Background
==========

The distribution range of elephant populations in southern Africa where elephants are not restricted by humans largely overlaps with the distribution of mopane (*Colophospermum mopane*) woodlands. Mopane is a principal food item in the diet of elephants, not only in northern Botswana \[[@B1]\], but also elsewhere in southern Africa \[[@B4],[@B20],[@B28],[@B30]\]. High utilization rates of plants by prolific elephant population may result with the alteration of vegetation structure and even the decline of species diversity \[[@B9]\].

Some mopane woodland areas in northern Botswana appear to sustain higher elephant utilization rates than neighbouring mopane areas. The availability of water from surface seasonal water sources can only partially explain the distribution of elephant browsing \[[@B1],[@B31]\]. Two findings relating to factors that affect the feeding habits of other large herbivores suggest possible additional explanations for the patchy browsing by elephants on mopane. First, large herbivorous species are influenced by elevated nutrient concentrations in the forage \[[@B7],[@B24]\]. And second, the feeding patterns and the differential distribution range of large herbivores can be explained by localized differences in soil factors and nutrient levels in the forage \[[@B5],[@B22]\].

The elephant population in northern Botswana has been growing steadily over the past two decades \[[@B8],[@B21]\]. Already, some reports cautioned against the spread of excessive elephant damage to woodlands \[[@B15],[@B23],[@B27]\]. This risk gives practical urgency to identifying the factors that determine the pattern in which food -- in this case mopane -- is used by elephants. In particular, we seek to test here whether high mopane utilization by elephants is predicted by the higher nutrient qualities of either mopane itself or of the soil on which it grows. In tackling this question we assume that if elephants showed selective feeding in northern Botswana, then elephants would also display feeding preferences within a principal and abundant food source.

Results
=======

Soils, mopane woodlands and elephant utilization
------------------------------------------------

Analysis of variance for the independent parameters measured in each plot revealed high variations of variables related to soil properties and vegetation structure, but low variation in leaf protein contents (Table [1](#T1){ref-type="table"}).

::: {#T1 .table-wrap}
::: {.caption}
######

Mean values and standard deviations for variables describing soil properties, plant densities, and extent of elephant utilization in Mopane woodlands of northern Botswana.
:::

                                             Mean     Std
  ------------------------------------------ -------- -------
  pH                                         6.6481   0.23
  Nitrogen (kg/acre)                         27.18    19.26
  Phosphorus (kg/acre)                       40.27    23.15
  Potassium (kg/acre)                        133.51   36.28
  Silt (%)                                   8.82     4.52
  Clay (%)                                   3.67     0.91
  Shrub (No/100 m^2^)                        12.26    9.98
  Tree (No/100 m^2^)                         0.60     0.44
  New elephant damage
  (No of utilized mopane shrubs /100 m^2^)   1.40     0.93
  Old elephant damage
  (No of utilized mopane shrubs /100 m^2^)   14.71    6.83
  Protein in leaves (%)                      11.45    0.93
:::

Figure [2](#F2){ref-type="fig"} describes the influence of the geographical position of plots on the levels of protein in mopane leaves. Although the range of leaf protein was narrow across the plots (10.2--13.5%), it is possible to distinguish apart between 3 different clusters of points representing plots that correspond to separate sites selected for the analysis. Accordingly, Savuti had the lowest protein contents in leaves. South Gate dominated the medium range and plots of the Khwai River site had the highest protein contents in mopane leaves. Multiple comparisons indicated significant differences between the sites (T = 2.96, α = 0.05), except for Savuti and South Gate.

![Protein levels in mopane leaves in relation to the distance between sampling plots from one threshold plot at 0 distance at the Khwai area.](1472-6785-2-3-2){#F2}

Further multiple comparisons indicated that the densities of shrubs did not differ significantly between the three sites. Similarly, the extent of new elephant damage to mopane plants did not differ significantly between the sites. Tree densities and the extent of old elephant damage however, differed significantly only between Savuti and South Gate. The match between the two observations can be explained in that mature trees showed signs of past elephant utilization. Levels of phosphorus in the soils did not differ significantly between the sites. Potassium levels differed significantly between the Savuti and Khwai sites. Soil nitrogen levels differed significantly between the three sites except for Savuti and South Gate (T = 2.96, α = 0.05).

The matrix of variables incorporated for the multivariate analyses included 10 rows (each representing the 10 individual plots) and 8 columns (each representing one environmental parameter). The environmental parameters were tree densities, shrub densities, nitrogen (**N**), potassium (**K**) and phosphorus (**P**) contents of soils, protein contents of mopane leaves (**Pr**), densities of mopane shrubs and trees in plots, and new (**NE**) and old elephant damage (**OE**) to mopane plants. Figure [3](#F3){ref-type="fig"} provides the summary analysis of these parameters. The ordination of the first principal axes described 90.2% of the variation in the analysis. However, the test of Monte Carlo permutations indicated that the associations between variables representing plots and environmental parameters were not significant (P = 0.11). The geographical classification of sites related to clusters of neighbouring plots was retained to some extent in the analysis. Plots in the Savuti area however, showed a wide dispersal range along the principal axes due to high variability among the parameters examined. Although protein levels in mopane leaves differed between plots the differences did not add much to the overall variation in the analysis. The proximity of the point representing protein levels to the origin of the two principal axes indicates that there were poor associations between elevated protein levels in mopane leaves and high elephant damage to mopane plants or any other variable included in the analysis.

![The first two axes of cannonical correlations representing mopane plots and environmental parameters. Plots are marked with +, and general locations are indicated. Environmental parameters:**Shrub:** density of shrubs.**Tree:** density of trees.**OE:** old elephant damage. NE: new elephant damage.**Pr:** protein contents in leaves.**K:** potassium contents in soils. N: nitrogen contents in soils.**P:** phosphorus contents in soils.](1472-6785-2-3-3){#F3}

A breakdown of associations between variables to linear correlations between pairs of variables revealed only two significant correlations among the environmental variables measured. The associations described elevated soil nitrogen and potassium levels with increasing protein contents in mopane leaves (R^2^ = 0.68 and 0.64, P = 0.002 and 0.04 respectively).

Discussion
==========

A simple assumption would be that herbivores maximize their energy intake by selecting the most nutritious food items available within their home range. In reality however, this assumption is confounded by many ecological factors that reduce the energy return of potentially nutritious feed items. We already know that mopane woodlands are principal food items for elephants in northern Botswana and that the proximity of water sources also influences feeding patterns \[[@B1]\]. These two factors were imbedded a priori in the sampling design in an attempt to assess the magnitude of other factors that may influence the choice of food sources by elephants.

The physiognomic diversity of mopane woodlands that vary from dense stands of shrubs to scattered tall canopied trees was not explained by either of soil factors examined. Nonetheless, soil factors seem to affect leaf chemical composition. High protein levels in mopane leaves correlated with elevated levels of nitrogen and phosphorus in the soils. Although nutrient intake by plants is generally independent of concentrations in the supporting substrate, this pattern suggests that mopane plants in some areas accumulate more nutrients than mopane plants elsewhere in northern Botswana. These differences were evident when comparing between mopane plots that were situated more than 30 kilometers apart. The question is whether the differential nutrient levels in mopane leaves influence the feeding patterns of elephants?

Local soil properties, shrub and tree densities and protein levels in mopane leaves appear not influence elephants\' choice of feeding sites dominated by mopane. Thus, the seemly patchy distribution of elephant utilization of mopane shrubs in northern Botswana was not corroborated by the results. It seems that the differences in protein levels are too subtle as far as the elephants are concerned, or do not offset other factors, such as distance to permanent water sources and the proximity of other food sources. Furthermore, other chemical properties in leaves, such as carbohydrate content and secondary chemicals that inhibit digestion may influence elephant feeding habits than initially perceived.

Management implications
-----------------------

The importance of mopane plants as a food source for elephants cannot be underestimated in northern Botswana where woodlands grow on much of the dry land area. The resilience of mopane both in terms of survival rates of individual plants and recovery of biomass was demonstrated before \[[@B2],[@B3]\]. Yet, some mopane woodland areas in the region are utilized intensively by elephants and damage to these adds credence to support the notion that a prolific elephant population in some parts of the region may be so numerous as to become unsustainable by local woodland habitats.

The results presented here indicated that elephants appear not to differentiate between areas with respect to the protein contents of mopane leaves. This to some extent diminishes the likelihood that elephants will concentrate feeding in a particular (high protein) area until they exhaust the available resources and destroy the vegetation in the process. Therefore, in contrast to early concerns, areas that are currently heavily utilized are unlikely to be further decimated by elephants. Insofar as this finding indicates a certain component of stability to the northern Botswana ecosystem \[[@B4]\].

Methods
=======

The study area
--------------

In this study, northern Botswana is defined as an area of more than 80,000 km^2^ between 18° and 21° south and 21° and 26° east, but excluding the permanent swamps of the Okavango Delta. Rainfall ranges from 400 mm in the south and 650 mm in the north-east. Rains occur during the summer from October to April and mainly in heavy storms. Temperatures vary between monthly mean maximum of 34° (October) and a mean minimum of 6°-7° (June) (Bhalotra 1987) \[[@B6]\].

The topography of the area is very flat and the altitude is around 950 m asl. The north easterly elongated Goha and Shinamba Hills, which rise to 150 m above the surrounding plain, form the only significant topographical feature. The area is covered with deep deposits of aeolian and alluvial sands and silts of the Kalahari beds. These soils are generally neutral or slightly acid, poor in nutrients, poorly structured and with low water holding capacity. More fertile vertisols and Molapo soils are mainly found on flood plains and in some depressions. The bedrock consists mainly of Karoo sediments, material of the Ganzi, Kwebe and Damara formations and granitoid gneisses of the old basement complex. Bedrock is exposed in the eastern and south-eastern parts of Botswana where the sand cover is thin. In the northern, central and western parts, solid rock is obscured by loosely consolidated deposits, known collectively as the Kalahari Beds, which attain thickness of 300 m or more \[[@B16]\]. Most of the area is covered by tree and shrub savanna, towards the north, changing into woodland and to dry deciduous forest with *Baikiaea plurijuga* dominating in the Chobe area while the south is dominated by shrub savanna, rich in *Acacia* species. Low lying areas and the edge of flood plains are often dominated by mopane woodlands.

Soil and vegetation plots
-------------------------

The relationship between soil factors, mopane plants and elephant utilization of plants was examined in 10 plots located in 3 sites within Chobe National Park and Moremi Game Reserve in northern Botswana, namely South Gate, the Khwai River and Savuti. To include a measure of the spatial relationships between plots, we chose one plot at the Khwai site to represent a benchmark point and measured the distances of the rest of the plots from that plot.

The distance between plots situated within a site varied from 1 to 7 kilometers. Plots were allocated to cover as much as possible the existing regional range in soil properties, judged by the appearance of soil colour and texture. To override the influence of surface water as a source for the attraction of elephants, all plots were not situated further than 10 kilometers away from a permanent water source. Additional criteria for the allocation of plots included varying densities and physiognomic forms of mopane plants and evidence of elephant damage to plants. The position of the 3 sites followed a regional stratification to vegetation communities \[[@B10]\] and also the annual estimates of elephant densities \[[@B21],[@B10]\].

In each plot, squares of 10 × 10 m were allocated within two elongated rectangular areas that were parallel to the direction and on each side of a nearby track. The size of each plot varied with respect to the density of the mopane plants within. In all, the size of plots did not exceed 500 × 300 m. The number of squares per plot was determined after conducting an analysis of variance for plots where at least 150 squares were sampled. The allocation of squares within a plot was not contiguous to assure the independent status of squares \[[@B18]\]. Sampling took place during the middle of the dry season (August) when herbivores are expected to be more selective and also to offset variations in the concentrations of minerals in mopane leaves because of seasonal fluctuations \[[@B28]\].

In each square, the number of woody plants of the respective dominating species belonging to specific height classes was counted. Height classes ascribed for shrubs (\>1 and \<3 m) and trees (\>10 m) were represented in terms of the mean number of plants/100^2^ m (Table [1](#T1){ref-type="table"}). The category of shrubs included fallen trees, the individual branches of which were considered as shrubs if the size criteria were met. Multi-stemmed plants with stem diameters of less than 6 cm were treated as a single shrub unless a gap wider than the width of 10 cm could be distinguished between the stems. Trees were defined as plants with a stem diameter of 6 cm or more and higher than 3 m. Woody plants that corresponded to the definition of a tree and had several stems that were jointed close to the ground were considered as one tree. In all, 9,858 mopane plants were sampled.

Elephant utilization
--------------------

The frequency of mopane plants utilized by elephants in the assigned plots was recorded in addition to the estimation of vegetation structure. Damage to woody plants as a result of elephant activity was estimated for individual plants within each square as being as either new or old elephant damage, or both. New damage is that which had occurred since the recent rainy season. Damage becomes characteristically greyish in colour after rain soaks into the exposed inner plant parts. Damage in this context is not only excessive vegetation destruction, but is all easily noticeable utilization of woody material. Only damage that was positively attributed to elephants was recorded. Hence, damage was estimated in terms of the number of mopane shrubs utilized per square of 10 × 10 m \[[@B1]\].

Soil properties and leaf protein
--------------------------------

Thirty soil samples were taken from the woodland plots in locations that were thought to represent the typical features of each plot, such as within tall tree stands, dense shrub formations, near plants that sustained high or low elephant utilization. Ten soil samples were taken within an area of 300 × 150 m to investigate the local variation in soil properties in one mopane woodland site. All samples were taken from 5 to 10 cm below the surface that represented the horizon at which plant root development is substantial \[[@B12],[@B20]\].

Each soil sample was subjected to five tests that included soil texture composition, pH and contents of nitrogen, phosphorus and potassium using a sampling kit \[[@B19]\]. The proportions of sand, silt and clay were derived by particle composition analysis using soil separation tubes and texture dispersing reagents \[[@B13]\]. Soil pH was determined through saturating the samples in a Tricon flocculating solution and indicator. Soil extracts were utilized for the determination of nitrogen, phosphorus and potassium contents \[[@B17]\]. Reagents for nitrogen included sodium bisulphate monohydrate sulfanilamide and ammonium sulphate. Stannous chloride, sodium molybdate and potassium chloride solutions indicated phosphorus levels. Sodium nitrite and sodium cobaltinitrite were the principal reagents for the extraction of potassium.

Mopane leaves were sampled in each of the selected 10 plots. Crude protein was extracted from leaves of 40 to 50 mopane shrubs in each plot. To eliminate the seasonal effects on protein levels, the leaves were cut within the span of a few days during the dry season. The leaves were cut from shrubs between 1--3 meter height. The reasoning behind the selection of this particular height range is because elephants prefer to feed upon mopane shrubs and less from trees and seedlings \[[@B1]\]. Total nitrogen was determined after a wet digestion with sulphuric acid, by the Kjeldahl method \[[@B17]\]. The method does not separate between the organic and inorganic quantities of nitrogen (mostly in the form of nitrate) in leaves. However, it is unlikely that under conditions of low soil nitrogen and ample light in northern Botswana that plants accumulate large quantities of inorganic nitrogen and that these quantities vary significantly between the different areas.

Data analysis
-------------

Records of soil properties, leaf protein, plant densities and number of plants utilized by elephants were subjected to ANOVA, primarily to examine the extent of variation within a sampling plot \[[@B26]\]. A multivariate analysis provided a comparative measure for the variance between records of individual plots. The analysis produced an overview of the associations between plots and variables related to soil properties, vegetation structure, and elephant utilization. Canonical correspondence analysis \[[@B29]\], an eigenvector ordination technique for displaying variables in a two-way contingency table was used to generate ordination axes. These axes are linear combinations of a matrix in which plots were represented by columns of categories for soil properties, densities of shrubs and trees, and elephant damage. The matrix of variables consisted of eight rows representing each sampling plot. A Monte Carlo permutation test examined the significance of the first canonical ordination axis. Essentially, the test identified whether the associations between plots and environmental parameters were significant. The results of multivariate analyses were complemented by multiple comparisons and linear regression analyses \[[@B25]\]. The former compared between the mean values derived for plots and sites (T critical = 2.96, α = 0.05).

Several multivariate analyses were performed on the same data set to examine the stability of associations between variables before deriving the final results. The analyses included Detrended Correspondence Analysis, Principal Component Analysis and Canonical Correlations \[[@B29]\]. Canonical Corrections with symmetric scaling of variables provided the optimal presentation of variables although the arrangement of variables along the principal axes remained consistent with the other analyses performed \[[@B29]\].

Authors\' contributions
=======================

RBS conducted a 10 years study on the ecology of elephants in northern Botswana and drafted the manuscript. DWM conducted many studies on the behaviour and ecology of carnivores and large herbivores.

![Location of three sites in northern Botswana where variables related to soil factors, mopane plants and elephant utilization rates were recorded.](1472-6785-2-3-1){#F1}

Acknowledgments
===============

RBS thanks the Office of the President and the Department of Wildlife and National Parks for a permission to do this research. RBS thanks P. Gadilmang and M. Bathami for their enthusiastic assistance in the collection of data. We thank Dr. Steven Hill for constructive advice. This project was funded by the National Geographic Society.


Background
==========

Delivering care to people with diabetes
---------------------------------------

There is broad, international agreement over what constitutes high quality health care for people with diabetes \[[@B1],[@B2]\]. This will be enshrined in a National Service Framework for people with diabetes, due in summer 2002. However, in the face of poor current performance \[[@B3],[@B4]\] the most efficient method of delivering care remains unclear \[[@B4]\]. Following a 1994 systematic literature review \[[@B5]\] suggesting structured care improved patient care, an editorial in the British Medical Journal concluded that more evaluative research was needed before widespread adoption of any of the models could be recommended \[[@B6]\]. A subsequent systematic review of routine surveillance of patients with diabetes by Griffin and Kinmonth \[[@B7]\] concluded *\"Computerised central recall, with prompting for patients and their family doctors, can achieve standards of care as good or better than hospital outpatient care, at least in the short term. The evidence supports provision of regular prompted recall and review of people with diabetes by willing general practitioners and demonstrates that this can be achieved, if suitable organisation is in place\'.* However, the evidence base on which these conclusions are based is limited in several ways. Firstly there are only five randomised controlled trials (RCTs) involving 1058 patients. All of these studies are \'patient randomised\" trials, thus potentially under-estimating the effectiveness of the intervention (see Study Design). They were all evaluating more or less selected patients and general practices and none of them were explicitly evaluating a UK National Health Service (NHS) service area wide intervention. Only one of the four UK based studies evaluated patient based outcomes and included an economic assessment and this study only involved patients from three general practices \[[@B8]\]. Thus, the effectiveness of an area wide, patient focussed, structured recall and management system (in terms of process of care, patient outcome and economic impact) remains unknown.

The current system
------------------

The current computerised diabetes management system runs in Hartlepool, Easington and Stockton, three Primary Care Group (PCG) areas, in the Northern and Yorkshire Region. It was introduced to all 36 general practices in Hartlepool and Easington Districts in mid-1995. Stockton (25 practices) agreed to join the system in 1999 and it was operational there by October 2000. There are three key components to the current system:

1\. A central register of patients with diabetes.

2\. A structured minimum dataset to be completed and returned to the central register.

3\. The provision of both patient specific and aggregated data to both patients and clinicians.

The system (developed by Westman Medical Software) allows three methods of collection of data at each contact with a patient with diabetes who is registered on the database. Two methods use a standard form completed by clinicians to collect data concordant with the UK minimum data set \[[@B9]\]. Within secondary care, forms are completed at every new patient or annual review. In primary care, forms are completed by the practice nurse (usually) or general practitioner, either opportunistically or at practice diabetic clinics. In both cases, the completed data forms are sent to the Diabetes Register Facilitator for data entry. Thirdly, the hospital laboratory provides a monthly download of laboratory test details (e.g. HbA1c). A patient can be identified as having diabetes and added to the register by any permutation of one or more of these three routes. Feedback of individual patients\' data, including review status, is provided to general practices quarterly. This feedback is \'passive\' in that it does not explicitly prompt either patients or doctors as to required actions. Audit packages within the software can audit on every variable collected. District wide audit is provided on anonymised aggregated data; individual practice audits (with comparisons to other practices) are provided to participating practices at least annually. Feedback of the data to the patient (for hospital patients only) is by a patient information sheet and to the GP as a standardised letter. A Diabetes Register Facilitator co-ordinates and updates the register. A steering group composed of GH, the Diabetes Register Facilitator and representatives of the PCGs and patients, oversees the register and deals with issues such as confidentiality.

### Impact of the system to date

Measures of the impact of the system to date relate only to Hartlepool, Easington and Stockton. The main impact on patient registration was in its first 12 to 18 months of operation: during 1995, 747 patients were registered on the system (0.4% prevalence) which had increased to 3867 (1.8% prevalence) by the end of 1996. The increase in registration has stabilised since then, reaching 4324 (2% prevalence) by 1999. During 1999, 70% of registered patients attended a clinic; 52% had their feet examined and 51% had their eyes examined. Seventy three per cent had an HbA1c result recorded and 69% a blood pressure measurement. These figures are similar to those reported by other centres using the same system \[[@B10]\].

### The need for an extended system

Recording of clinical measures increased during the first few years of operation of the system but began to plateau more recently (for example, 50% of patients had an HbA1c recorded during 1996, compared to 60% in 1997 and 63% in 1998). This plateauing of performance has been reported by others \[[@B10]\]. We believe that this is due to a lack of coordination (patients being lost to follow up) and lack of prompting of clinicians to deliver appropriate clinical interventions. Furthermore, given that most patients with diabetes are primarily seen in primary care the greatest potential impact is from optimising and extending the system in primary care. In order to address these shortcomings the additional key components, over and above those already in the system, will be:

1\. Locally adapted evidence based guidelines for the management and follow up of patients with diabetes.

2\. Automated prompts to patients and primary care clinicians that a review consultation is necessary.

3\. A structured management sheet (including patient specific management suggestions based on (1)).

4\. An enhanced monitoring system to follow up reasons for non-attendance from both patients and clinicians and to re-schedule appointments, based on nonreturn of a completed management sheet.

5\. Patient feedback for patients in primary care.

There is some limited supportive trial evidence for these developments, although the existing studies involved small sample sizes and may not be generalisable to the NHS \[[@B11]-[@B13]\]. In evaluating the system with these extended features this study will also address the design shortcomings of previous studies of shared care in diabetes \[[@B6]\]. It will be tailored to each practice, PCG defined areas will be studied, rather than an unrepresentative sample of general practices; and the system will be transparent and replicable in other areas.

Methods
=======

Design of the study
-------------------

The study design is a pragmatic two-arm cluster randomised controlled trial. The unit of randomisation will be the general practice. Simple patient randomised trials are rightly considered the most robust method of assessing most health care innovations \[[@B14]\]. This design, however, cannot be regarded as the gold standard for evaluating systematic approaches to chronic disease management, an essentially behavioural field of research \[[@B15],[@B16]\]. If both intervention and control patients were to be cared for within the same practice there is the risk that the management of control patients would be influenced by the practitioners knowledge of the care of intervention patients. This would result in an underestimation of the effect of the intervention \[[@B17]\]. Therefore, practices rather than patients are the appropriate unit of randomisation and analysis.

As the current system has been in place for different lengths of time within the three participating PCGs, we will stratify the randomisation by PCG. Randomisation will be performed by a statistician independent of the research team using computer generated numbers to avoid allocation bias \[[@B18]\].

Study setting and recruitment of practices
------------------------------------------

The study will be based in the general practices of the three PCGs of Easington, Hartlepool and Stockton. Since the recent merger of Hartlepool and North Tees Acute Trusts all three PCGs are now exclusively served by one secondary care diabetes service (and thus the one diabetes register). GH is the lead clinician for diabetes services in the new Trust.

The 61 general practices in the three PCGs constitute the target practices for the study and we will attempt to recruit all practices. The PCG diabetes leads or the PCG clinical governance leads in all three PCGs have provided letters confirming their support for the project. We do not envisage major difficulties with recruitment, given the need to agree local guidelines as part of the process involved in the Trust merger, the likely requirements in the forthcoming National Service Framework for diabetes, and the 100% practice coverage with the current diabetes system. We will (through the PCGs) write to all practices, giving information about the project to the senior partner or diabetes lead and practice manager of practices. Practices will be invited to opt out if they do not wish to be included in the study -- this is an approach we have used successfully before. The PCG diabetes lead, clinical governance lead and GH will be co-signatories of this letter. If practices do decline we will collect data on characteristics of non-participating practices to assess the impact on the generalisability of the trial\'s findings. Finally, if there are significant problems with recruitment, there are other practices which could be approached in a nearby PCG (South Tyneside) which uses the same software for its diabetes register.

Details of the intervention
---------------------------

### Local guidelines and management prompts

A guideline development group will be established to develop local guidelines for the management of diabetes, based upon available evidence based guidelines (Scottish Intercollegiate Guidelines Network \[[@B19]-[@B22]\](SIGN, 1996, 1997a, 1997b, 1997c), and Effective Care Bulletins \[[@B23],[@B24]\]. They will also use the forthcoming national diabetes guidelines as these become available. The group will be multidisciplinary and contain primary and secondary care doctors and nurses, patients and the Diabetes Register Facilitator \[[@B25]\]. The group will define review periods for specified patient groups (e.g. patients with diabetes satisfactorily controlled on diet alone should be reviewed every 12 months), referral criteria for patients moving from primary to secondary care and back and simple decision rules for the management prompts. These would be of two types. The first would prompt for actions to be performed and only require their performance to be documented (e.g. asking for a foot examination to be performed in a patient who does not have a recorded foot examination). The second would be more complex and suggest alterations to clinical management on the basis of data in the database (e.g. patients with persistently raised blood pressure should have their anti-hypertensive medication increased). These decision rules will be integrated into the recall and management system.

### Running the system

The proposed enhancements to the system are designed to require the primary care team to perform no additional work over and above the current configuration. The current database has a patient identifier, a minimum dataset and retrieval systems to support the structured recall of patients. Westman Medical Software has agreed to amend the system as required. A \'circle of information exchange\' will be established between the participating general practices and the database. The local guidelines will be used to adapt the current centralised database, along with the practices\' preferred method of following up patients (for example, within consultations in routine surgeries or within special clinics). The central database system will identify when patients are due for review (based upon the local guidelines) and will generate a letter to the patient asking them to make an appointment for a review consultation. Patient information or educational materials could be included with the letter. At the same time, the central database will generate a letter to the practice stating that the patient should be making a review appointment in the near future. The letter to the practice will include a management sheet (to be held in the patient\'s record) to capture an agreed minimum data set to be collected during the consultation. This management sheet will also contain the relevant prompts (as described above).

When the patient is seen in the practice, the primary care professional (currently this is usually done by the practice nurse) will complete the management sheet and return a copy for entry onto the central register within a designated period of time. This circle of information is broken if the patient does not visit the general practice as planned or the general practice does not return the management sheet to the central register. If this happens, the central register would alert the Diabetes Register Facilitator who will ascertain the reason for failure and take appropriate action, (e.g. send a reminder to the patient, prompt the practice to return the management sheet).

A range of educational activities will be provided for intervention practices, as part of the usual local structures for contact with practices, with some additions, These will include: distribution of information about the trial in local newsletters; meetings with practice clinical governance leads; evening meetings for practice nurses (with small group discussion of the practical implications for intervention practices); and a telephone meeting with the practice diabetes lead (usually the practice nurse) in each intervention practice.

Practices in the control arm will continue to receive the recall system as currently configured.

### Logistical considerations

From the prevalence of patients with diabetes on the current register, there will be about 7500 patients on the system if 61 practices are recruited. Half of these will be in intervention practices. On current patterns of usage, we anticipate there being the need for 1.5 recalls per annum per patient on the register, resulting in about 6000 recalls per year for the intervention group. Assuming a 40 week working year, the system will need to dispatch, receive and process about 150 forms per group per week.

### Identification of patients

Patients for the structured recall and management system are already identified on the Hartlepool and North Tees database. As some practices have children registered on the system, who are under the care of an exclusively secondary care adolescent service, an age limit of 18 years or over will be set for inclusion. Practices will be asked to check lists of their patients on the database regularly throughout the study. The central database will remove patients from the recall system who are known to have died or moved away.

### Patient consent

Patients have already consented, or are being consented, to their data being held within the current diabetes register. The study will involve no extra \'routine\' data being collected, and this data will be anonymised before being sent for analysis; all data held for analysis will be held in accordance with the Data Protection Act. For the patient-based questionnaire study, we will seek additional patient consent to complete one survey. The three relevant Local Research Ethics Committees have approved the trial.

Data collection
---------------

The main study outcome measures will be rates of performance of process of care and the patient based measures of functional and psychosocial wellbeing. Data will be collected for 15 months after the start of the intervention. Fifteen months was chosen to allow for patients who are reviewed every 12 months but fail to attend on initial invitation.

### Process of care variables

Process of care variables will be collected via the computerised database. The exact data to be collected will be determined by both the current content of the database and the guidelines but will include such data items as rates of attendance at clinics and annual reviews, conduct of eye and feet examinations, performance of investigations and prescribing. We will also collect data on clinical measures (e.g. HbA1c, and blood pressure levels).

### Outcome of care measures

Outcome of care data will be collected, by postal questionnaire, 15 months after commencement of the study. A portfolio of validated \[[@B26]-[@B30]\] and responsive \[[@B30],[@B31]\] generic and disease specific instruments will be used to measure functional and psychosocial variables that will be potentially influenced by the intervention. These will include:

i\) The SF36 health status profile which we will use to generate Mental (MCS) and Physical Component Summary Scales (PCS) \[[@B26],[@B27],[@B32]\].

ii\) The Newcastle Diabetes Symptoms Questionnaire \[[@B28]\].

iii\) The Bradley Treatment Satisfaction Questionnaire \[[@B29]\].

Patient costs questions will be developed by the study health economist. We have successfully used such packages of questionnaires within trials before and have achieved response rates in excess of 70% in similar surveys in this region. \[[@B33]\].

Sample size considerations
--------------------------

On the basis of previous work we have made the following assumptions. The mean number of patients per practice for whom we will be able to collect process data will be 30 and the ICC (a measure of the lack of independence of responses from patients from the same practice) calculated from our local data is 0.14 for measures of process (whether a blood pressure measurement and whether an HbA1c measurement has been recorded in a 12 month period). Standard methods for determining the sample size requirements for a cluster randomised trial \[[@B34]\] indicate that we need 60 practices to detect a difference of 15% (42.5% v 57.5%) with 80% power assuming a significance level of 5%. Assessment of outcome of care will be based on health status scales such as the SF-36. Previous work has shown that this type of intervention is likely to produce an effect size of approximately 0.25 in such measures \[[@B32]\] and that the ICCs for such measures will be approximately 0.07. The most efficient study design (that minimises the number of patients required) is one that makes use of all the available practices. A sample of 27 patients from each of 61 practices will give us 85% power to detect an effect size of 0.25 assuming a significance level of 5%. With a predicted response rate of approximately 70% (based on our experience in the COGENT study \[[@B33]\]) after two reminders, our starting sample size will need to be 2379 patients (approximately 39 patients per practice).

Principles of data analysis
---------------------------

Analysis will be by intention to treat. Multilevel modelling (using the MlwiN package \[[@B35]\]) will be used to take into account the clustering of patients within practices \[[@B36]\]. Both binary variables (when a process was undertaken or not) and continuous variables (such as the physical health component of the SF-36) can be analysed using these techniques. For both types of variable, variation between practices will be fitted as a random effect and the difference between intervention and control practices will be fitted as a fixed effect. In the case of binary variables, a logit link function will be used.

Economic evaluation
-------------------

The economic impact of implementing the new structured recall and management system will be evaluated in terms of the marginal costs of adapting and running the system; the costs of developing and disseminating the guidelines; the educational activities for intervention practices; the implications for the use of health care services; and the costs to the patients and their carers. The benefits will be measured as described earlier on in the clinical study. The estimation of health service resource use will relate to diabetes-specific clinical visits, tests, investigations, and procedures. This data will be routinely collected as part of the management system implementation and subsequent costing, using health service pay and price data, will be undertaken using a mixed approach based on micro-costing and gross-costing methods \[[@B37]\]. Use of drugs, referrals to secondary care and the impact of the intervention on the change of use of patients\' and their carers\' time will also be monitored through postal questionnaires at the end of the follow-up period. A sensitivity analysis will be undertaken to test the robustness of the results to the uncertainty not related to sampling variations and to enhance the generalisability of the results \[[@B38]\]. We are aware that the costs of the system might be balanced only in the longer term against the cost savings related to averted complications \[[@B39]-[@B41]\]. However, the assessment of the benefits in terms of final outcomes (e.g lives saved, or QALYs) and long term costs is beyond the objective of the present study.

Competing interests
===================

None declared

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

<>

Acknowledgements
================

The study is funded by Diabetes UK and the Northern and Yorkshire NHS Executive Research and Development Programme. We thank our collaborators at North Tees and Hartlepool NHS Trust, particularly Dr Carr, Dr MacLeod, Joanne Clayton and John Fitzsimmons; at Easington Primary Care Group and North Tees Primary Care Trust; and at South Tyneside Primary Care Group and South Tyneside Healthcare NHS Trust, particularly Drs Bradshaw and Parr, Clare Beard and Wynn Schembrie.


Background
==========

The beta-thalassemia syndromes (thalassemia major and beta-thalassemia intermedia) originate from the absence of or from a reduction in the synthesis of structurally normal beta-globin chains. The beta-globin chain deficiency contributes to the pathogenesis of anemia by impairing the formation of functional Hb A and by causing ineffective erythropoiesis. The anemia stimulates the secretion of Epo and leads to erythroid hyperplasia of the bone marrow and to an accelerated erythropoiesis \[[@B1]\]. As a result, erythroblasts expressing the fetal globin gene are generated and fetal hemoglobin (Hb F) is synthesized in the postnatal life \[[@B2]\]. Two more factors, the amplification of the cellular clones producing F-cells and the slower turnover of these cells in circulating blood favour the Hb F presence in thalassemia \[[@B3]\]. In these affections the persistent synthesis of Hb F is very important because it restores the alpha/non-alpha globin ratio, thus reducing the bone marrow hyperplasia and ineffective erythropoiesis and therefore the severity of disease.

Genetically, the beta-thalassemia syndromes are heterogeneous. The severity of these disorders is determined primarily by the type of the gene defect as well as the gene dosage.

The molecular analysis has uncovered that different clinical variants of beta-thalassemia are associated with a wide spectrum of different mutations of the beta-globin gene; thalassemia major patients, characterized by a severe transfusion-dependent anemia and a marked increase in the synthesis of Hb F, are either homozygous carriers of mutations that abolish the beta-globin synthesis (beta° thai) or compound heterozygotes for beta° thal and severe beta^+^ thal defects which drastically reduce it. These mutations are referred to as severe beta-thal mutations. beta-thal int patients which have a less severe anemia and a modest increase in the synthesis of Hb F, are carriers mainly of beta^+^ thal mutations that reduce slightly beta-globin synthesis. These mutations are referred to as mild beta-thal mutations.

Furthermore, deletions of the non-alpha globin gene cluster encompassing the beta- and delta-globin genes are associated with a genetic variant of beta-thalassemia (deltabeta thal), which is characterized by a marked increase in the synthesis of Hb F even in the heterozygous state. In these patients the gamma-globin genes remain intact and are expressed in the postnatal life, thus reducing the ineffective erythropoiesis and ameliorating the severity of the disease.

It is worth noting that also genetic factors unlinked to the beta globin cluster, favour the Hb F synthesis in the post-fetal life \[[@B4]\]. Particularly interesting for its frequency is the polimorphic single-base substitution C→T at Ggamma -158, recognizable for the creation of a XmnI restriction site, that appears to cause an increase of Hb F expecially during erythropoietic stress.

Recently, it has been suggested that the increased synthesis of Hb F associated with beta-thalassemia syndromes is directly or indirectly dependent from the Epo level \[[@B5],[@B6]\].

The aim of this study was to investigate the relationships between the genetic defects and the severity of the disease in patients affected by a thalassemic disease. In particular, the relationships between the genetic defect, the total Hb, and between the synthesis of Hb F and the Epo level have been examined in patients with beta thalassemia intermedia (beta thal int). In addition, the distribution and frequency of point mutations of the promoter region of gamma-globin genes were analysed and the relationship between these mutations and the content of Hb F was investigated. The analysis of the beta-globin gene and gamma-globin gene mutations was extended also to patients affected by Cooley\'s disease.

Subjects
--------

Seventy-four patients with clinical picture of beta thal int have been examined: 33 of them were affected by a mild or sub-silent form of the disease, and 41 by an evident clinical picture (Tab. [1](#T1){ref-type="table"}). In addition, 58 patients carried a beta thal defect and also a triplicated or quadruplicated alpha locus. Twenty-four patients were affected by thalassemia major, and 15 by Hb H disease. The control group included 53 healthy individuals and 42 iron-deficient, non thalassemic patients.

::: {#T1 .table-wrap}
::: {.caption}
######

Hematologic and hemoglobinic data and Epo levels in thalassemic patients and control subjects.
:::

  **Group of patients**                                                **N. cases**   **Hb g/dl**   **Hb F %**                      **EPO mU/l**                     **Glob. synth. alpha/non-alpha**   **Ret. ‰**
  -------------------------------------------------------------------- -------------- ------------- ------------------------------- -------------------------------- ---------------------------------- ------------
  Sub-silent beta thal int                                             33             10.1 ± 1.0    20.5 ± 18.2 3.0 → .70.6         41.22 ± 32.00 11.66 → 136.6      2.10                               31
  Evident beta thal int                                                41             8.3 ± 1.16    33.40 ± 28.04 6.80 → 85.0       95.83 ± 64.33 17.84 → 218.97     2.20                               60
  beta thal int in double heterozygous beta thal + triplicated alpha   58             10.0 ± 1.38   5.15 ± 3.9 2.0 → 19.3           28.45 ± 17.11^\*^ 7.85 → 60.72   2.50                               30
  Hb H disease                                                         15             9.4 ± 1.2     \< 1                            32.88 ± 12.11^\*\*^              0.52                               28
  Thal major                                                           24             10.4 ± 1.0    7.7 ± 8.5^\*\*\*^ 1.60 → 40.9   46.83 ± 40.78 9.82 → 129.59                                         17
  Healthy non-thal subjects                                            53             14.4 ± 1.1    \<1                             11.90 ± 3.3                      \~1.0                              3
  Iron-deficient non-thal subjects                                     42             8.7 ± 1.2     \<1                             160.10 ± 312.6 2.7 → 1960.0                                         8

^\*^ The measurement of Epo has been detected only in 17 patients. ^\*\*^ The measurement of Epo has been detected only in 10 patients. ^\*\*\*^ This value is not informative as patients were routinely transfused.
:::

Patients with an homozygotic condition for a delta beta thal deletion were excluded from this study, since only Hb F is synthesized. The patients homozygotes for severe beta-globin defects, and carriers of an alpha thal defect, were also excluded.

All of the individuals examined in this study were in the post-puberal age.

Methods
=======

Blood samples were collected in EDTA. Hematologic parameters were determined using a Technicon H1 Analyser (Bayer). Hb A~2~, Hb F and Hb H values were determined using an electrophoretic method \[[@B7]\]. Concentrations of Hb F higher than normal were confirmed by radial immunodiffusion (Quick Plate kit, Helena Laboratories, Beaumont, TX, USA). Serum iron and ferritin levels were measured by standard methods. The serum level of erythropoietin (Epo) was determined using the Epo-Trac kit (INSTAR Corporation, Stillwater, MN, USA). The average value of 53 healthy individuals was assumed as the normal Epo concentration. Reticulocyte counts were performed on fresh blood preparations stained with brilliant-cresyl-blue. The synthesis of globin chains in vitro was determined following the method of Clegg \[[@B8]\], using a high pressure liquid chromatograph.

The Amplification Refractory Mutation System (ARMS) was used to detect mutations of the beta- and gamma-globin genes \[[@B9],[@B10]\] and a modified PCR method previously described in our laboratories \[[@B11]\], was used to detect alpha-globin genes defects. The patients affected by thalassemia major or intermedia were screened for all of the beta- and alpha-globin gene mutations present in the Italian population (Tab. [4](#T4){ref-type="table"}, ref. \[[@B4],[@B12]\]) and the mutations analysis was also extended to mutations of the promoter region of the gamma-globin genes which are until reported to occur in the Italian population (Ggamma-158 C→T; Agamma -196 C→T; Ggamma -175 T→C; Agamma -117 G→A; AgammaT and the deletion Agamma -225/-222 bp). The Ggamma -158 C→T and AgammaT substitutions were searched in all of the subjects reported in Table [1](#T1){ref-type="table"}. The remaining mutations of the gamma genes promoters were searched in 286 more chromosomes of beta-thalassemia patients or control subjects.

::: {#T4 .table-wrap}
::: {.caption}
######

Distribution of beta thal alleles in beta thal int and thal major patients.
:::

  **Type of thalassemic disease**                                      **N. cases**   **n. of identified alleles**   **Alleles beta thal silent**   **Alleles beta+ thal mild**   **Alleles beta+ thal severe and beta ° thal**
  -------------------------------------------------------------------- -------------- ------------------------------ ------------------------------ ----------------------------- -----------------------------------------------
  Sub-silent thal int                                                  33             61                             22 (37%)                       8 (13%)                       31 (50%)
  Evident thal int                                                     41             81                             9 (12%)                        36 (44%)                      36 (44%)
  beta thal int in double heterozygous beta thal + triplicated alpha   58             100                            0                              0                             100 (100%)
  Thal major                                                           24             47                             1 (2.1%)                       7 (15%)                       39 (83%)

The silent, mild, severe beta-thal defects present in Italy are: 1. Silent: -101 C→T; -92 C→T; IVS II-844 C→G; triplicated alpha^anti\ 3.7^; triplicated alpha^anti\ 4.2^: ; they have a hematologic-hemoglobinic picture completely normal or with some mild thal characters. 2. mild or sub-silent: -87 C→G or C→T; -86 C→G or C→A; IVS-1 6 T→C that have a constant but very mild thal hematologic picture and a mild increase of Hb A~2~. 3. severe: beta°39 C→T; IVS 1--110 G→A; IVS II-745 C→G; frameshift 6 (-A) that have all very marked hematologic and hemoglobinic characters.
:::

The statistical significance was evaluated using the Student\'s t-test. Linear regressions were fitted by the least square deviations method and the correlation coefficient r was calculated according to a standard formula. A p value \< 0.05 was assumed to be statistically significant, as only few tests were performed.

Results
=======

The analysis of the hematologic data and the clinical symptoms of 74 patients affected by beta-thal int indicate that this disease can be classified into two clinical variants referred to as sub-silent and evident beta-thal int (Table [1](#T1){ref-type="table"}). The patients with sub-silent beta-thal int have a reduced total Hb content and show no or very mild clinical symptoms. The patients with evident beta-thal int show severe clinical symptoms, often associated with massive splenomegaly, and 41% of them have undergone splenectomy. The Hb content is significantly lower than that of the patient with sub-silent beta-thal int (t = 7.44; gl 72; p \< 0.001). The Hb F level is higher in with evident than in patients with sub-silent beta-thal int, and the difference is statistically significant (t = 2.28; gl 72; p \< 0.05). Thus, the severity of clinical symptoms of beta-thal int correlates with a decrease of the total Hb content and an increase of the Hb F content. Normally the Hb F content is positively correlated with the total Hb content, as illustrated in Fig [2](#F2){ref-type="fig"}.

![Epo level and Hb F content are not related in patients with β-thalassemia intermedia. The logarithm of the Epo level was plotted against the corresponding total Hb content of each patient. The patients showing an increase of Hb F higher than 40% are represented by red squares, whereas the blue squares denote the patients showing an increase of Hb F equal to or less than 40%.](1471-2326-2-2-1){#F2}

In the 42 iron-deficient but not thalassemic subjects, the Epo level ranging from 2.7 to 1960 mU/1 with a mean value of 160.10 ± 312.60 mU/1, was negatively correlated (r = -0.74) to the Hb content (Fig. [1](#F1){ref-type="fig"}). The correlation is statistically significant (n = 42; p \< 0.001).

![Negative correlation between total Hb content and Epo level in iron-deficient patients. The logarithm of the Epo level plotted against the corresponding total Hb content of each patient is denoted by a blue square. The linear regression (solid line) fitting the data is described by the following equation: log \[Epo\] = 4.78--0.33 \[Hb\]. The correlation coefficient r is -0.74.](1471-2326-2-2-2){#F1}

The mean Epo level of beta-thal int patients is apparently lower than that of iron-deficient patients (Tab. [1](#T1){ref-type="table"}). Regarding to total Hb, only between the groups of sub-silent beta-thal int patients and iron-deficient patients the difference is statistically significant (t = 2.17; gl 73; p \< 0.05).

The mean values of the Epo level and the Hb F content in the two groups of beta-thal int seem to be positively related, but an accurate analysis of single cases indicates (Tab. [2](#T2){ref-type="table"}) that the Epo level and Hb F content are unrelated: subjects showing a low content of Hb F and an high level of Epo as well as subjects showing an high content of Hb F and a low level of Epo were frequently observed. This observation is reinforced by the analysis of groups of subjects in which the severity of anemia is similar. Within the group of evident beta tal int patients (the only adapt for this analysis) subjects showing an Hb F level higher or lower than 40% \[[@B5],[@B6]\] were selected. As illustrated in Fig. [2](#F2){ref-type="fig"} and Tab. [3](#T3){ref-type="table"}, subjects having a very similar Epo level and Hb content fall in both classes of Hb F content, indicating that Hb F content and Epo level are not related.

::: {#T2 .table-wrap}
::: {.caption}
######

Total Hb, Hb F, and Epo level in patients with evident beta thal int.
:::

  **Case**   **Hb g/dl**   **Hb F %**   **EPO mU/l**
  ---------- ------------- ------------ --------------
  1          10.5          87           23
  4          10.6          70           55
  11         7.2           21           132
  19         8.2           7            51
  20         7.4           24           126
  21         9.0           12           233
:::

::: {#T3 .table-wrap}
::: {.caption}
######

Epo level and Hb F content in a group of evident beta thal int patients showing a similar degree of anemia and divided for Hb F \< or \> 40%.
:::

  **Patients\' group**                     **N. cases**   **Hb F and EPO mean levels**
  ---------------------------------------- -------------- ---------------------------------------- -----------------------------------------
  Evident beta thal int with Hb ≤ 9 g/dl   32             HbF = 13.6% EPO = 92.9 mU/l (25 cases)   Hb F = 59.9% EPO = 118.8 mU/l (7 cases)
:::

Different sets of mutations of the globin genes are associated with groups of patients characterized by a different severity of the disorder. The spectrum of the beta-globin gene mutations commonly found in the Italian population can be divided in three categories, the first of which includes the -101 C→T, -92 C→T, and IVS II 844 C→G single nucleotide substitutions; the second the -87 C→G, -86 C→G or C→A, and IVS I C→T single nucleotide substitutions, and the third the beta° 39 C →T, IVS I 110 G→A, IVS II 745 C→G single nucleotide substitutions and the frameshift 6 (-A) mutation. These three categories are referred to as silent beta-thal, mild beta-thal, and severe beta-thal mutations, respectively.

In the group of 33 patients with sub-silent beta-thal int the frequencies of silent beta-thal, mild beta-thal, and severe beta-thal mutations were 37%, 13% and 50%, respectively (Tab. [4](#T4){ref-type="table"}). In the group of 42 patients with evident beta-thal int, the most common and equally frequent (44%) beta-globin gene defects were mild and severe beta-thal mutations. The frequency of silent beta-thal mutations was 12%. Conversely, severe beta-thal mutations were found at high frequency (83%) in thalassemia major patients. Silent beta-thal mutations were detected only in a small fraction (2.1%) of thalassemia major patients.

In the 58 patients both heterozygous for a mutation of the beta-globin gene and heterozygous or rarely homozygous for the presence of three or four copies of one alpha-globin locus, the beta-globin gene defects were invariably beta° or severe beta^+^ thal mutations (Tab. [4](#T4){ref-type="table"} and ref. \[[@B13]\]); the alpha-globin gene mutation was in 54 out of 58 patients a triplicated alpha anti 3.7 I gene cluster; in 3 patients a quadruplicated alpha anti 4.2 gene cluster, and in one patient the homozygous condition for a triplicated alpha anti 3.7 I gene cluster. In these patients, the average Hb content was 10.0 ± 1.38 g/dl (Table [1](#T1){ref-type="table"}), the content of Hb F was slightly higher than normal, ranging from 2.0 to 19.3 with an mean value of 5.15 ± 3.9 %, and the average Epo level was 28.45 mU/1, ranging from 7.85 to 60.72 mU/l. This value is lower than that of regularly transfused thalassemia major patients. Although the Hb F content appears to be low in all these patients, their clinical picture is highly heterogeneous, 50% of the patients having a sub-silent beta thal int, 30% an evident beta thal int and 20% a severe anemia with 5 transfusion dependent patients. Ten out of 58 patients (17.2%) have undergone splenectomy.

The patients with Hb H disease have a normal Hb F level, and a Epo level lower than that of patients with beta-thal int.

In the beta-thalassemia patients and in the control subjects, screened for the six mutations of the promoter region of the gamma-globin gene observed in Italy, only the Ggamma -158 C→T and AgammaT single nucleotide substitutions were detected. In all cases except 7 the AgammaT mutation was to the Agamma -225--222 bp deletion. The frequencies of the Ggamma -158 C→T and AgammaT mutations are significantly different in the various groups of beta-thalassemia patients (Tab. [5](#T5){ref-type="table"}), with the lowest frequency of the Ggamma -158 C→T mutation (2.1%) in thalassemia major patients.

::: {#T5 .table-wrap}
::: {.caption}
######

Distribution of the Ggamma -158 C→T and AgammaT mutations in thalassemic patients and in control subjects.
:::

  **GROUP OF PATIENTS**                                     **Allele frequencies**
  --------------------------------------------------------- ------------------------ ------
  Sub-silent beta tal int                                   8.6                      27.3
  Evident beta tal int                                      18.3                     26.6
  beta tal int with genotype triplicated alpha + beta tal   16.6                     17.6
  Hb H disease                                              37.5                     5.5
  Tal major                                                 2.1                      35.4
  Normal controls                                           25.0                     18.0
:::

In the group of patients with evident beta-thal int the frequency of the Ggamma -158 C→T mutation was 6.2% in 7 patients having a total Hb content \> 9 g/dl and 1.6% in 32 patients having a total Hb content ≤ 9 g/dl.

Discussion
==========

It is well known that the severity of thalassemic diseases (thalassemia major and thal int) syndromes is primarily determined by the nature and type of mutations as well as gene dosage of the globin genes underlying these disorders. The results reported in this study extend this conclusion to two clinical variants of beta-thal int, referred to as sub-silent and evident beta-thal int. Distinct sets of mutations of the beta-globin gene have been found associated with these two variants of beta-thal int. Silent mutations of the beta-globin gene were detected at high frequency in patients affected by sub-silent beta-thal int, whereas mild and severe mutations of the beta-globin gene were the most common mutations amongst the patients affected by evident beta-thal int (Table [4](#T4){ref-type="table"}). This notion is reinforced by the high frequency (83%) of severe beta^+^ and beta° mutations in patients affected by thalassemia major.

Several hematologic parameters of beta-thalassemia reflect the diversity of mutations at beta-globin locus. The total Hb content was found to be significantly different between patients affected by thalassemia major and patients with beta thal int, as well as between patients affected by sub-silent and patients with evident beta-thal int (Table [1](#T1){ref-type="table"}). These last two groups of patients showed also significant differences in the content of Hb F (t = 2.28; df = 72; p \< 0.05).

The comparative analysis of the serum Epo level in groups of patients affected by thalassemia diseases or iron deficiency shows first of all that the synthesis of Epo, as predicted, is always directly related to the severity of anemia and, consequently, inversely related to the total Hb content. Furthermore, in the thalassemic diseases mean Epo level is consistently lower than that of iron deficient patients, probably as a result of the different pathophysiology of the anemia in the two conditions. No firm relationship could be established between the Epo and Hb F levels. The variations of these two parameters in each patient with evident beta thal int appear to be erratic (Tab. [2](#T2){ref-type="table"}); and in patients having a similar level of both total Hb and Epo, the amount of Hb F is indifferently higher or lower than 40% (Tab. [3](#T3){ref-type="table"}). Therefore, the data do not support the hypothesis that Hb F content is regulated by the serum level ofEpo. The relationship between total Hb content, Hb F content and Epo level were not studied in patients with Hb H disease, since Hb F is in these patients below the detection limit.

In patients with a compound heterozygosity for a triplicated copy of a alpha globin gene cluster and a beta globin gene mutation, only a moderate increase of the Hb F level (mean 5.5 ± 3.9% within a range from 2.0 to 19.3%) was observed. This observation indicates that the excess of alpha globin chains, as assessed by the unbalanced alpha/non-alpha globin ratio (2.5) is not sufficient to increase the gamma-globin chains synthesis.

Only the Ggamma -158 C→T and AgammaT single nucleotide substitutions out of 6 gamma gene promoter region mutations explored in this study were detected, and their frequencies varied in different groups of patients (Table [5](#T5){ref-type="table"}). The role of this mutation in determining the Hb F amount is not yet definitively cleared. The demonstration, already reported by other Authors \[[@B14]\], that the frequency of Ggamma -158 mutation is very low in beta thal major patients (Tab. [5](#T5){ref-type="table"}), and, inside the beta thal int patients, higher in those with a higher amount of total Hb and lower in those with a lower amount of total Hb, seems to indicate that this mutation is important in ameliorating the severity of the disease.

Conversely, other obstervations \[[@B15]-[@B17]\] report that this mutation does not influence the Hb F content of subjects which are heterozygous for a beta thal mutation \[[@B16]\] as well as of heterozygous compounds for a beta thal mutation and a triplicated or quadruplicated copy of the alpha-globin cluster.

At variance with these observations, in sickle cell disease patients carrying some beta^s^ haplotypes which show a high frequency (up to 100%) of the Ggamma -158 C→T mutation, show also an increase of the Hb F content that leads to a reduction of the severity of the disease \[[@B18]\].

The relationship between the content of Hb F and point mutations of the promoter region of the gamma-globin genes in thalassemic patients needs then to be further investigated. The analysis must be extended to those mutations of the gamma-globin gene promoter region that have not yet been detected in the population of Italy or other Mediterranean countries. The hypothesis \[[@B19]\] that the Ggamma -158 C→T mutation when linked in cis to the sequence motif (AT)~x~(T)~y~ mapping at position -530 in the 5\' untranslated region of the beta-globin gene could interact to generate a high expression of Hb F under the erythropoietic stress, has to be ulteriorly evaluated. Finally, the role played by loci unlinked to the beta-globin locus in the regulation of Hb F synthesis remains to be elucidated. Two candidate regulatory loci have been mapped at Xp22 \[FCP locus \[[@B20],[@B21]\]\] and 6q22.3-23.2 \[[@B22]\].

A study regarding all these factors, obviously necessary also for the various heterozygotic thalassemic conditions, is now in progress in our laboratories.

Conclusions
===========

The severity of beta-thalassemia syndromes including the increased synthesis of Hb F is primarily determined by the genotype of the beta-globin locus. The synthesis of Hb F does not appear to be regulated by the serum level of Epo. The Ggamma -158 C→T mutation is frequent among patients with beta-thal int, and very rare in patients with thalassemia major, but at variance with sickle cell disease, increased synthesis of Hb F in beta-thal int patients cannot be firmly related to the linkage in cis of this mutations.

Competing interests
===================

None declared.

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

<>

Acknowledgements
================

The Authors are grateful to Dr. Giacomo D\'Agostaro (ENEA, Rome) for his cooperation and helpful comments in preparing the manuscript; Dr. Paolo Cianciulli Director of the Day Hospital for Thalassemics of the Sant\'Eugenio Hospital of Rome, to have made possible the study of his thalassemic patients; to the Mrs. Brunella Sidorini and Mrs. Nadia D\'Arcangeli for their technical assistance; to Mr. Marco Ballante for his excellent contribution to the making of the manuscript.


Background
==========

On the 25 January, 1999 an earthquake with an intensity of 6.2 points on the Richter\'s scale occurred in a focus located only 20 kilometers away from Armenia, Colombia. Although 28 towns or municipalities, including Armenia, were severely damaged by the earthquake, the worst effects occurred in Armenia itself and in its surrounding towns. The consequences of the catastrophe were devastating, mainly because of the loss of people\'s property and belongings: around 70% of Armenian houses were destroyed and in some neighboring towns the percentage was greater than 80%. In the rural area, the proportion of lost houses was also 80%. The death toll was estimated at 1184, and the number of people injured was more than 5,000 \[[@B1]\].

The region known as \"Departmento del Quindío\" includes, according to the political and administrative division of the Colombian territory, several small towns and its capital city, Armenia. The whole area of influence of the telluric movement is known as \"Eje Cafetero\" (Coffee Axis), because it is here that most of the world famous Colombian coffee is produced. This area is located within the country\'s central mountain range. Before the tragedy, Armenia had 296,330 inhabitants, with the whole Department of Quindío having approximately 500,000.

The devastating effects on the houses of Armenia induced the organization of transitory housing facilities in tents and \"alojamientos\" (multi-family spaces), which caused difficulties in the provision of basic sanitary services, water supplies and food. Fears about epidemics were high, but no outbreaks of infectious diseases, including diarrhea, dengue or malaria occurred \[[@B2]\]. Temporary housing facilities remained until two years after the disaster, which raised concerns about public health conditions in this people. The University of Quindío undertook epidemiological studies in order to determine which health disorders were occurring. This included the effect on mental and physical health morbidity \[[@B2],[@B3]\]. One of the pathological aspects that was studied was the prevalence of intestinal parasites in this people. During this survey it was found that giardiasis was the most prevalent intestinal parasitic infection. Therefore, a study was designed in order to ascertain the epidemiological factors related to this high prevalence. In this paper we report the findings of the laboratory examination of stools and their association with data collected by questionnaire.

Methods
=======

Study design, characteristics of the camps and questionnaire
------------------------------------------------------------

A prospective study of the prevalence of *Giardia* in stool specimens of children living in temporary houses in Armenia was carried out. A total of 26,780 people lived in temporary housing camps built under the supervision of non-governmental organizations. This study was conducted in 18 camps (\"asentamientos\"), multi-family spaces which grouped temporary houses or tents, and were representative of a total of 86 camps which were established in Armenia after the January 25, 1999 earthquake. A census population of the families living in the 18 camps found 816 children aged between 3 and 13 years. A random sample of 193 targeted households was selected for a systematic household survey conducted between January 2000 and July 2001. Thus, 217 children aged between 3 and 13 years were sampled. The children were included only if their father or other adult with legal custody of the children agreed to give written informed consent.

A household was defined as a unit of persons residing in one tent or temporary house. The median household size was made up of 5 persons. All households had municipal garbage disposal services. Electricity was available for all households.

From each selected household one adult was interviewed using a standardized questionnaire which focused on both the demographic characteristics of children such as age and school years, as well as on the presence of clinical symptoms such as diarrhea (passing more than two liquid stools daily) or flu symptoms (rhinorrea without pharyngitis and with only minor or no fever at all) with a duration of less than eight days at the moment of interview. The weight of each child was recorded. Interviewers were selected from university students of biology who had previous experience in community work, were from the same city and knew the terms used by people to describe their typical symptoms and living conditions; they were previously trained by an epidemiologist.

Stool samples of children were collected the day after the questionnaire was filled. The source of drinkable water and the type of toilet used were recorded for each child. Water supply varied between camps: in some camps water was provided by the municipal water ducts, in others there were individual tanks for each house. Municipal water (piped water) was the major source of drinking water and was used in 15 camps. The source of municipal water was surface water (\"Río Quindio\") provided through the aqueduct system which existed before the earthquake. Water chlorination concentrations were between 2,5 and 3 mg/liter. This is a closed water delivery system; however after the earthquake some of the ducts collapsed and leakages occurred. For this reason, in 3 camps individual tanks were used as a source of drinkable water. The tanks were made of plastic material and had a 2,000 liter storage capacity. Individual tanks were filled by a tank car. In addition, municipal health services distributed 1,217 kg of sodium isocianyde dichloride in all camps to be used in individual tanks. There were two types of hygienic room services. One was the communal toilet service that consisted of one latrine for each 9--10 families placed in the center of camp; other type of toilet was the individual sanitary service located in each temporary house and used only by the members of that household. The researcher also registered the \"clean status\" of the temporary house; a house was considered \"clean\" if no particular odors were detected, the food was stored in proper recipients, and food waste was disposed of in garbage cans. The place for food storage was also recorded. No questions about animal contact were done.

Specimen collection and stool examination
-----------------------------------------

Stool samples were collected early in the morning in sterile plastic containers supplied by the researchers and conserved on ice until laboratory examination. Samples with water or urine contamination were discarded. A second sample was requested from all children at day 5 after the first sample and a third sample one month later. Stool samples were stained with ferric hematoxilin and examined after centrifugation; this test has a sensitivity of 61% on the first sample and a cumulated sensitivity of 83% when three different serial samples are examined \[[@B4]\].

Statistical Analysis
--------------------

Statistical significance tests for comparison of rates were performed when sample sizes were insufficient and asymptotic tests when sizes were sufficient. All tests were two-sided, and a p value of \<0.05 was considered as significant. Exact 95% confidence intervals (Cl) were calculated \[[@B5]\].

Results
=======

In total, stools from 217 children were examined, 97 were girls (44.7%) and 120 boys (55.3%). In the first sample, prevalence of *Giardia* cysts was 60.4% (131 children) and *Giardia* trophozoite forms 4.6%. A second sample was obtained in only 53 of 217 children. Families were not informed about the result of the first sample before collecting the second one. Collection was requested on a voluntary basis. From these 53 children with a second sample, 40 were positive and 13 negative on the first sample. Thirty children were positive for *Giardia* cysts on the second sample (56%), only three of them being negative at the first sample. Only six children provided a third sample; three of them, who were all previously positive for *Giardia* cysts, were positive.

Analysis of infections by other parasites was also performed (Table [1](#T1){ref-type="table"}). There were no statistically significant differences in the prevalence of giardiasis in children infected with other parasites. No cultures for bacteria were performed during this study.

::: {#T1 .table-wrap}
::: {.caption}
######

Parasitic infections in stool samples of children (n= 217) living in temporary housing after the 1999 earthquake disaster in Armenia.
:::

  **Organism**                                                **Number of infected children**   **Percent of sampled children**   **Number of infected children also infected with giardiasis**
  ----------------------------------------------------------- --------------------------------- --------------------------------- ---------------------------------------------------------------
  *Ascaris lumbricoides*                                      36                                16.6%                             19
  *Balantidium coli*                                          4                                 1.8%                              3
  *Endamoeba coli*                                            71                                32.7%                             40
  *Endolimax nana*                                            125                               57.6%                             56
  *lodamoeba buschlii*                                        31                                14.3%                             19
  *Entamoeba histolytica/dispar* (determined by micrometry)   116                               53.4%                             75
:::

The analysis of epidemiological characteristics (Table [2](#T2){ref-type="table"}) showed that the presence of *Giardia* cysts was associated with the use of communal toilet versus use of individual toilet (OR 3.9). The water provision by municipal water services was associated with a higher risk for giardiasis, in contrast with people that used individual water tanks (OR 3.5). The finding of mucus in stool was higher in children with *Giardia* cysts (OR 2.3). A \"clean house\" was a protective factor for the presence of *Giardia* cysts (OR 0.4).

::: {#T2 .table-wrap}
::: {.caption}
######

Association between epidemiological and laboratory findings with the presence *Giardia* cysts in stool samples from children living in temporary housing after the 1999 earthquake disaster in Armenia.
:::

  **Risk factor**                                        **With factor**   **Without factor**   **OR**   **95% Confidence Intervals**   **P**
  ------------------------------------------------------ ----------------- -------------------- -------- ------------------------------ -----------
  Communal toilet (versus individual toilet)             21/25             110/192              3.9      1.2--16                        **0.01**
  Municipal water service versus individual water tank   27/58             104/159              3.5      1.1--14                        **0.02**
  Mucus in fecal sample                                  20/26             111/191              2.39     0.9--6.7                       **0.049**
  Leukocytes in stool                                    16/22             115/195              1.85     0.70--5.35                     0.15
  Plastic stock of food versus carton stock of food      49/93             82/124               1.7      0.9--3.1                       0.06
  Yeast in stool                                         57/93             74/124               1.06     0.61--1.82                     0.46
  Diarrhea                                               23/42             108/175              0.75     0.4--1.4                       0.84
  Flu symptoms                                           79/136            52/81                0.7      0.4--1.4                       0.45
  Lipids in stool                                        120/201           11/16                0.67     0.18--2.2                      0.65
  Clean lodgment                                         62/118            69/99                0.48     0.27--0.84                     **0.014**
:::

In total 199 of the 217 children studied attended school. The source of water at school was municipal water and communal toilet was the type of sanitary service there. As data were collected concerning the putative factor (water source) and/or disease (*Giardia*), data were also analyzed on the presence or absence of the confounding variable (attendance at school). The data were then divided into strata for confounding variable (attendance at school) in a series of 2 × 2 tables, one for each level of the confounding variable (Figure [1](#F1){ref-type="fig"}). Using Mantel-Haenszel odds ratio method the OR was 15.1 (CI95% 8--36) with a p value of 0.00000000. This analysis indicate that drinking municipal water at camps was strongly associated with giardiasis even with the confounding variable \"school\" taken into account.

![Stratified analysis of children who did and did not attend school and presence of *Giardia* cysts in stool samples from children living in temporary housing after the 1999 earthquake disaters in Armenia.](1471-2458-2-5-1){#F1}

Presence of *Giardia* trophozoites was higher in patients with municipal water service and with lipids in stools (Table [3](#T3){ref-type="table"}); however due to the small number of children with trophozoites, no difference attained statistical significance. Other demographic factors including weight, age and the number of years at school were not associated with giardiasis (Table [4](#T4){ref-type="table"}).

::: {#T3 .table-wrap}
::: {.caption}
######

Association between epidemiological factors, laboratory findings and the presence of *Giardia* trophozoites in stool samples from children living in temporary housing after the 1999 earthquake disaster in Armenia.
:::

  **Risk factor**                                        **With factor**   **Without factor**   **OR**   **95% Confidence Intervals**   **P**
  ------------------------------------------------------ ----------------- -------------------- -------- ------------------------------ -------
  Municipal water service versus individual water tank   10/159            0/58                 ?        ?-1.1                          0.06
  Lipid drops in stools                                  2/16              8/201                3.41     0.45--16.5                     0.16
  Flu symptoms                                           6/81              4/136                2.62     0.69--10.8                     0.11
  Plastic stock of food versus carton stock of food      1/19              9/198                1.16     0.050--7.71                    0.97
  Leukocytes                                             1/22              9/195                0.98     0.04--6.44                     0.66
  Yeast in stools                                        4/93              6/124                0.88     0.21--3.0                      0.69
  Communal toilet (versus individual toilet)             1/25              9/192                0.84     0.03--5.50                     0.71
  Mucus in fecal sample                                  1/26              9/191                0.80     0.03--5.24                     0.72
  Diarrhea                                               1/42              9/175                0.45     0.01--2.86                     0.88
  Clean lodgment                                         3/118             7/99                 0.34     0.07--1.34                     0.97
:::

::: {#T4 .table-wrap}
::: {.caption}
######

Mean age, school years and weight and presence of cysts of *Giardia* in stool samples from children living in temporary housing after the 1999 earthquake disaster in Armenia (Colombia).
:::

                 ***Giardia* Cysts**   **Mean ± SD**   **P**
  -------------- --------------------- --------------- -------
  Mean age       No                    7.3 ± 2.9       0.79
                 Yes                   7.4 ± 3.1
  Weight (Kg)    No                    23.1 ± 9.1      0.89
                 Yes                   23.3 ± 9.9
  School years   No                    1.6 ± 1.9       0.87
                 Yes                   1.7 ± 2.2
:::

Discussion
==========

Two previous studies carried out by the Colombian National Institute of Health found that *Giardia* was the most prevalent intestinal parasite. In 1965 a prevalence of 9.4% was found and in the 1980 survey the rate was 21.4%. These rates were the reported for children in the 5 to 14 year-age group living in the central region of Colombia, where the department of Quindío is located. In both studies (in 1965 and in 1980) diagnostic methods were the same as those used in the present study, microscopic examination and the rates were estimated from the examination of only one sample \[[@B6]\]. We found a higher prevalence. Our findings could indicate that giardiasis emerges during events which alter the existing sanitary conditions. We found that a higher prevalence was associated mainly with communal toilets and municipal duct provision at camp, which indicates that both factors favored dissemination of giardiasis in temporary camps after the disaster. As demonstrated by stratified analysis, drinking municipal water at camps was strongly associated with giardiasis even when attendance to school was taken in account as confounding variable. In future in similar conditions it will be important to make efforts addressed to guarantee individual toilet for each household and to control the water source by checking the presence of *Giardia* in water at temporary camps.

In the present study, clinical symptoms were not associated with the presence of *Giardia* cysts or trophozoites in stools, but presence of cysts were significantly associated with mucus in stool. Mucus in stool can indicate inflammatory responses and our finding could indicate that chronic infection may induce inflammatory changes without major clinical symptoms. However, a longitudinal prospective study comparing infected and non-infected children with *Giardia* would be necessary in order to establish if chronic *Giardia* infection has an impact on growth or nutritional status in this particular population. In humans, the clinical effects of *Giardia* infection range from the asymptomatic carrier state to a severe malabsorption syndrome \[[@B7]\]. Factors possibly contributing to the variation in clinical manifestations include the virulence of the *Giardia* strain, the number of cysts ingested, the age of the host, and the state of the host immune system at the time of infection.

Giardiasis was most prevalent in children who drank municipal water. *Giardia* survives in chlorinated water \[[@B8]\]. To protect against transmission all drinking water should receive chemical pretreatment, preferably with sedimentation, and filtration in addition to disinfection \[[@B9]\]. Municipal water should supply water with a concentration of less than 0.7--70 cysts per 100 liters \[[@B10]\]. There are two previous reports of outbreaks of giardiasis after natural disasters, both linked to flooding after heavy water run-off. One occurred in Montana (United States) where an outbreak of gastrointestinal illness affected 780 persons \[[@B11]\]. In Utah (United States) 1,230 people were affected with diarrhea \[[@B12]\]. Water systems providing unfiltered surface water and contaminated with flooding were identified as the origin of both epidemics. In Armenia the earthquake damaged the municipal system of water ducts \[[@B2]\]; this probably induced their contamination, explaining the high rate of giardiasis that we found in children. It is interesting to note that individual tanks were filled with municipal water. One explanation for the lower prevalence of giardiasis in children that used individual tanks is that in tanks there is additional sedimentation that can reduce the contamination of water with *Giardia*\[[@B10]\]. Health municipal services also distributed chlorine to be used in individual tanks. Although we could not establish the extent to which this chlorine was used, it is possible that additional chlorination contributed to reduce the probability of *Giardia* infection. There are no data available on the prevalence of giardiasis in general population in Armenia. It will be necessary to begin studies in order to establish the public health impact of this parasite infection in the city.

Conclusions
===========

Giardiasis was the most prevalent intestinal parasite found in children living in post-earthquake camps in Armenia, Colombia. Our results suggest that this is an emerging disease in post-disaster situations. However, data on prevalence of giardiasis before the earthquake and in population living away from camps is lacking. The use of communal toilets instead of individual household toilets was found to be an epidemiological risk factor associated to giardiasis. Adequate prevention measures which include efforts to make individual toilets available are also important to prevent dissemination of this parasite. The present study also found a relationship with the water provided by municipal closed ducts that were affected by the earthquake. This highlights the need for implementing adequate surveillance systems for the presence of *Giardia* in water sources in similar circumstances.

Competing interests
===================

None declared

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

<>

Acknowledgements
================

We thank Dr. Santiago Nichols from the \"Instituto Nacional de Salud\" at Bogota, who help us checking english spelling of this paper. This work was financed by research funds of the University of Quindio, a public Colombian University.