterial communities and ammonia oxidizing and denitrifier gene diversities in a Cerrado field site in Central Brazil. A high impact of land use was observed in soil under the common burnt cane management. The green cane soil also presented different profiles compared to the control soil, but to at a lesser degree.
Sugarcane is an efficient substrate for bioethanol production, wich is currently largely used in Brazil as a substitute for fossil fuels. Traditionally, sugarcane crops are burnt before harvest, in order to remove leaves, thus facilitating easier manual harvest. However, this procedure results in high emissions of particulate matter and smoke, which can be harmful to humans and livestock. Current regulation of bioethanol production is leading to a transition towards mechanical harvest. Several authors have reported the positive effects of unburnt harvest (green cane) on soil fertility, soil structure, soil C levels and biological activity [1-3]. Most of these data have been generated in studies in the Atlantic Forest biome, however none has addressed the microbial community structures and diversities in soils under burnt versus green cane management in Cerrado Biome.
The Cerrado is the second largest terrestrial biome in Brazil and it is characterized by a savannah-like vegetation on ancient and plain soils [4]. Currently, cultivation of sugarcane is increasing in this region, with some states showing a 300% expansion of cropped areas over the last few years [5]. Due to high concentrations of endemic plant species and the accelerated pace of deforestation, the Cerrado region has been classified as a high priority area for biodiversity conservation [6]. Therefore, there is a need to develop studies that address the effects of sugarcane expansion in Cerrado soils.
The use of agricultural land for cropping generally results in modifications of the soil biological and physicochemical properties, which, in turn, affect soil biogeochemical processes such as nutrient cycling and gas emissions, influencing ecosystem productivity and sustainability [7-11]. Brazil is the fifth largest contributor to the global emission of greenhouse gases (GHG). A major part, up to 75%, is the consequence of unsustainable agricultural practices next to deforestation, which include removal of crop residues, exposure of the soil surface to erosion, excessive plowing and the introduction of nitrogen fertilizers in excess [12-14]. In areas under sugarcane, there is no consensus regarding the effects of burning or maintenance of crop residues on the soil microbiota and the emission of GHG, nor on the chemical and physical factors of the soil [15-17].
Some soil properties respond relatively rapidly to land use and soil management changes, which makes these suitable to serve as soil quality indicators [18]. For instance, the light, labile fraction of soil organic matter, dissolved C and N contents, soil microbial biomass and activity, and bacterial diversity, have all been proposed to represent suitable early warning indicators of soil quality degradation or improvement [2,11,19-23]. However, we are far from having a consolidated set of soil quality indicators, which might allow such monitoring across a range of different soils [24,25].
Specific groups, such as ammonia oxidizing and denitrifying bacteria, play basic roles in the N cycling. The study of these groups is very important, mainly in agricultural soil, since nitrification coupled with denitrification are major sources of soil N loss. The use of molecular tools targeting key genes such as amoA and nirK have been widely used to improve the knowledge about this issue. Their ecology can be more readily understood by exploring the abundance and diversity of key marker genes than through cultivation based approaches [26].
The great majority of studies on effects of different cropping systems evaluates just one or a few parameters in soil; thus, stable isotopes are used to better understand C and N dynamics [3], bacterial communities to establish soil quality bioindicators [17] and greenhouse gas fluxes to evaluate impacts on global warming [15]. On top of this, there is a paucity of knowledge with regard to parameters that might serve as quality indicators for Cerrado soil under sugarcane cultivation, that is, what parameters might serve as quality indicators.
Since physical, chemical and biological factors in soil are not independent from each other, it is important to evaluate them together in one system and to attempt to establish the links between them. The main goal of our study was therefore to evaluate the impact of the different management strategies of sugarcane (burnt cane and green cane) on the soil chemical, biological and physical properties (including GHG flow) and to analyze the relationships between these features.
The study area (17° 55' 35" S 50° 08' 36" W) was located in the municipality of Porteirão, state of Goiás, Brazil. The region´s climate is classified as Aw (Köppen), with annual average rainfalls exceeding 1500mmyear-1 and annual average air temperatures of 23.1°C. The soil type is a eutrophic Latossolo vermelho (Ferralsols), which is characterized by high levels of base saturation (>50%). Although the area was very flat, petroplinthite (lateritic nodules or concretions) were found in the subsurface, which may restrict drainage and exhibits a concretionary character.
The field had been previously used for cotton, soy and sunflower production, and was converted to sugarcane cultivation in 2002. The samples were collected in September 2008, during the sugarcane growth stage, approximately 7 to 8months after bud germination (after six yearly harvest cycles). The field was divided into three treatments (split-plot) in which three different regimes were applied:
(i) Burnt sugarcane – Before harvest, the sugarcane crop was burnt to remove the leaves. The stem was then manually harvested. After harvest, the soil remained uncovered.
(ii) Green sugarcane – Harvest was performed using a machine that separates the sugarcane leaves from the stems. The leaves are then returned to the soil. After harvest, the soil remained covered by the vegetal residues.
(iii) Control – covered with trees interspersed with open areas, contiguous to the sugarcane treatments.
The sugarcane treatments had 6years of implementation until the sampling. The fertilization regime of the area was composed by the addition of 400kgha-1 of NPK (5-25-15) during the implementation of the sugarcane crop (6years before the sampling), and an annual addition of 400Kg of NPK (20-0-20), after each harvest (8months before the sampling). Monoammonium phosphate was used as nitrogen source during the first fertilization and urea in all other subsequent ones. To allow replication, per treatment, five 5x5m subplots were defined randomly (approximately 10m of distance from each other). The soil was collected as five replicates per subplot (which were pooled) approximately to 10cm depth, using a core borer (total up to 2.5kg). The sizes of the burnt sugarcane, green sugarcane and control treatments were 23.5, 9.9 and 2.9ha, respectively. The native vegetation was chosen as control because it represents the soil's natural condition; it received no addition of fertilizers. This control was a small fragment of native Cerrado (Cerradão-type, characterized by a dense formation of trees up to 4 meters tall) [4]. The three treatments were very close to one another, less than 300m apart.
Subsamples of soils from each site were air dried, sieved (2mm) and analyzed chemically. Exchangeable nutrients: Ca2+, Mg2+ and Al3+ extracted by 1M KCl; P, Na and K by Mehlich-1 extractant – 0.05molL-1 in HCl in 0,0125molL-1 H2SO4) and pH (soil:water, 1:10); Potential acidity: H+Al extracted with calcium acetate 1N (pH 7), titrated with 0.0125 N NaOH, were analysed according to Embrapa [27]. Inductively coupled plasma apparatus for Ca2+, Mg2+ and Al3+, flame emission (K and Na) and photocolometry (for P) were used for nutrient determinations. All analyses, except bulk soil density and potential denitrification (where samples were pooled), were conducted with all five replicate samples per treatment.
Soil granulometry was determined using the aerometer method, after chemical dispersion [27]. Soil bulk density (2.5-7.5cm) was determined in undisturbed samples, collected with 5cm diameter and 5cm height stainless steel rings, from three samples per treatment. The water-filled pore space percentage (%WFPS) was determined by converting soil gravimetrical water content values (θg gg-1), using the equation:%WFPS=(100θgd)/[1 – (d/pd)], where d is the soil bulk density and pd is the particle density [28].
The isotopic organic C to N ratio was used to infer the C and N turnover in this environment. Since the previous vegetation at the sites were plants with C3 metabolism and sugarcane is a plant with C4 metabolism, we could measure the turnover of organic matter by measuring the differences in the isotopic ratio values. Soil total C and N contents and 13C/12C and 15N/14N isotopic ratio variations were determined by use of an elemental analyzer coupled to a mass spectrometer (Carlo Erba/Delta Plus). Results were expressed in the form of δ 13C (‰) in relation to the international PDB standard and as δ 15N (‰) in relation to the atmospheric N [29].
On the day of sampling, inorganic N was extracted from the soil samples using a KCl (2M) solution (time 0). Moreover, the soil was extracted after a 7-d incubation period [30]. Phenyl mercury acetate (0.1mL) was added to the filtrate to preserve the samples. The ammonium (NH4+) and nitrate (NO3-) contents in the extracts were determined using an automatic flow injection analysis system. Ammonium was quantified colorimetrically using the Solorzano method [31], and nitrate estimated by conductivimetry in the form of nitrite (NO2-), after reduction with a cadmium base catalyst [32]. The net N mineralization rates of the soil samples were calculated by the difference between the concentrations of NH4+-N and NO3--N before and after 7days of incubation. The net nitrification rates were calculated by the differences between final and initial NO3--N contents in the incubated soil samples.
To determine the fluxes of CO2, N2O and CH4, gaseous samples were collected from 10-L static chambers installed in the field. We installed six chambers per treatment, and samplings were done for three consecutive days (at 10p.m.). Thus, in the sugarcane treatments, to cover the different soil conditions in relation to the plant influence on gas flux, two chambers were placed along the cultivation rows, two in between the rows (0.45m from the row) and two in an intermediate region between the rows and the space between the rows (0.225m from the row). The samples were obtained through nylon syringe (50mL; BD) at intervals of predetermined time (1, 10, 20 and 30 minutes). The gas collected was immediately transferred to glass vials (20ml) pre-evacuated and sealed for storage and further analysis. The N2O concentration was determined with an electron capture detector (ECD) detector, using a Haysep Q 3m, 1/8" column and the CO2 and CH4 concentrations were determined with a flame ionization detector (FID) detector using a Porapak Q 2m, 1/8" column. The gas fluxes were calculated by the change in the concentration of the gases inside the chambers over the incubation period (30min).
Denitrification rates were determined as described by Smith and Tiedje [33]. Fifty grams of soil were incubated in hermetically sealed glass (1.8L) bottles, containing a nutrient solution with NO3- (100mgNl-1), glucose (40mgl-1) and chloramphenicol (10mgl-1). The atmosphere in the bottle was replaced by pure N2 and approximately 10% of acetylene was added. Gas samples were removed after 0, 30, 60 and 90min. Tests were conducted in triplicate. The N2O concentrations were quantified with a gas chromatograph (Shimadzu GC17A).
Soil DNA was extracted in triplicate (only three soil samples randomly chosen from the five replicate subplots) by using the FastDNA® Spin Kit for Soil and a FastPrep® equipment (Bio 101, CA, USA), according to the manufacturer's instructions.
To analyze total bacterial community structure and diversity, we used a pair of universal primers for the domain Bacteria, which amplify the gene fragment coding for a fragment of the 16S rRNA subunit (U968-GC and L1401) [34]. Specific primers for the functional genes amoA (AmoA1F-Clamp and AmoA-2R-TC) [35] and nirK (F1aCu and R3CuGC) [26] were used to study the ammonia oxidizing and denitrifying bacteria, respectively. A CG-rich clamp was added to the end of one primer for each system [36].
Amplifications were carried out by PCR in 50 μL reactions containing approximately 10ng of DNA, Taq buffer 10X, MgCl2 (2.5mM), dNTPs (0.2mM), primers (0.2μM), BSA (bovine serum albumin) (0.1gl-1), formamide (1%v/v) and Taq DNA polymerase (Fermentas; 2.5 U).
The bacterial PCR was run as follows: initial DNA denaturation step at 94°C for 4min, followed by 35 cycles of 1min at 94°C, an annealing step of 1min at 55°C, and amplification during 2min at 72°C, with a final extension of 10min at 72°C. The amoA gene-specific PCR was run with an initial denaturation at 94°C for 3min, followed by 35 cycles of 30s at 94°C, 1min at 57°C, 1min at 72°C, with a final extension of 10min at 72°C. The denitrifying gene-specific PCR was run with an initial denaturation at 94°C for 3min, followed by 5 cycles of 30s at 94°C, 1min at 60°C and 1min at 72°C; 30 cycles of 30s at 94°C, 1min at 62°C, and 1min at 72°C; with a final extension of 10min at 72°C.
The amplified fragments were analyzed via DGGE [37] on a Universal Dcode™ Mutation Detection System (Bio-Rad, Richmond, California, USA). We prepared the polyacrylamide gels (6%) using a mixture of 37.5:1 acrylamide/bisacrylamide (w:w) in a TAE 1X buffer (10mM Tris-acetate, 0.5mM EDTA pH 8.0), with denaturing gradients of: 45 to 65%, 45 to 65%, and 55 to 70%, for bacterial, ammonia oxidizing and denitrifying gene amplicons, respectively.
Approximately 30 μL of the PCR products (approximately 20–30μg of DNA) were applied to each slot, and electrophoresis was performed at 75V at 60°C for 16h. Gels were stained with SybrGreen® (Molecular Probes, Oregon, USA) and observed on a Storm® scanner (GE Healthcare).
All data were tested for normality and homoscedasticity. When these conditions were met, analysis of variance (ANOVA) followed by Tukey tests for the significance of the differences were used. Otherwise, the non-parametric Kruskal-Wallis ANOVA & Median, followed by two-sided Kolmogorov-Smirnov tests were applied. All analyses were performed using the program STATISTICA 7 (StatSoft).
To analyze the difference between microbial community structures, N transformation gene diversities, and their interactions with abiotic factors, we used non-metric scaling (NMS) with the aid of the PC-ORD statistical package V5 (MjM Software, Gleneden Beach, OR).
Matrices containing all physicochemical properties and bacterial community and functional gene data were assembled to carry out the ordinations. The DGGE band profiles were digitalized and inserted into the data matrices by use of the Bionumerics v6.0 package (Applied Maths), according to the manufacturer's instructions.
The matrices were ordered by NMS [38,39], employing a Bray-Curtis distance matrix. NMS was performed using a random initial configuration, and the data matrices were analyzed using 250 runs with real data and compared with the Monte Carlo test with 250 runs of random data. The final result of the NMS analyses was restricted to two dimensions to simplify data analyses and discussions (stability criterion=0.00001; interactions to evaluate stability=15; maximum number of interactions=250). The stability of the standards of ordination in reduced size was developed by plotting the values of stress by numbers of interactions. Despite the fact that all variables are present in the ordination analysis, only those that were significantly correlated with the microbial ordination are presented.
To confirm the existence of the groupings generated by NMS analysis we performed a Multi-Response Permutation Procedure (MRPP) that tests the hypothesis that no difference exists between two or more groups of entities [40].
To evaluate the association between the generated matrix and the data from the physicochemical properties and the matrices from the DGGE profiles, we used a Mantel test [41], which evaluates the hypothesis that a relationship between two matrix distances does not exist. All Mantel tests were employed using the asymptotic approximation of Mantel and the Sørensen distance [42].
The three field sites studied were homogeneous and belonged to the same soil class. Briefly, all three sites were very similar in their mineralogical composition, constituted mainly by kaolinite, gibbsite, hematite and goethite (data not shown). The clay content was variable across the samples of all three fields, between 300 (minimum) and 480g Kg-1 (maximally). Other soil properties differed between the fields where the different treatments were applied (Table (Table1).1). Thus, the burnt soils showed slight acidification and decrease of exchangeable Ca, exchangeable Mg, total P and SB (sums of bases) values and CEC (cation exchange capacity) levels.
Moreover, significant differences between treatments regarding soil bulk density and water filled pore space (WFPS) were noted. Both green and burnt cane soils had significantly higher bulk densities as compared to the control, i.e. 1.25 and 1.31, respectively, versus 0.96. We did not observe any major differences in soil moisture content, although the control showed a significantly decreased WFPS value (Table (Table11).
The increase of soil bulk density under sugarcane cultivation is commonly observed when soil passes from its natural to a cultivated condition [3]. It occurs due to the breaking up of aggregates caused by soil tilling, the use of agricultural machines and the loss of organic matter [43].
The data showed lower values for total C and total N in the green cane (p<0.05) versus the burnt treatment. In addition, the C:N ratio was significantly higher in the green cane soil (Table (Table1)1) than in other treatments. Moreover, raised values of δ13C and δ15N were observed in green cane, in comparison with the other treatments. Collectively, these data suggested that, in the green cane soil, a larger contribution to soil organic matter was provided by sugarcane (C4 photosynthetic cycle plant), next to a more intense and open N cycling.
The lower C and N contents in the green cane soil were unexpected, and appear to contradict previous reports [3]. However, other studies on different cultivation and agricultural management systems [44], including sugarcane [45], reported that the recovery of the soil C stock depends on the time elapsed after changes in the agricultural practices were made. The relatively short time given in the current study to the green cane management was likely insufficient to positively affect the C content in the soil. Possibly, during the transition to this system, more labile organic matter was incorporated than that incorporated in the form of burnt compounds, resulting in higher soil respiration rates, which may have reduced C contents in this treatment. Moreover, the maintenance of crop residues may have created better conditions for microbial activity, resulting in an increased cycling of soil organic matter. This hypothesis is supported by the higher values of δ13C and δ15N found in the respective soil (Table (Table1).1). The soil δ13C detected in all treatments was between −20‰ and −23‰, suggesting that the soil OM is a combination of the OM from previous cultivation (C3 plants) and also from the current sugarcane cultivation (C4 plants). However, the more enriched signal found in green cane indicates that the detected C derives primarily from the C4 route. Moreover, the higher δ15N also indicates a more intense N cycling.
The C contents of the soil under the two regimes were on the order of those found in other sugarcane plantings [3]. However, studies in the same soil under natural vegetation or agricultural use previously reported higher organic C contents [46,47]. Further studies should attempt to assess the extent to which land use affects soil C stocks.
Ammonium was the predominant form of mineral N in the control soil, whereas the two soils under sugarcane showed a predominance of nitrate (Table (Table2).2). Such changes of the predominant soil N form promoted by land use change have been reported earlier [10]. With respect to the N cycle, the net rates of N mineralization and nitrification were significantly lower in the two soils under sugarcane cultivation, when compared with the control (Table (Table2).2). Such effects of the use of soil have been observed before [10,48,49]. However, the changes in sugarcane harvest management did not result in an alteration of the patterns of N transformations, agreeing with previous published results [50].
Variations in the NH4+-N:NO3--N ratio values may result from distinct processes [51]. In our study, the main factor that interfered with the ratio values was the denitrification rate. As the highest rate of nitrification, found in the control soil, was associated with higher ammonium content, this is not the most plausible mechanism. Additionally, the potential soil denitrification rates were higher in the control soil, as compared to the two planted treatments (Table (Table2).2). The suppression of the soil potential denitrificaton rate can provide higher N-NO2 content, and could be explained by a shift in soil microbiology. Denitrification enzyme activity (DEA) value distributions correlated significantly (p<0.01) with changes in the soil bacterial community and ammonia oxidizing and denitrifiers gene structures. It corroborates work of other authors that stressed the link between shifts on specific bacterial communities with changes in the denitrification process [52,53].
We analyzed the in situ fluxes of several selected gases to understand the effect of land use on greenhouse gas production. The data showed that the N2O and CO2 fluxes had similar behavior (Figure (Figure1),1), and differences were not observed between the different treatments. However, the flux of methane suffered an inversion in its direction in both sugarcane soils (Figure (Figure11).
Probably, the lower density and WFPS measured on Cerrado plays an important role in the flux dynamics for CH4 and N2O gas, because it means that the Cerrado Soil (letra maiuscula) offers a more aerobic environment, inhibiting both methane production and denitrification enzyme activity. However, the fluxes of N2O and methane were low in the period of measurement, and therefore might be negligible as contributors to greenhouse gas emission, even considering their higher effect on global warming.
Regarding the spatial variation of the fluxes within the sugarcane cultivated soils, higher emissions were detected in the chambers that had been placed on the planted rows when compared with the region between the rows (data not shown), showing the influence of the rhizospheric soil and the root respiration. It is important here to point out that these conclusions were obtained from a single sampling of three days. To confirm the observations, a more comprehensive study including different sampling times, possibly over different seasons, is needed.
The bacterial DGGE profiles of all treatments were complex, with high numbers of bands and no clear dominance. The profiles of the three samples of each treatment revealed great similarity. The analyses of the structure of the bacterial communities (Figure (Figure2)2) showed that these were significantly impacted by both the use (cultivation of sugarcane) and the management (burnt versus green cane) of the soil, according to pairwise comparisons (MRPP analysis; p<0.03). The ordering generated by the NMS grouped the replicates of each treatment in a distinct region, and the three treatments (centroids) practically equidistant from each other. The sensitivity of soil bacterial communities to changes in land use and management has already been shown by different authors in various settings [11,54-56], including DGGE analyses carried out in Brazilian Cerrado soils [20].
Several factors correlated with the NMS ordination. In particular, the total P and exchangeable Mg contents and soil density were associated with the bacterial community structures in the control soil, while the (reduced) C and N contents were correlated with the bacterial communities in the green cane treatment. Finally, the (decreased) value of the sum of bases (SB), the degree of saturation of the bases (V), the cation exchange capacity (CEC) and exchangeable calcium (Ca) were correlated with the communities from the burnt cane treatment (Figure (Figure2).2). The soil properties that correlated with the segregation of the bacterial community structures were consistent with observations from Atlantic forest soils under different agricultural production systems [11,17,20].
The amoA gene based DGGE (ammonia oxidizing bacteria) showed relatively simple profiles in all treatments (4–10 bands), with relatively similar patterns between the triplicates. The control soil revealed a higher number of bands in comparison to the green and burnt cane soils. The analysis of these communities indicated a diffuse distribution, with some within-treatment variability (Figure (Figure3).3). However, as reflected in the X axis, these communities responded significantly to the change in land use management (MRPP<0.05), being the burn treatment a factor that exacerbated the response.
None of the parameters tested correlated with the grouping of the amoA communities in the green cane soil, with the exception of the C:N ratio in one replicate. The clear distinction between the bacterial communities in the control soil and in the burnt cane soil was correlated with the high exchangeable Mg content and the low WFPS value in the former. Moreover, it was associated with low values of the sum of bases, cation exchange capacity, exchangeable Ca and the degree of saturation of the bases in the burnt cane soil (Figure (Figure33).
The nirK gene based DGGE profile (denitrifying bacteria) showed more complex patterns (8–15 bands) than that of the ammonia oxidizing bacteria. The triplicate profiles were similar between each other. Much like the total bacteria, the nirK based patterns (Figure (Figure4)4) showed significant differences between treatments (MRPP<0.03). However, there was great variation in community structure. There was a distinction between green cane and control samples along the Y axis and a marked distinction between the burnt cane and the other samples along the X axis, that contained the major percentage of variance (74%).
None of the soil parameters tested showed significant correlation with the alterations in the structure of the denitrifying community in the green cane soil. In the burnt cane soil, the factors involved in the process were the same as described above. The communities in the control soil were also strongly influenced by the high exchangeable Mg value and the low WFPS (Figure (Figure44).
Ordination of the physicochemical data as primary matrices classified the treatments as three distinct groups (data not shown), which is the same basic grouping found with the bacterial community. In contrast, the two functional communities did not follow the same pattern as the bacterial communities, perhaps because these groups were subjected to more specific selective forces, such as caused by different levels of NH4+-N and/or NO3--N. The Mantel correlation data (not shown), that test the correlation and the significance between two matrices, provided evidence for the latter hypothesis, because the largest correlation value found was that of the ammonia oxidizing community with the denitrifier community (r=0.70), while the correlation of these groups with soil properties was respectively at r=0.45 and r=0.63. In spite of the fact that all correlations were significant (p<0.01), the high value found between the two groups of N cycle bacteria emphasized the interdependence of the two different bacterial groups involved in the N cycle with soil N chemistry. It may hint at the importance of biological factors in the structure of these communities. A change in density, reflected in the respective community, may directly affect the others. Du et al. [57] also demonstrated (in vitro) a strong correlation between ammonia oxidizing and denitrifier bacteria, and this relationship can apparently also be detected in agricultural soil.
Sugarcane land use significantly impacted the structure of soil bacterial communities and ammonia oxidizing and denitrifier gene diversity in a Cerrado field site in Central Brazil, with significantly correlations (p≤0.01) with several soil properties. Different factors, but especially the DGGE and the DEA activities were very sensitive to the management practices. A high impact of land use was observed in soil under the common burnt cane management, where the shifts were correlated with soil bulk density and water-filled pore spaces. The green cane soil had also changed from the control soil, but to at a lesser degree. Both treatments showed positive correlations between the make-up of the respective communities and soil fertility indicators (sum of bases, CEC and degree of base saturation), with the green cane treatment showing a negative correlation with C and N contents in the bacterial community structure, possibly due to increased biological activity and C oxidation.
Given the fact that soil nitrification is known to be a phylogenetically restricted process, it is important to assess the effects of land use on its diversity. We here found that the use of Cerrado soil for sugarcane cropping results in a community structure shift as compared to a control treatment. Importantly, the burn treatment resulted in the largest change in this microbial structure for both ammonia oxidizing and denitrifying gene diversity, as could be noted by the reduction of band numbers in the DGGE profiles and higher community differentiation on NMS analysis. We believe that answers obtained by the evaluation of bacterial community structure can be as important as the number of microorganisms, and that is important to quantify the size of these communities in this environment. Therefore, a complex study to answer this question is being carried on.
It is clear that we have provided just a snapshot of potential changes in soil resulting from the changed management (burnt to green cane). Thus, further research is required in which soil samples from different sites of the Cerrado are used, possibly comprising different seasons, in order to address the changes due to changes in management over the years.
The authors declare that they have no competing interests.
CTCCR: Conception and design, field work, laboratory analysis, data interpretation and analysis, manuscript writing; MCP: Conception and design, field work, coordination, data interpretation and manuscript review; DCAL: Laboratory analysis; FCB: Conception and design, field work, data interpretation and manuscript writing; HLCC: Data interpretation and manuscript review; JDVE: Data interpretation and manuscript writing; RSP: Data interpretation and manuscript writing; ASR: Data interpretation, coordination and manuscript review. All authors read an approved the final draft.
This work received funding from the Inter-American Institute for Global Change Research (IAI), through the CRN II Program (Project 2031), which is supported by the US National Science Foundation (Grant GEO-0452325), the IAI Project 104358, supported by the International Development Research Center (IDRC). This work was also funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). The authors would like to thank Dr. Guillermo Montandon Chaer (Embrapa Agrobiologia) for his knowledge on multivariate analyses and Dr. Vinícius de Melo Benites (Embrapa Solos) for his support with the logistics and the fieldwork.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Iran (Persian: ایران - Irān), also known as Persia, officially the Islamic Republic of Iran (جمهوری اسلامی ایران – Jomhuri ye Eslāmi ye Irān), is a sovereign state in Western Asia. It is bordered to the northwest by Armenia and Azerbaijan; to the north by Kazakhstan and Russia across the Caspian Sea; to the northeast by Turkmenistan; to the east by Afghanistan and Pakistan; to the south by the Persian Gulf and the Gulf of Oman; and to the west by Kurdistan and Arabia. It is the only country with both a Caspian Sea and an Indian Ocean coastline. The country's central location in Eurasia and Western Asia, and its proximity to the Strait of Hormuz, make it of great geostrategic importance. Tehran is the country's capital and largest city, as well as its leading cultural and economic center.
Iran is heir to one of the world's oldest civilizations, beginning with the formation of the Proto-Elamite and Elamite kingdoms in 3200–2800 BC. The area was first unified by the Iranian Medes 625 BC, who became the dominant cultural and political power in the region. Iran reached its highest during the Achaemenid Empire founded by Cyrus the Great in 550 BC, which at its greatest extent stretched from parts of Eastern Europe in the west, to the Indus Valley in the east, making it the largest empire the world had yet seen. The empire collapsed in 330 BC following the conquests of Alexander the Great, but reemerged shortly after as the Parthian Empire. Under the Sassanid Dynasty, Iran again became one of the leading powers in the world for the next four centuries.
Beginning in 633 AD, Rashidun Arabs conquered Iran and largely displaced the indigenous faiths of Manichaeism and Zoroastrianism by Sunni Islam. Iran became a major contributor to the Islamic Golden Age that followed, producing many influential scientists, scholars, artists, and thinkers. The rise of the Safavid Dynasty in 1501 led to the establishment of Twelver Shia Islam as the official religion of Iran, marking one of the most important turning points in Iranian and Muslim history. During the 18th century, Iran reached its greatest territorial extent since the Sassanid Empire, and under Nader Shah briefly possessed what was arguably the most powerful empire at the time. Through the late 18th and 19th centuries, a series of conflicts with Russia led to significant territorial losses and the erosion of sovereignty. Popular unrest culminated in the Persian Constitutional Revolution of 1906, which established a constitutional monarchy and the country's first legislative body, the Majles. Following a coup d'état instigated by the United Kingdom and the United States in 1953, Iran gradually became closely aligned to with the United States and the rest of the West but grew increasingly autocratic. Growing dissent against foreign influence and political repression led to the 1979 Revolution and the establishment of an Islamic republic.
Iran played a major role as they joined Anti-Berber Coalition in 1980, by declaring Morocco as one of worst enemies among with United States and starting Great War Against Berbers via Atomic Bombing of Anfa in 1986.


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