Monitoring of platinum surface contamination in seven Dutch hospital pharmacies using inductively coupled plasma mass spectrometry

Objective: To develop, validate, and apply a method for the determination of platinum contamination, originating from cisplatinum, oxaliplatinum, and carboplatinum.
Introduction
Cytotoxic drugs are widely used for the treatment of cancer. Occupational exposure to these drugs has been recognised as a potential health hazard since 1970s (Donner 1978; Falck et al. 1979). Because cytotoxic drugs can affect the DNA, RNA, or protein synthesis, many of these drugs are classified as being carcinogenic, mutagenic, or teratogenic to humans (International Agency for Research on Cancer (IARC) 1997). Skin contact with cytotoxic drugs, due to contamination of the work area or contamination of packaging material, seems to play an important role in the uptake of these drugs by hospital personnel (Fransman et al. 2004; Sessink et al. 1994). Therefore, strict health and safety rules have been established and applied for the handling of these agents. Evidently, the potential health risks for persons manipulating cytotoxic drugs, such as pharmacists, pharmacy technicians, nurses, and cleaners, however, still are a concern. This concern is consolidated by a number of recent publications demonstrating workplace contamination (Crauste-Manciet et al. 2005; Hedmer et al. 2005; Leboucher et al. 2002; Mason et al. 2005; Schmaus et al. 2002; Zeedijk et al. 2005; Ziegler et al. 2002) and contamination of packaging of cytotoxic drugs (Connor et al. 2005; Hedmer et al. 2005; Mason et al. 2003; Nygren et al. 2002). Moreover, detection of cytotoxic agents in urine (Ensslin et al. 1994a, b, 1997; Minoia et al. 1998; Pethran et al. 2003; Schreiber et al. 2003; Sessink et al. 1994; Turci et al. 2002) and blood (Nygren and Lundgren 1997) of personnel who were involved in preparation or administration has been reported with increasing frequency.
The relationship between prolonged exposure to small quantities of cytotoxic drugs and harmful effects is difficult to establish. Based on current scientific knowledge, it is impossible to set a level of exposure that, beyond doubt, will not cause adverse effects. Because no regulations on the maximal acceptable amount of contamination for these drugs have been set so far, hospitals should aim for the lowest contamination as is reasonably achievable. Monitoring of contamination, therefore, is essential. This can aid in the identification of the main exposure routes and in assessing the effectiveness of cleaning and working procedures. Evaluation of environmental contamination will, moreover, lead to an increase of the consciousness among personnel, concerning the handling of the chemotherapeutic agents. This can lead to an improvement of and the compliance to working and cleaning procedures. Wipe sampling is a common method to monitor surfaces for the presence of cytotoxic drugs. Hence, sensitive and validated methods are indispensable to be able to detect the relatively low quantities of drug present on surfaces.
Platinum coordination complexes, such as cisplatinum, oxaliplatinum, and carboplatinum play a major role in the treatment of a variety of tumours. As a result, large amounts of these agents are processed in hospital pharmacies. Several wipe sample methods for platinum containing drugs have been used in earlier studies and platinum was detected as a surface contaminant in many of the workplaces (Leboucher et al. 2002; Mason et al. 2005; Schmaus et al. 2002; Ziegler et al. 2002) or drug vials (Connor et al. 2005; Nygren et al. 2002) investigated. A description of the validation of the analytical methods, however, has been scarce. Validation results were mentioned briefly for the method of Ziegler et al. (2002), using electro thermal vaporisation coupled to inductively coupled plasma mass spectrometry (ICP-MS). Raghavan et al. (2000) described the validation of a high-performance liquid chromatography method for the determination of cisplatinum in cleaning validation samples. The limit of quantification of this method, was 500 ng l−1, which is high compared to the limits achievable with for example ICP-MS or voltammetry. Schmaus et al. (2002) reported the validation of a voltammetric method with a limit of quantification of 40 pg of platinum per sample.
In the present study, we describe the development and validation of an ICP-MS method for the evaluation of surface contamination by platinum originating from cisplatinum, oxaliplatinum, and carboplatinum. ICP-MS assures an ultra high sensitivity and specificity and requires relatively simple sample pre-treatment procedures. The validated method has been applied to measure surface contamination in seven Dutch hospital pharmacies.
Experimental
Chemicals
Cisplatinum and carboplatinum reference standards were purchased from Calbiochem (San Diego, CA, USA). Oxaliplatinum was obtained from Sigma-Aldrich (St Louis, MO, USA). Chloroplatinic acid, containing 1,000 mg l−1 platinum in 3.3% HCl, used for preparation of calibration solutions, was obtained from Inorganic Ventures/IV Labs (Lakewood, NJ, USA). Iridium chloride, containing 1,000 mg l−1 iridium in 3.3% HCl, used for internal standardisation, was also purchased from Inorganic Ventures/IV Labs. Nitric acid (HNO3) 70% and hydrochloric acid (HCl) 35% Ultrex II ultrapure reagents were obtained from Mallinckrodt Baker (Philipsburg, NJ, USA). Water used for the ICP-MS analysis was sterile water for irrigation (Aqua B. Braun Medical, Melsungen, Germany). Ethanol 80% was purchased from Fresenius Kabi (Den Bosch, The Netherlands). A multi-element solution containing 10 mg l−1 of Ba, Be, Ce, Co, In, Mg, Pb, Th, Tl (VAR-TS-MS) was purchased from Inorganic Ventures/IV Labs. Hoek Loos (Schiedam, The Netherlands) provided argon gas (4.6) with 99.996% purity.
Instrumentation
Analyses were performed on an ICP-quadrupole-MS (Varian 810-MS) equipped with a 90° reflecting ion mirror (Varian, Mulgrave, Victoria, Australia). The sample introduction system consisted of a Micromist glass low flow nebuliser (sample uptake 0.4 ml min−1), a peltier-cooled (4°C) double pass glass spray chamber and a quartz torch. The spray chamber was cooled to reduce the vapour loading on the plasma, increasing the available energy for atomisation and ionisation of the elements of interest. Sample transport from the SPS-3 autosampler (Varian) to the nebuliser was performed using a peristaltic pump (Watson-Marlow Alitea, Stockholm, Sweden). The instrument was cooled by using a Kühlmobil 142 VD (Van der Heijden, Dörentrup, Germany). Data were acquired and processed using the ICP-MS Expert Software version 1.1 b49 (Varian). Further data handling was performed using Excel 2000 (Microsoft, Redmond, WA, USA). All measurements were carried out in a dedicated temperature-controlled, positively pressurised environment in order to maintain optimum instrument performance and minimise exogenous contamination. All solutions were prepared using pipettes (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ, USA) and polypropylene tubes 10 ml (Plastiques-Gosselin, Hazebrouck Cedex, France) and 30 ml (Sarstedt AG&Co, Nümbrecht, Germany). Filters (Minisart) used for filtration of wipe samples were obtained from Sartorius (Hannover, Germany). Prior to method development, tubes were checked thoroughly for platinum, iridium, and hafnium contamination and appeared to be suitable for platinum analyses.
Determination of platinum by ICP-MS
To optimise the ICP-MS signal for the high masses and to reduce the formation of oxides, a solution containing 1,000 ng l−1 of Th, In, Ce, Ba and Pt was used. Typically this 1,000 ng l−1 solution gave readings of 115In, 7 × 105 c s−1; 232Th, 1 × 106 c s−1 and 194Pt, 2 × 105 c s−1. The production of CeO+ was less than 1.0% of the total Ce+ counts. The formation of doubly charged Ba++ was less than 3%. Performance was checked daily.
The platinum isotope used for calculation of platinum concentrations was 194Pt. Iridium was used as internal standard. Detection of platinum can be subject to the interference of hafmium oxides (Lustig et al. 1997). Therefore, hafmium signals were monitored for all samples. The detection mode for all isotopes was based on peak jumping with peak dwell times of 50 ms, 25 scans per replicate, and three replicates per sample. Quantitation was based on the mean concentration of three replicates analysed against a calibration curve using weighted linear regression analysis.
Assay development
The most suitable wipe material, desorption solvent, and wipe solvent were selected using one surface sampling and desorption procedure. This will be described below. Recovery data were assessed for the three most commonly used platinum agents; cisplatinum, oxaliplatinum, and carboplatinum. The different molecular structures and, as a result, the variable physical characteristics, might possibly lead to a variation in absorption and desorption characteristics. Therefore, we decided to evaluate all three compounds instead of choosing one reference compound.
Surface sampling and desorption procedure
Each wipe tissue was moistened with 500 μl wipe solvent. In general, sampling was performed by wiping a defined surface area of 10 cm × 10 cm. However, for surfaces for which it was not possible to take a 10 cm × 10 cm sample, the complete top of the device was sampled and the area was estimated. All wipe samples were collected with a uniform sampling procedure by wiping in three different directions (vertical, horizontal, and diagonal). Wipe samples were stored in 50 ml disposable polypropylene flasks (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ, USA) at −20°C until further processing. Prior to analysis, 10 ml of desorption solvent was added to the sample and flasks were kept in an ultrasonic bath for 60 min. Then, samples were filtered to remove particles which could possibly obstruct the ICP-MS nebuliser, or could interfere with the analysis. Two millilitres of sample were, after addition of iridium as internal standard, introduced directly into the ICP-MS. Samples of locations which were expected to be highly contaminated, were diluted prior to analysis to prevent contamination of the sample introduction system of the ICP-MS.
Wipe material
A variety of wipe tissues are available for collecting samples of surface contaminants. These vary in type of material, surface area, and content of platinum contaminants. Three types of wipes were evaluated for this study; Kimtech Science precision wipes (Kimberley-Clark Professional, Irving, TX, USA), Whatman glass fibre filters (Schleicher&Schuell Microscience GmbH, Dassel, Germany), and Klinion non-woven gauzes (Medeco, Oud-Beijerland, The Netherlands). The tissues were checked for platinum contamination and for their ability to release platinum from stainless steel surfaces.
Desorption solvent
One percent HNO3 (v/v), 5% HNO3 (v/v), and 1% HCl (v/v) were evaluated as desorption solutions. Kimtech Science precision wipes were spiked with cisplatinum, oxaliplatinum, and carboplatinum and the platinum recovery was determined after desorption with 10 ml of each solvent.
Wipe solvent
Initially, water, 1% HCl, and 80% ethanol were selected as wiping solutions. To investigate the capability of these solutions to effectively wipe surfaces, 100 cm2 stainless steel surfaces were spiked with cisplatinum, oxaliplatinum, and carboplatinum. These surfaces were subsequently wiped and samples were desorbed using a 1% HCl solution.
Validation procedures
Linearity
For calibration, the chloroplatinic acid reference solution containing 1,000 mg l−1 platinum was diluted with 1% HCl to obtain working solutions with concentrations ranging from 50.0 to 5.00 × 103 ng l−1 platinum. Working solutions were diluted with 1% HCl to obtain calibration standards, ranging from 0.500 to 100 ng l−1 platinum. Before analysis, 15 μl of iridium internal standard solution was added to 1.5 ml of each calibration standard (final internal standard concentration 100 ng l−1). The seven non-zero calibration standards were processed and analysed in singular in three separate analytical runs. The calibrations were back-calculated from the responses. Deviations from the nominal concentration were evaluated.
Recovery and precision
Quality control (QC) samples were prepared to obtain information on the recovery and precision of the desorption method and platinum analysis. These samples were analysed in the validation runs and subsequently also during the analysis of the wipe samples of each hospital pharmacy. Therefore, stock solutions of the platinum agents in water, each containing a concentration of drug equivalent to 400 mg l−1 platinum, were prepared. These stock solutions were further diluted to obtain spiking solutions with concentrations ranging from 10.0 to 2.00 × 103 ng l−1. Tissues were spiked with these solutions serving as QC samples at the following concentration levels; 5.00 × 10−3, 2.5 × 10−2, 0.100, and 1.00 ng platinum on the tissues, corresponding to 0.500, 2.50, 10.0, and 100 ng l−1 platinum in the final solution. These tissues were processed as described earlier. Three replicates of each sample were analysed in three analytical runs. Recovery was expressed as a percentage of the nominal concentration. Within-run and between-run precisions were calculated by analysis of variances (ANOVA) for each test concentration using the analytical run as the grouping variable.
Two of the most important surfaces, stainless steel and linoleum, were used to obtain information on the recovery and within-run and between-run precisions of the complete sampling procedure, including both wiping and desorption. Therefore, 1.00 ng of cisplatinum, oxaliplatinum, or carboplatinum was pipetted in triplicate on a 100 cm2 stainless steel and linoleum surface. After drying overnight, surfaces were wiped following the previously described procedure and analysed using ICP-MS. Three replicates of each sample were analysed in three analytical runs for both surfaces. Recovery was expressed as a percentage of the nominal concentration. Within-run and between-run precision were calculated by ANOVA for each test concentration using the analytical run as the grouping variable.
Sensitivity
The lower limit of quantitation (LLOQ) was defined as the concentration at which the analyte response was at least five times the response of a blank wipe sample. Besides, the LLOQ, when spiked on blank tissues, had to be determined with a precision less than 20% and the mean value should not deviate more than 20% of the actual value.
Stability
Stability of cisplatinum, oxaliplatinum, and carboplatinum spiked to tissues, at two concentration levels, was evaluated at ambient temperatures for 1 week and under storage conditions (−20°C) for up to 3 weeks. From each storage condition two wipe samples were analysed. Samples were considered stable when 80–120% of the initial concentration was recovered.
Platinum determination in pharmacy facilities where no cytotoxic agents are processed
Platinum is an element that not only appears in the environment due to contamination with platinum containing cytotoxic drugs, but also due to pollution by car exhaust catalysts. As a consequence, road dust also contains platinum (Barefoot 1997). Even though, in the pharmacy facilities, precautions (use of slippers/clogs) are taken to reduce the chance of contamination of the facility, road dust contamination might occur. Because ICP-MS does not differentiate between the sources of elemental platinum, this should be taken into account when considering this technique for evaluation of environmental contamination by cytotoxic platinum agents. Therefore, two additional locations were included in this study to set a threshold below which it was not possible to address the source of the contamination. The first location was the laminar-airflow (LAF) hood in a clean room in which no platinum contamination was expected. The second location was a preparation unit with LAF hood of a public pharmacy.
Monitoring of surface contamination in seven Dutch hospital pharmacies
The wipe samples were taken in seven hospital pharmacies in The Netherlands. Characteristics of the facilities are shown in Table 1. The facilities were selected to provide a representation of the diversity in hospital pharmacies in The Netherlands in terms of size and amount of platinum compounds handled. The facilities of each hospital consisted of a preparation room with at least one LAF hood and a room for storage and checking of the prepared drugs and administration purposes. In each facility, samples were taken at locations that were prone to contamination. For good comparison of the results 15 standard locations (Fig. 1) were selected: (1) the middle of the bench-top of the LAF hood, (2) front edge of LAF hood, (3) floor in front of LAF hood, (4) handle of service hatch, (5) door handle, (6) waste bin top, (7) bench-top on which materials are placed in storage/checking room, (8) floor in front of (7), (9) mouse computer, (10) handle telephone, (11) storage shelve of cisplatinum, (12) storage shelve oxaliplatinum, (13) storage shelve carboplatinum, (14) transport box, (15) handle refrigerator. Locations 1, 2, and 3 were sampled in duplicate to get an impression of the overall contamination of these locations. A new pair of gloves was used for each wipe sample. For each facility three blank samples were prepared by moistening tissue with 500 μl water. All samples were stored and processed as described earlier. The storage time from sampling until work-up procedure was less than 2 weeks.
Table 1Amount of platinum agents processed and years that the facilities are in serviceSite1234567Cisplatinum use in 2005 (in g)52.36.8064.014716.829.9104Oxaliplatinum use in 2005 (in g)47.58.6544.110923.498.862.0Carboplatinum use in 2005 (in g)25648.212463556.5223217Total amount of platinum processed in 2005 (in g) 19234.012948352.1185212Number of years in service510182.51.51015Wipe sampling frequencyOnce per year2005 first timeSporadic: last in 2004Once per yearOnce per yearOnce per year Twice per yearFig. 1Sample locations in pharmacy facilities
Wipe sampling was announced in each facility in advance and was performed after the daily cleaning procedure of the LAF hoods, but before the daily cleaning procedure of the rest of the facility. Wipe sampling in all the facilities was performed by the same person.
Results
Assay development
Wipe material
As a result of high platinum backgrounds (10–20 pg platinum per filter depending on the batch analysed), Whatman glass fibre filters were found to be not suitable for platinum wipe sampling. Kimtech Science precision wipes and Klinion non-woven gauzes did not show platinum contamination. However, Kimtech Science precision wipes showed better recoveries of platinum compared to Klinion non-woven gauzes and consequently Kimtech Science precision wipes appeared to be the best choice.
Desorption solvent
The most effective desorption of platinum from the wipe materials was achieved by 1% HCl (94–99%). As a result 1% HCl was selected as the desorption solution of choice.
Wipe solvent
Recoveries were inadequate for 80% ethanol (<40% for all three compounds) and acceptable for water (50–77%) and 1% HCl (63–78%). Because 1% HCl appeared to be corrosive for some types of stainless steel, water was selected as wipe solution.
Validation procedures
Linearity
The calibration curve was best described by linear regression, using 1/(sd of triplicate sample reading) as weight-factor, to avoid bias in favour of samples with high standard deviations. Deviations from the nominal concentration were between −10.0 and 10.2 for all concentration levels. Relative standard deviations for the calibration samples were up to 7.84%. Correlation coefficients were higher than 0.99999.
Recovery and precision
Within-run and between-run precision data for spiked tissues, which served as QCs are summarised in Table 2. Precision data showed that, for all QC concentration levels, the reproducibility of the desorption procedure and platinum analysis was excellent. Recoveries for cisplatinum, oxaliplatinum, and carboplatinum were between 86.7 and 103% for all concentration levels. These results indicated sufficient recovery.
Table 2Within and between-run precision data for quality control samplesAmount of platinum spiked to tissue (in ng)Final platinum concentration (in ng l−1)CisplatinumOxaliplatinumCarboplatinumWithin-runBetween-runWithin-runBetween-runWithin-runBetween-run5.00 × 10−30.5007.75a8.53a8.018.752.50 × 10−22.504.104.051.661.152.39a0.10010.01.357.441.751.272.862.501.001001.077.791.63a0.841.96aNo statistically significant additional value was observed as a result of performing the assay in different runs (mean square within runs is greater than mean square between runs)
For recovery and within-run and between-run precision data from the spiked stainless steel and linoleum surfaces see, respectively, Tables 3 and 4. Precision data showed that the reproducibility of the method, including the wiping procedure was good. Recoveries from the spiked stainless steel surface were 50.4% for cisplatinum, 73.8% for oxaliplatinum, and 77.2% for carboplatinum (Table 3). Recoveries for the linoleum surface were, respectively, 76.8, 77.9, and 81.4% (Table 4).
Table 3Recovery of 1.00 ng platinum from a stainless steel surfaceCisplatinumOxaliplatinumCarboplatinumMean recovery (%)50.473.877.2Within-run precision (in %)2.214.632.53Between-run precision (in %)3.36aaNumber of days333Number of samples per day333aNo statistically significant additional value was observed as a result of performing the assay in different runs (mean square within runs is greater than mean square between runs)Table 4Recovery of 1.00 ng platinum from a linoneum surfaceCisplatinumOxaliplatinumCarboplatinumMean recovery (%)76.877.981.4Within-run precision (in %)3.622.123.35Between-run precision (in %)12.25.416.82Number of days333Number of samples per day333
Sensitivity
The LLOQ of the assay was set at a platinum concentration of 0.5 ng l−1 in 1% HCl, corresponding to 5 pg per wipe sample or 0.05 pg cm−2 taking into account a surface of 10 cm × 10 cm. Signal to noise ratios at the LLOQ level exceeded 5 during all the experiments, which was in accordance with the requirement. The acceptance criteria, that the LLOQ was determined with a precision less than 20% and that the mean value should deviate no more than 20% from the actual value, were met for all three compounds (Table 2).
Stability
Stability has now been established up to 3 weeks, but further testing is still ongoing. Sample storage at room temperature for 1 week was not possible. Tissues which were spiked with cisplatinum showed a decrease in platinum levels of 30% after 1 week. Oxaliplatinum and carboplatinum spiked tissues did not reduce under these conditions. Sample storage at −20°C was possible for at least 3 weeks. Platinum concentrations of cisplatinum spiked tissues were decreasing more obvious with time than oxaliplatinum and carboplatinum spiked tissues. However, no decrease of more than 20% of the initial concentration was observed after 3 weeks at −20°C.
Platinum determination in pharmacy facilities where no cytotoxic agents are processed
No platinum was detected in wipe samples from the LAF hood of the public pharmacy. Platinum levels of the LAF hood in the clean room and the floor in the public pharmacy ranged between 0.430 and 0.922 ng l−1 (or 0.0430–0.0922 pg cm−2). Therefore, it was recommended to set a threshold of 1.00 ng l−1 platinum (0.100 pg cm−2 when wiping a surface of 100 cm2), below which it was not possible to address the source of the contamination. All surfaces in the preparation units with platinum levels above this threshold were considered as being contaminated by platinum containing drugs.
Monitoring surface contamination in seven Dutch hospital pharmacies
In February 2006, wipe samples were collected from seven Dutch hospital pharmacies with centralised units dedicated to the preparation of intravenous mixtures of cytotoxic drugs. The amount of platinum which was processed in these facilities ranged from 34.0 to 483 g per year (Table 1). Surface contamination of all sample locations is depicted in Table 5 in pg cm−2. It is important to consider that recoveries of the samples, as assessed in the validation study, deviate from 100% dependent on the type of surface sampled and on the type of compounds present on the surface. Therefore, results depicted in Table 5 represent ≥50.4% of the actual contamination present on the surface.
Table 5Platinum contamination in seven Dutch hospital pharmaciesS.no.Sampled surfacePlatinum contamination (in pg cm−2)Site12345671Middle of bench LAF hood0.221800.5432.70.3607.222.94Duplicate of 10.1891240.64518.70.3288.222.282Front edge of LAF hood–a3563.3299.50.13328.25.12Duplicate of 2–a2688.34180–a37.05.193Floor in front of LAF hood3.141738241,1070.2282.4821.7Duplicate of 33.202327282,2110.1861.9112.54Handle of service hatch–a22.7–a2,055–a1.9611.85Door handle–a3.17–a16.8–a21.416.16Waste bin top–a1.020.39210.1–a7.380.098c7Bench-top on which materials are placed0.8290.9490.29890.60.2020.37563.48Floor in front of bench0.105c19.758.938.1–a0.311c11.99Mouse computer0.2520.8161.3410.2–b0.7585.4110Handle telephone–a3.220.5912.1–a3.065.1211Storage shelve cisplatinum0.176c1.140.157c4.760.5364.0433612Storage shelve oxaliplatinum0.3680.9160.141c4.1082.71.152.2113Storage shelve carboplatinum3.251.530.1473.130.1860.9895,76014Transport box74.5–a0.2854.44–a0.828–b15Handle refrigerator0.45226.31.4636.00.9481.425.71aRecovered platinum concentrations were below the thresholdbDevice was not present of available for wipe samplingcHafnium oxide might have accounted for up to 20% of the platinum content
Platinum was detected in 94% of the wipe samples and 88% of the samples contained levels above the threshold set. Six of the 126 samples showed raised hafmium signals which, considering a maximum oxide formation of 1%, might have accounted for up to 20% of the platinum signals of these samples. None of blank samples prepared for each facility by moistening tissues with wipe solvent, contained levels of platinum exceeding 20% of the LLOQ standard. The variation in the level of contamination between pharmacies was high. Pharmacies of site 1 and 5 showed overall low platinum contaminations. For these sites, respectively 33 and 39% did not contain platinum levels above the threshold set. Platinum levels detected at pharmacy 3 were relatively low as well, although the wipes taken from the floor were high at this site. These high values were, most probably, the result of a calamity in 2005 with a cisplatinum infusion mixture, which was spilled on the floor. Most locations wiped at the hospital pharmacies of site 2, 4, 6, and 7 showed high contaminations. Only one sample from these sites did not contain any detectable platinum. The high contamination of site 2 seemed to run counter to the quantities of drugs handled, because in this pharmacy relatively low amounts of platinum were processed. This site, however, was occasionally used, for preparation of larger amounts of cytotoxic drugs to serve another hospital. Therefore, the amount of drugs processed in 2005, was not fully representative for the amount of drugs processed in the 10 years that this site was in use. Site 4 showed the highest contamination, which paralleled the relative amount of drug handled in this unit.
As expected, platinum was found in most wipe samples taken from the middle of the LAF hood bench. Notable was that, in general, wipe samples of the front edge of the LAF hood were more contaminated than samples taken from the bench-top of the LAF hood. Furthermore, floor samples usually contained the highest platinum levels. Other locations showing substantial contamination were storage shelves, door handles, and handles of service hatches. Duplicate samples of locations 1, 2, and 3 showed similar results, indicating a homogeneous distribution over de surface area.
The number of years that the seven units were in use, did not parallel contamination levels and the amount of drug handled in 2005, overall, did not predict the level of contamination either.
Discussion
The presence of cytotoxic drug contamination in hospital pharmacies is recognized as a potential health risk. Therefore, it is important to monitor this contamination. Because platinum coordination complexes belong to the most extensively used anticancer agents, it is relevant to focus on the occupational exposure of these drugs. The rationale for evaluation of platinum contamination is also illustrated by several studies showing increased levels of platinum in blood (Nygren and Lundgren 1997) and urine (Ensslin et al. 1994a, 1997; Nygren and Lundgren 1997; Pethran et al. 2003; Schreiber et al. 2003; Turci et al. 2002) of hospital personnel working with these agents.
To be able to accurately assess the platinum contamination originating from cisplatinum, oxaliplatinum, and carboplatinum at different locations, we developed and validated a wipe sampling method. ICP-MS was used for quantification of platinum, because this technique assures a high sensitivity and relative simple sample pre-treatment. The sensitivity of the method was excellent. The LLOQ was set at a platinum concentration of 0.5 ng l−1, corresponding to 5 pg per sample or 0.05 pg cm−2 when wiping a surface of 100 cm2. To our best knowledge, the method described here is 2–300 times more sensitive than other methods described for determination of platinum in wipe samples (Connor et al. 2005; Mason et al. 2003, 2005; Raghavan et al. 2000; Schmaus et al. 2002; Ziegler et al. 2002).
Sample pre-treatment only involved surface sampling, desorption, and filtration. After filtration, samples could be analysed immediately. During method development it was shown that, in addition to tissue material and desorption solvent, also the wipe solvent affected the recovery to a considerable extent. This was in contrast with results described by Turci et al. (2003), who mentioned that the type of wipe solvent would not influence the recovery, since contaminants would be wept away from the surfaces independent of the composition or the pH of the solution itself. Best recoveries were achieved by wiping with Kimtech Science precision wipes moistened with 500 μl water and subsequent desorption with 1% HCl.
Validation of the method was performed for the three, in oncology most prominently used platinum agents, cisplatinum, oxaliplatinum, and carboplatinum. We decided not to choose one reference compound, because the different molecular structures of the platinum agents and, as a result, the variable physical characteristics, might possibly lead to a variation in absorption and desorption characteristics. Excellent reproducibility (imprecision up to 8.75%) and recoveries (86.7–103%) were demonstrated with spiked tissues, for all concentration levels and compounds. Up to 13.3% of the initial amount of platinum added to the tissues was not recovered after desorption and analysis. This could be due to variation in analysis, as well as loss due to adsorption to the tissues. Recoveries from the spiked stainless steel surface were 50.4% for cisplatinum, 73.8% for oxaliplatinum, and 77.2% for carboplatinum. Recoveries, for the three compounds, from the linoleum surface were, respectively, 76.8, 77.9, and 81.4%. These results showed that for stainless steel, depending on the compound analysed, up to 49.6% of the initial amount of platinum spiked to the surface was lost. This was, for the greater part, caused by the inability of the wipe procedure to remove all the added platinum and, for a minor part, by the variation in analysis and loss due to adsorption to the tissues. The lower recovery that was observed for cisplatinum, is, most probably, a consequence of its superior reactivity compared to oxaliplatinum and carboplatinum. This might lead to a stronger binding affinity of cisplatinum to materials and surfaces. For linoleum up to 23.2% of the initial amount of platinum spiked to the surface was not recovered. For this surface, recoveries were similar for all compounds.
To evaluate the stability of spiked samples, recoveries were assessed after storage at room temperature and −20°C. Storage of spiked tissues at −20°C for at least 3 weeks was possible. However, storage of tissues spiked with cisplatinum for 1 week at room temperature led to considerable decrease in platinum levels. Even though platinum concentrations from oxaliplatinum and carboplatinum spiked tissues did not reduce under these conditions, storage at room temperature was not recommended, also because the source of elemental platinum is not known in wipe samples performed in pharmacies. Differences in recovery of the cisplatinum and the oxaliplatinum and carboplatinum spiked samples, again, could be explained by the higher reactivity of cisplatinum.
For a correct interpretation of surface sampling results, it is relevant to take into account that platinum is an element that not only appears in the environment due to contamination with platinum containing cytotoxic drugs, but also due to pollution by car exhaust catalysts. By wipe sampling two locations where no cytotoxic drugs were handled, we, therefore, determined that below a threshold of 1.00 ng l−1 platinum (0.100 pg cm−2 when wiping a surface of 100 cm2), it was not possible to address the source of contamination. All surfaces in the preparation units with platinum levels above the threshold were considered as being contaminated by platinum containing drugs.
Taking this threshold into consideration, platinum contamination was reported in 88% of the samples taken in the seven Dutch hospital pharmacies. It is important to consider that recoveries of the samples, as assessed in the validation study, deviate from 100% dependent on the type of surface sampled and on the type of compounds present on the surface. Therefore, results depicted in Table 5 represent ≥50.4 of the actual contamination present on the surface.
The results of this study indicate that there is substantial variation in surface contamination of the pharmacies tested and that the amount of platinum processed in the pharmacies did not always parallel the level of contamination. The number of preparations with platinum drugs was, however, not assessed and might also be related to surface contamination. This suggests that variation in the application of or compliance to cleaning and working procedures and the incidence of calamities, rather than the amount of platinum processed, caused variation in surface contamination.
In general, results reveal that the LAF hoods, the floor in front of the LAF hoods, door handles, and handles of service hatches were often contaminated. This demonstrates that contamination is often spread throughout the pharmacy. Notable was that wipe samples of the front edge of the LAF hood were more contaminated than samples from the bench-top of the LAF hood. This is thought to be due to incorrect application of working procedures or insufficient cleaning.
By taking duplicate wipe samples of the LAF hood, the front edge of the LAF hood, and the floor, we demonstrated that these locations were overall contaminated and that contamination did not appear to be spotty as was mentioned by Zeedijk et al. (2005).
We also investigated storage shelves and, in most pharmacies, considerable platinum contamination was found. These elevated levels could be a consequence of elevated levels of platinum on packaging material (Connor et al. 2005; Mason et al. 2003; Nygren et al. 2002). Contamination of packaging can lead to a spread of cytotoxic drugs to locations where the drugs are stored or processed. Therefore, it was not surprising that the storage shelves of platinum agents were contaminated. Furthermore, it was noticed that in at least one of the hospital pharmacies (at site 4), secondary packaging and caps of the vials were discarded onto the floor during preparation, most probably to prevent the packaging from interfering with preparation activities. This pharmacy indeed showed considerable contamination of the floor.
In our study, surfaces showed platinum contamination of up to 5,760 pg cm−2. The results are in the same range as findings of some other studies describing surface contamination of platinum in hospital pharmacies (Leboucher et al. 2002; Schmaus et al. 2002). In the study of Leboucher et al. (2002), however, no platinum was found outside the LAF hood, which could be due to the high detection limit of the atomic absorption spectrometry method used (10 μg l−1). Schmaus et al. (2002) performed a study in 14 hospital pharmacies and all samples tested positive for platinum, even though the LLOQ of their method was eight times higher than of the method described here. Yet, although in our study not all samples tested positive for platinum, the highest contamination found (5,760 pg cm−2) was comparable to the highest contamination found by Schmaus et al. (2,700 pg cm−2). Mason et al. (2005) showed lower contamination levels than found in our study, despite that the amount of drug handled in these pharmacies was higher than in the pharmacies in our study.
When comparing the amounts of platinum detected on different locations in this study (up to 0.576 μg per wipe sample) with the platinum content of one vial (between 6.50 and 237 mg platinum), contamination seems to be relatively low. Furthermore, the extremely sensitive technique used in this study, leads to a high percentage of positive samples. Interpretation of these results is rather complicated. It is important to consider that the total area of the contamination is large and that pharmacy personnel are at risk to be exposed to the contamination daily. Hence, for safety precautions, it is recommended to attempt to achieve the lowest possible contamination. Environmental monitoring therefore, may be used to monitor and control contamination and thereby evaluate working and cleaning procedures, rather than to interpret potential health risks.
In general, when a minimal contamination level is desired, the results of this study demonstrate that cleaning and working procedures do not sufficiently prevent contamination in most hospitals. This could be due to an inadequate compliance of personnel to these procedures. Moreover, contamination can spread out unconsciously by hands or feet of the personnel. It is also likely that cleaning procedures as applied in the different pharmacies are not fully optimised and validated, leading to contamination due to sub-optimal cleaning. With respect to the physical properties of the different cytotoxic drugs, it is recommended to consider cleaning techniques appropriate for specific agents. As was shown in this study for platinum, for instance, 80% ethanol did not effectively remove platinum from a stainless steel surface. Although water gave better recoveries for platinum, it was not capable of removing all of the added platinum from the stainless steel surface. This illustrates the importance to evaluate several cleaning procedures for the different cytotoxic agents handled and to optimise a procedure which does remove all drugs with acceptable recoveries.
In conclusion we developed and validated an ultra sensitive and reliable ICP-MS method for the determination of platinum in surface samples. This method was successfully applied in the evaluation of platinum contamination in the preparation units of seven Dutch hospital pharmacies. It was demonstrated that pharmacy personnel is at risk to be exposed to platinum, despite the use of cleaning and safety procedures. As long as the consequences of long-term exposure are not known, the aim should be to achieve a contamination levels as low as possible. This study, therefore, highlights the need to further evaluate cleaning and safety procedures. Wipe sampling can be applied to quantify improvements made through changes in procedures.

platinum
surface contamination
hospital pharmacies
validation
icp-ms


Spider mite (Acari: Tetranychidae) mitochondrial COI phylogeny reviewed: host plant relationships, phylogeography, reproductive parasites and barcoding

The past 15 years have witnessed a number of molecular studies that aimed to resolve issues of species delineation and phylogeny of mites in the family Tetranychidae. The central part of the mitochondrial COI region has frequently been used for investigating intra- and interspecific variation. All these studies combined yield an extensive database of sequence information of the family Tetranychidae. We assembled this information in a single alignment and performed an overall phylogenetic analysis. The resulting phylogeny shows that important patterns have been overlooked in previous studies, whereas others disappear. It also reveals that mistakes were made in submitting the data to GenBank, which further disturbed interpretation of the data. Our total analysis clearly shows three clades that most likely correspond to the species T. urticae, T. kanzawai and T. truncatus. Intraspecific variation is very high, possibly due to selective sweeps caused by reproductive parasites. We found no evidence for host plant associations and phylogeographic patterns in T. urticae are absent. Finally we evaluate the application of DNA barcoding.
Introduction
Species identification is the basis for understanding species diversity, phylogenetic patterns, and evolutionary processes. Only correct identifications allow for comparisons between studies and the repetition or expansion of earlier experiments. In pest species, species identification is also important in the development of (biological) pest control strategies.
Identification and delineation of species within the Tetranychidae has been an issue of debate for the past few decades. Within the family about 1200 different species are described, many of which are of agronomical importance (Bolland et al. 1998). The genus Tetranychus is well studied and includes two common major agricultural pest species with a worldwide distribution: Tetranychus urticae Koch, 1836 and Tetranychus kanzawai Kishida, 1927. Morphological identification of tetranychid species is difficult. The number of potential diagnostic characters is limited (partly due to the small size of the mites) and key traits often exhibit large phenotypic plasticity. As a result, many species cannot be distinguished on the basis of external morphology. For example, in Japan 10 Tetranychus species are recognized (Ehara 1999). However, morphological identification using adult females is possible for only two of the species. The remaining eight species can only be identified by microscopic examination of the shape of the aedeagus (part of the male genitalia). Another example that shows our inability to identify species on the basis of morphology is the well-studied two-spotted spider mite species T. urticae. This species is considered a species complex (Navajas et al. 1998) and as many as 44 synonymous names are known (Bolland et al. 1998). The question whether red T. urticae mites should be considered a separate species (T. cinnabarinus) has occupied taxonomists for many years (Dupont 1979; Gotoh and Tokioka 1996; Zhang and Jacobson 2000). The fact that there are only few taxonomists specialized in morphological identification of mites and that their number is decreasing adds to the problem of spider mite identification.
DNA sequences are currently an indispensable tool for delineating and identifying species. In this context it is important to distinguish between DNA taxonomy and DNA barcoding. DNA taxonomy concerns the circumscription and delineation of species using evolutionary species concepts (Vogler and Monaghan 2007). DNA barcoding aims at the identification of pre-defined species and does not address the issue of species delineation per se (Monaghan et al. 2005). In DNA barcoding a short standardized DNA sequence, usually the 5′ end part of the mitochondrial cytochrome c oxidase subunit I (COI) gene, is used to identify species. DNA barcoding can be used to (i) identify and assign unknown specimens to species that have been previously described and (ii) enhance the discovery of new species using a threshold of sequence divergence (Hebert et al. 2003; Moritz and Cicero 2004). DNA taxonomy may be based on one or several mitochondrial as well as nuclear DNA regions and can serve as a database for DNA barcoding. DNA taxonomy is an offshoot of phylogenetics, in which the evolutionary relationships between taxa (e.g., species) are investigated. Throughout this paper we use the term ‘species delineation’ when it concerns DNA taxonomy and ‘species identification’ when it concerns DNA barcoding.
The usefulness of the COI region for delineating tetranychid species has been investigated in several studies (Hinomoto et al. 2001; Hinomoto and Takafuji 2001; Lee et al. 1999; Navajas et al. 1994, 1996a, 1996b, 1998; Toda et al. 2000; Xie et al. 2006a). Recently, a DNA barcoding approach was used to identify tetranychid species (Hinomoto et al. 2007). Each of these studies used a different or sometimes partially overlapping subset of tetranychid sequences. Many studies extended their dataset with one or several tetranychid sequences from the GenBank database, serving as a reference for phylogeny reconstruction or species identification. However, sequence diversity within T. urticae is substantial (e.g., Navajas et al. 1998) so that different T. urticae sequences are available from GenBank, some of which bear an incorrect species name (due to misidentification). As a consequence, different T. urticae reference sequences were used in above-mentioned studies, leading to the emergence of variable taxonomic groupings and phylogenetic patterns. This, combined with the analyses of restricted subsets in each study, gives an incomplete and fragmented view of species delineations and phylogenetic relationships within the family Tetranychidae.
In this study we create an extensive COI dataset of the family Tetranychidae, with a wide coverage of the species T. kanzawai and T. urticae (including T. cinnabarinus, which is currently considered synonymous to T. urticae). We have collected all currently available mitochondrial COI sequences from GenBank and added data on mites collected from Europe and North America. We critically evaluate the assembled data and perform an overall phylogenetic analysis. This approach reveals novel patterns on species delineation and phylogenetic relationships. We discuss the use of COI for DNA barcoding purposes by considering the intra- and interspecific variation. In addition, we discuss the observed variation in COI in relation to associated host plant, phylogeographic patterns and the presence of endosymbionts (e.g., Wolbachia, Cardinium). Finally, we provide guidelines for future phylogenetic studies on (tetranychid) mites.
Material and methods
Additional tetranychid samples
Tetranychid mites were collected in Europe (six locations), in North America (one location), and from two cultures maintained in our lab for 10 years (Table 1). Mites were not identified morphologically to the species level. DNA was extracted from single individuals using a modified CTAB extraction method (Doyle 1991). A single adult female was ground in 5 μl of proteinase K (20 mg/ml) and 100 μl CTAB (2% CTAB w/v in 100 mM Tris-HCl [pH8], 20 mM EDTA, and 1.42 M NaCl) buffer was added. After vortexing, samples were incubated at 55°C for 1 h. Next, 100 μl chloroform: isoamylalcohol (24:1) was added and contents were gently mixed for 2 min. Tubes were centrifuged for 10 min. at 15,800 g. After centrifugation, 80 μl of the supernatant was transferred to a clean tube and DNA was precipitated by adding 200 μl ice-cold 96% ethanol. Tubes were incubated at −20°C for at least 1 h prior to centrifugation at 15,800 g for 15 min at 4°C. The supernatant was removed and the DNA pellet was washed with 70% ethanol. Next, the DNA was air dried for at least 15 min, eluted in 30 μl sterile water, and stored at −20°C.
Table 1Overview of samples sequenced in this study and the primer sequences used for COI amplificationStrainCountryLocalityHost plantCollection date PrimerbCommon nameScientific nameFRNL1NetherlandsCastricumEuropean SpindleEuonymus europaeusSep-0612NL2NetherlandsCastricumEuropean HoneysuckleLonicera periclymenumSep-0612F1FranceVireuxBlackthornPrunus spinosaJul-0612T1UnknownUnknownCucumberaCucumis sativusa12T2NetherlandsAalsmeer (greenhouse)RoseaRosa spec.a12US1United StatesTucson (AZ)UnknownUnknownMay-0534P1PortugalCaldas de MonchiqueCitrusCitrus spec.Feb-0534S1SpainMont-roig del campOrangeCitrus spec.Apr-0434PL1PolandRabkowaPlumbPrunus spec.Aug-0534aMaintained in the lab on bean (Phaseolus vulgaris) for over 10 yearsb1 = GGAGGATTTGGAAATTGATTAGTTCC (Navajas and Boursot 2003); 2 = AAWCCTCTAAAAATRGCRAATACRGC (modified from Hinomoto and Takafuji (2001); 3 = TGATTTTTTGGTCACCCAGAAG (Navajas et al. 1994); 4  = TACAGCTCCTATAGATAAAAC (Navajas et al. 1994)
Part of the mitochondrial COI gene was amplified using various primer combinations (Table 1). Depending on the primer combination, this yielded a fragment size of 410–863 basepairs (bp), excluding the primer annealing sites (Fig. 1). PCR was performed in a 25 μl reaction mix containing 2.5 μl 10X Super Taq buffer (HT BioTechnology, Cambridge, U.K.), 1.25 μl bovine serum albumin (10 mg/ml), 1.25 μl MgCl2 (25 mM), 5 μl dNTP mix (1 mM of each nucleotide), 0.2 μl of each primer (20 μM each), 0.2 μl of super Taq (5 u/ μl) (HT BioTechnology), 11.9 μl water and 2.5 μl of DNA extract. PCR cycling conditions were 4 min. at 94°C, followed by 35 cycles of 1 min at 94°C, 1 min at 48°C and 1 min at 72°C, and a final extension at 72°C for 4 min. Products (2 μl) were visualized on a 1% agarose gel stained with ethidium bromide in 0.5X TBE buffer (45 mM Tris base, 45 mM boric acid, and 1 mM EDTA, pH 8.0).
Fig. 1Overview of the COI fragments sequenced in different studies and their relative position after alignment. The position of the fragment analyzed in this study and of the standard DNA barcoding fragment (Folmer fragment) on the total mitochondrial COI gene (position 1474–3009) are indicated on top. Base pair numbers correspond to the Drosophila melanogaster mitochondrial DNA sequence (GenBank accession nr. U37541). For each study, thick horizontal lines represent the fragment sequenced in all specimens and thin lines indicate the maximum sequence length. Number of sequences (between parentheses) and references are listed at the right. For references of unpublished studies, see Appendix
PCR products were purified using a DNA extraction kit (Fermentas, St. Leon-Rot, Germany). The purified products were directly sequenced using the ABI PRISM BigDye Terminator Sequence Kit (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands) according to the manufacturer’s instructions but diluted 16 times. Both strands of the products were sequenced using the same primers as used in the PCR amplification. Sequences were run on an ABI 3700 automated DNA sequencer. Obtained sequences were aligned using ClustalX v 1.8.0 (Thompson et al. 1997) and compared to the sequences obtained from GenBank (see below).
Database compilation
Sequence collection
A single database was constructed comprising all available tetranychid COI sequences from GenBank and the sequences obtained in this study. Sequences were collected from GenBank on September 8th, 2006 (keywords for search were ‘cytochrome oxidase subunit I AND Tetranychidae’). This yielded a total of 165 sequences (156 from GenBank, 9 from this study), of which 79 (48%) were published in peer reviewed journals. An overview of all sequences, their GenBank accession numbers, assigned species names, references, sample locations, and associated host plants (if known) is given in the Appendix. Sequences were aligned using ClustalX. Due to the use of different primer combinations in the various studies, the sequences differed in length and in position on the COI region (Fig. 1). A central part of 390 bp was chosen for subsequent analysis (highlighted region in Fig. 1). Considering this central part, 25 sequences were found more than once (see Appendix). Prior to phylogenetic analysis, identical sequences (except one) were removed from the dataset, resulting in 96 unique sequences. In addition, six sequences with accession numbers AF131105–AF131110 (Lee et al. 1999) were excluded from analysis because of too many missing data in the region of overlap (272 bp of the 390 bp part are missing; Fig. 1). The dataset was further adjusted for wrongly submitted sequences (see next paragraph) leading to the addition of one corrected sequence, yielding a final number of 91 aligned COI sequences. Of these, 71 were published in peer reviewed journals and 68 belong to the genus Tetranychus according to the GenBank submission info. A Clustal alignment of the 91 unique sequences can be obtained from the corresponding author upon request.
Data validation: Incongruencies in the database
When compiling the dataset, two discrepancies were encountered between sequence information submitted to GenBank and the description in the associated articles. The first one concerns accession X80860. Its sequence was wrongly submitted to GenBank. In GenBank, accessions X80859 and X80860 are listed as T. neocaledonicus and T. gloveri respectively. According to the associated article, these sequences should differ 10% (Navajas et al. 1996b). However, accessions X80859 and X80860 show identical sequences, both concurring with the sequence T. neocaledonicus from the article. The correct sequence of T. gloveri was obtained from the original article and added to the dataset. The second discrepancy concerns accessions X99873, X99874 and X99875. According to the description in GenBank, accession X99873 was obtained from the Amphitetranychus quercivorus strain Sapporo and accession X99874 from the A. quercivorus strain Tsukuba. Comparing this to sequences in Navajas et al. (1997), the X99873 GenBank sequence concurs with A. quercivorus strain Tsukuba in the article. GenBank sequence X99874 concurs with the sequence of A. viennensis in the article (and therefore is identical to the GenBank sequence of X99875, previously named T. viennensis). This means that the sequences of accessions X99873 and X99874 are different from the article sequences. We included accessions X99873 as A. quercivorus and X99875 as A. viennensis in the dataset. Accession X99874 was excluded from the dataset.
Phylogenetic analysis
PAUP* version 4.0b10 (Swofford 2002) and DAMBE version 4.1.15 (Xia and Xie 2001) were used to calculate numbers of variable sites, uncorrected pairwise divergences, nucleotide composition, and transition and transversion ratios. PAUP was used to perform a chi-square test of base frequency homogeneity across all taxa.
Phylogenetic analyses were conducted in PAUP using Neighbour-Joining (NJ) algorithms (p-distance) and Maximum Likelihood (ML) algorithms (TBR heuristics, random addition sequence with five replicates, reconnection limit of 10). Both PAUP and Modeltest 3.6 (Posada and Crandall 1998) were used to select the optimal evolution model for the ML analysis. The selected model was further optimized by critically evaluating the selected parameters (Swofford and Sullivan 2003) using the Akaike Information Criterion (AIC; Akaike 1974). Because COI is a protein coding gene, we tested if the likelihood of models with the lowest likelihood score could be further improved by incorporating specific rates for each codon position (Shapiro et al. 2006). Under the selected model, parameters and tree topology were optimized using the successive approximations approach (Sullivan et al. 2005). For the NJ analyses robustness of nodes was assessed with 1,000 NJ-bootstrap replicates. For the ML analyses bootstrap support was assessed by performing a NJ bootstrap (1,000 replicates) with distances calculated according to the selected ML model (because of computational constraints).
Phylogenetic analyses were performed for i) the family Tetranychidae and ii) the genus Tetranychus separately. The analysis of the family Tetranychidae included all species. As the species T. kanzawai, T. urticae and T. truncatus are over-represented, eight strains were selected representing these three species (Fig. 4). The dataset for this analysis included 37 sequences. The final tree was rooted using the species Petrobia harti and Bryobia kissophila. These two species belong to a separate subfamily (Bryobiinae) from all other species (subfamily Tetranychinae). The analysis of the genus Tetranychus included 68 sequences. For this analysis, two Panonychus sequences and one Petrobia and one Bryobia sequence were used as an outgroup.
Because saturation of the third codon position is frequently observed for COI (Gleeson et al. 1998; Söller et al. 2001), an analysis excluding this position was performed to see if this improved the resolution of the phylogeny.
Results
Data acquisition: New sequencing
Each COI sequence that we obtained in this study was identical to several sequences already present in the GenBank dataset. Two samples, from the USA (Arizona) and Portugal, were identical to Eutetranychus banksi. The sample from citrus in Spain was identical to Panonychus citri from Japan and the sample from Poland (from Prunus spec.) to P. ulmi from Japan. The remaining samples (the two lab strains and a sample from France) were identical to an Asian T. urticae strain (sampled from Japan, Thailand, and Taiwan) (see Appendix).
Alignment and analysis of patterns of molecular evolution
All sequences could be unambiguously aligned; no insertions or deletions were found. Translation of all sequences into amino acids revealed no stop codons. The total alignment of the 91 tetranychid sequences was 390 bp (minimum sequence length was 304 bp; Fig. 1); 146 sites were phylogenetically informative, 31 sites were variable but uninformative, and 213 sites were constant. On average across all taxa, the AT content was 75% (32%A, 43%T, 11%C, and 14%G). This high AT content is a general feature of the COI region in arthropods, and is comparable to other studies on insect and mite taxa (Lunt et al. 1996; Navajas et al. 1996b). However, the distribution of bias in base composition was not uniform with respect to the three codon positions (Fig. 2). First, second, and third codon positions showed AT biases of 69, 64, and 94% respectively. In some haplotypes, no C or G base was found at the third codon position. Nevertheless, a chi-square test of base frequency homogeneity revealed no significant differences across taxa for the overall data set or for the three base positions separately (Fig. 2). Note that this test ignores correlation due to phylogeny and therefore tends to reject the null hypothesis too easily, so that failure to reject can safely be taken as evidence of homogeneity (Frati et al. 1997).
Fig. 2Base compositions for each codon position of the 390-bp aligned COI region, averaged over all tetranychid samples. Error bars depict minimum to maximum range. Results of the homogeneity test are given for each codon position
The extent of saturation was assessed by plotting the transition and transversion rates against uncorrected p-distance divergences (Fig. 3). At the third codon position, transversions outnumber transitions and the number of transversions begins to plateau (Fig. 3), indicating saturation and making this position unsuitable for resolving more basal branching patterns. However, removing the third codon position from the analysis did not result in a more resolved phylogeny (results not shown). This is probably due to a conserved amino acid sequence (limiting the amount of variation in first and second base pair positions as changes in these positions in most cases change the amino acid sequence).
Fig. 3Saturation plots of transversion and transition rates against uncorrected p-distance at each codon position
The model selected by Modeltest for the tetranychid dataset was the General Time Reversible Model with invariable sites and a gamma distribution of rate heterogeneity (GTR+I+G). However, implementing the rate class ‘a b a b e f’ significantly improved the likelihood (AIC) and was therefore used for parameter and tree topology estimation. For the Tetranychus dataset the General Time Reversible Model (GTR) with site-specific rates for the three coding positions was further optimized by incorporating the following rate class: a b c d e a. This slightly simpler model significantly improved the likelihood (AIC) and was used for parameter and tree topology estimation.
Phylogenetic relationships between tetranychid genera
The ML tree of the overall analysis is shown in Fig. 4. The phylogenetic relationship among the taxa is not well resolved. This is probably due to the strongly biased nucleotide composition and the saturation at the third codon position. It shows that this portion of the COI gene is not suitable for resolving the branching order of the genera and the more distantly related species. P. harti and B. kissophila, both belonging to the subfamily Bryobiinae cluster together, and group outside the subfamily Tetranychinae. There is however no high support for monophyly of any of the genera. The NJ tree shows a similar non-resolved phylogeny (not shown) with the only difference that the genus Eotetranychus appears as a monophyletic group with high bootstrap support (78%). Differences between genera range from 8 to 22% and between species within genera from 1 to 13%.
Fig. 4Maximum likelihood tree of the tetranychid dataset based upon COI sequences. GenBank accession numbers and associated species names are given. If a haplotype is found more than once, the accession number is followed by the haplotype number (see Appendix) and the number of times the haplotype is found between parentheses. Numbers on the branches indicate the percentage bootstrap values (>50) based on NJ bootstrapping with ML settings (1,000 replicates). Bar at the lower left corner depicts the branch length corresponding to 10% maximum likelihood distance
The genus Tetranychus
Just over 50% of the sequences in GenBank are unpublished. Hong, Xie and colleagues have submitted 27 sequences (accessions DQ437542 through DQ437568, submitted March 7, 2006) as T. cinnabarinus. It is unclear why all these accessions were named T. cinnabarinus. The fact that these T. cinnabarinus accessions are scattered all over the phylogeny shows that these sequences do not concern a single species. Besides, the species name T. cinnabarinus is not generally accepted (Dupont 1979; Gotoh and Tokioka 1996), and is also not mentioned in the World Catalogue of the spider mite family (only as synonym of T. urticae) (Bolland et al. 1998). In the remainder of this paper we will not use the name T. cinnabarinus.
For the genus Tetranychus, the ML tree is shown in Fig. 5. The NJ tree shows a similar topology as the ML tree, bootstrap support values are slightly lower in the ML tree (NJ tree not shown). Several clades emerge, although the exact branching order remains unresolved (Fig. 5). The species T. kanzawai, T. urticae and presumably T. truncatus (see below) have been widely sampled and intraspecific variation is substantial. These species form a monophyletic group (bootstrap support 65%). The relationship between all other Tetranychus species remains unresolved, except that T. pacificus and T. mcdanieli cluster together (bootstrap support 83%). Two potentially new species are found (DQ437551 and DQ437566). Sequence divergence between these two accessions is 9.2%, which is of the same order as found between other species. Although these accessions are described in GenBank as T. cinnabarinus, this seems incorrect (see above).
Fig. 5Maximum likelihood tree of the genus Tetranychus based upon COI sequences. GenBank accession numbers and associated species name are given (except for T. cinnabarinus, see text). If a haplotype is found more than once, the accession number is followed by the haplotype number (see Appendix) and the number of times the haplotype is found between parentheses. Accessions deposited on GenBank as T. turkestani are marked in grey. Accessions followed by the letter A or B indicate samples belonging to clade A and B respectively, deduced from Navajas (1998) and Navajas et al. (1998). Numbers on the branches indicate the percentage bootstrap values (>50) based on NJ bootstrapping with ML settings (1,000 replicates). Bar at the lower left corner depicts the branch length corresponding to 10% maximum likelihood distance
Tetranychus urticae, Tetranychus kanzawai and Tetranychus truncatus
The species T. urticae and T. kanzawai have been investigated in several different studies and were sampled from all over the world (see Appendix). The analysis of COI variation reveals the existence of very divergent lineages (Fig. 5). Clade 1 contains all T. kanzawai specimens (bootstrap value = 71%). This clade contains two subclades that were previously described by Hinomoto and Takafuji (2001). On the other hand, T. urticae specimens form a highly diverse group in which several well-supported clades are recognized. One clade (clade 3 in Fig. 5) comprises T. urticae specimens all originating from China (bootstrap value = 100%). Hinomoto et al. (2007) renamed this clade T. truncatus. All other T. urticae specimens form a group of highly divergent lineages (clade 2), which fall into several more or less supported subclades. Moreover, within this group several specimens have been identified as T. turkestani, but these do not form a monophyletic group.
Discussion
The phylogenetic analysis of all COI sequences available in GenBank revealed novel patterns, which alter current views on species delineation and phylogeographic patterns in spider mites. In addition, we found that a number of accessions are probably registered under a wrong species name. This may in the past have led to erroneous interpretations of phylogenetic patterns that included these GenBank accessions.
One application of phylogenetic analysis is the identification of natural groupings in phylogenetic trees that represent biological species (DNA taxonomy). Our most inclusive assemblage of data shows new, and previously unnoticed, groups that most likely concern different species (Fig. 5). In particular, the phylogenetic patterns within T. urticae differ from these of previous studies and provide new insights in the evolutionary history of this group. Up to now, two clades within T. urticae were recognized, named clade A and B by Navajas et al. (1998) and Hinomoto et al. (2001) and lineage I and II by Xie et al. (2006a). The latter concluded that lineage I and II were consistent with the two clades A and B. However, this conclusion is not supported by our analysis. Our clade 2 (Fig. 5) contains specimens of lineage II (Xie et al. 2006a) and clade A and B (Navajas et al. 1998). Moreover, clade A and B disappear in our total analysis and new groupings emerge. Clade 3 contains specimens of lineage I. In fact, clade 3 is a well supported clade restricted to China that clusters outside the other T. urticae samples. It is unclear from the study of Xie et al. (2006a) whether mites from clade 3 were morphologically different from other T. urticae samples. Clade 3 presumably represents T. truncatus, as suggested by Hinomoto et al. (2007), based on morphological identification of newly sampled Japanese mites with highly similar COI sequences.
Tetranychus urticae and Tetranychus turkestani
GenBank specimens listed as T. urticae and T. turkestani do not form separate monophyletic clades (Fig. 5). This is in agreement with a study by Navajas and Boursot (2003) that was based on a smaller dataset. Although Navajas and Boursot (2003) were able to separate the two species based on ITS2 sequence, this distinction was based on three diagnostic sites only. Moreover, intraspecific and intra-individual variation within ITS2 was found, which further questions the recognition of two different species. In addition, there are no discrete morphological differences between the two species. Taxonomic identification is based on continuous traits (e.g., the shape of the aedeagus of males) and there is no thorough study describing variation of these traits within and between these species. The current data do not support the maintenance of T. turkestani as a separate species.
Host plant relationships
Host race formation is another evolutionary process studied in spider mites that may explain the diversity in this group of mites. Phylogenetics is one approach to assess spider mite––host plant associations. Most Tetranychus species are reported from many different host plant species. For example, Bolland et al. (1998) described 911 different host plant species for T. urticae, belonging to 121 plant families. We found no correlation between COI divergence and associated host plant species (Fig. 5 and Appendix), similar to what was found by Navajas (1998). Even strains with identical COI haplotypes can be found on very different host plant species. Also the other two relatively well sampled species T. kanzawai and T. truncatus do not show host plant associations.
Phylogeographic patterns
Phylogenetic analysis is also used for determining phylogeographic distribution patterns (Avise 2000). Phylogeographic information is important for assessing historic migration and colonization routes and can also be used for tracing the origin of accidental introductions. For T. kanzawai, two main clades are distinguished originating from eastern Asia (Japan, Taiwan and China), except one sample that originates from Congo. This suggests that T. kanzawai has a mainly eastern Asian distribution. However, Bolland et al. (1998) reported T. kanzawai from all over the world, but it is not known if these samples fall within the clades found so far. Clade 3 (T. truncatus) appears restricted to China (Fig. 5).
Within T. urticae no phylogeographic pattern is apparent with respect to COI variation. Samples form Europe, Asia, and North and South America are scattered over the tree. The phylogeographic patterns previously described by Navajas et al. (1998) completely disappeared. They found an entirely Mediterranean clade (clade A) and a clade of mixed origin (clade B). Because the Mediterranean clade had the highest diversity, they argued that this region served as a source from which other non-Mediterranean regions of the northern hemisphere were recently colonized by a subset of the Mediterranean clades (Navajas et al. 1998; Hinomoto et al. 2001). However, inclusion of all currently available sequences does not support their conclusion. Clade A and B fall apart and moreover, many more clades are found. There are several possible explanations for the absence of clear phylogeographic patterns. First, such patterns may simply not exist. Second, T. urticae is a pest species on many crops and ornamentals and it is likely that the international trade in crops has influenced the distribution of the mites around the world. This will obscure any correlation between geographical location and phylogeny. Finally, selective sweeps can greatly influence phylogenetic patterns (Ballard and Rand 2005). Evidence is accumulating that selective sweeps are often associated with the presence of reproductive parasites such as Wolbachia and Cardinium (Hurst and Jiggins 2005).
Phylogenetic inferences and DNA barcoding
The analyzed COI region shows considerable variation among the tetranychids examined. The diversity within species is especially high with a maximum of 7.2%. This is a mixed blessing: it makes COI suitable for investigating intraspecific variation, but its usefulness for resolving phylogenetic species relationships remains limited. The latter is due to a strongly biased nucleotide composition at the third codon position and consequently saturation at this position. Variation at first and second codon positions is very low. As a result, relationships between taxa are difficult to resolve, especially at the deeper nodes. An extremely high AT content and saturation at the third codon position was also encountered in other studies, for the same COI fragment as used in this study in parasitengona mites and for the adjacent COI region in velvet worms (Onychophora) (Gleeson et al. 1998; Söller et al. 2001).
The COI fragment analyzed in this study is different from the usual DNA barcoding fragment, which is located at the 5′ end side of our fragment (Fig. 1). Because this barcoding fragment is amplified by primers developed by Folmer et al. (1994) it is referred to as the Folmer fragment (Erpenbeck et al. 2006). Substitution patterns may differ between gene partitions and may result in different phylogenetic signals for these partitions (Erpenbeck et al. 2006). To determine whether our fragment had the same phylogenetic signal as the commonly used Folmer fragment we compared the substitution patterns of both fragments. We did not find different substitution patterns when investigating 294–448 bp of the Folmer fragment for samples for which this fragment was available (results not shown). A dataset of 27 unique sequences (representing 46 samples) revealed a highly similar substitution pattern with transversions outnumbering transitions resembling the patterns in Fig. 3. We therefore assume that analyzing the Folmer fragment for tetranychid mites will reveal similar patterns as found in this study.
Currently, the Folmer fragment is widely used as a gene partition for barcoding species (e.g., Gómez et al. 2007; Hebert et al. 2003, 2004), although other fragments have also been proposed (e.g., in plants; Kress et al. 2005). DNA barcoding assumes that genetic distances between species are greater than within species. In that way, clusters of similar sequences represent species, clearly separated from other clusters (species). Hebert et al. (2003) proposed the use of a standard threshold (divergence value) to identify species. Up to now, several studies have reported successful barcoding of species (e.g., Barret and Hebert 2005; Gómez et al. 2007; Hebert et al. 2003, 2004). However, often, intraspecific variation was not at all or not thoroughly investigated, because only one or two individuals per species were analyzed or geographic sampling was restricted (Dasmahapatra and Mallet 2006; Prendini 2005). This may result in significant underestimation of the amount of intraspecific variation. Additionally, interspecific variation might be overestimated if closely related sister taxa are not included in the analysis. Therefore, it is necessary to analyze samples from more than one geographic region and to include closely related sister species. Our study comprises an analysis of three closely related species that were widely sampled and showed extensive amounts of intraspecific variation. Moreover, in several cases intraspecific variation exceeded interspecific variation between the species, as is illustrated by overlapping frequency distributions of intra- and interspecific pairwise p-distances (Fig. 6). For example, differences within T. urticae reach up to 7.2%, exceeding the minimum 3.7% difference between T. urticae and T. kanzawai. Thus the general barcoding assumption that intraspecific variation is smaller than interspecific variation is violated in tetranychids, indicating that simply relying on genetic distances is not sufficient for species identification. This clearly illustrates the importance of including samples of various, geographically different populations for each species, and to include comparisons with sister species, when investigating the efficacy of barcoding. It also shows the need to include phylogenetic information to delineate species groupings, instead of simply relying on sequence divergences (Prendini 2005; Rubinoff et al. 2006). A careful analysis of the DNA phylogeny, preferably in a multi disciplinary approach (including multiple gene data, morphological, ecological or other relevant data), can assist in defining or delimiting species, but the use of single sequences in combination with a threshold seems insufficient to simply identify species.
Fig. 6Histogram of pairwise differences (p-distance) between 91 COI sequences within the family Tetranychidae. Pairwise differences are separated into three categories: 1. between individuals in the same species; 2. between individuals in the same genus (excluding intraspecific differences); 3. between individuals in the same family (excluding intraspecific and intrageneric differences). n = number of pairwise comparisons
Reproductive parasites and selective sweeps
There are a number of additional problems associated with the use of a single mitochondrial gene for barcoding. Hybridization can result in reticulate evolutionary relationships between species and disturb groupings into species based on mtDNA. Selective sweeps of mtDNA can both homogenize or increase mtDNA diversity. The widespread occurrence of reproductive parasites in arthropods can both influence the frequency of hybridization between host species and indirectly cause selective sweeps of mtDNA (Hurst and Jiggins 2005). These parasites can cause homogenization of biological species after hybridization followed by spreading of the intracellular reproductive parasite. The mitochondrial haplotype is dragged along with these parasites resulting in replacement of the original mitochondrial haplotype and reducing mitochondrial diversity. In a recent study, Whitworth et al. (2007) found a lack of species monophyly in the blowfly genus Protocalliphora due to introgressive hybridization associated with Wolbachia infection. On the other hand, the presence of different reproductive parasites co-infecting the same host species may increase the levels of mitochondrial diversity within that host species if each parasite is tightly linked to a different haplotype (Schulenburg et al. 2002). Infection with reproductive parasites may thus increase or decrease mitochondrial diversity and severely influence the patterns of mitochondrial DNA variation.
Intracellular reproductive parasites such as Wolbachia, Cardinium, and Rickettsia, are widespread in tetranychid mites (Breeuwer and Jacobs 1996; Gotoh et al. 2003; Hoy and Jeyaprakash 2005; Xie et al. 2006b). They can cause cytoplasmic incompatibility (CI) and hybrid breakdown in spider mites (Breeuwer 1997; Gotoh et al. 2003, 2006; Vala et al. 2000). It is possible that the COI variation found within and between closely related mite species is a result of selective sweeps caused by infection with reproductive parasites. Variation within species is relatively high. It is not linked to geographical location nor associated with the host plant. An interesting next step would be to investigate the link between haplotype variation within COI and variation in reproductive parasites. We should be especially cautious with the use of mitochondrial genes for delineating and barcoding biological species in light of the presence of reproductive parasites.
Conclusions and recommendations
Accurate species delineation and identification is important for our ability to understand and interpret evolutionary processes and ecological diversity in mites. It is also clear that mites are a difficult group to identify morphologically, as many key traits exhibit large phenotypic plasticity and lack suitable characters for identification. Many ecological, behavioral, genetic and pest-control studies have been conducted on various tetranychid species or strains, without the concurrent storage of voucher specimens, leading to subsequent uncertainty about the identity of the investigated specimens. In such cases, DNA barcoding can be an important and powerful tool to assist in species identification (Will et al. 2005). However, the use of a single (mitochondrial) gene for DNA barcoding or DNA taxonomy seems inappropriate. An integrative approach is needed combining nuclear and mitochondrial genes, morphological characters, and ecological information (and if possible crossing experiments).
A combined analysis of mitochondrial and nuclear markers is commonly used to avoid the problem that gene trees are not necessarily congruent with species trees and for the detection of hybridization. The challenge is to find suitable nuclear markers and robust geographic sampling designs that allow for the assessment of intra- and interspecific variation. Navajas and Fenton (2000) and Cruickshank (2002) have investigated the suitability of various molecular markers, but there is still a need for nuclear markers suitable for distinguishing closely related species. Recently, Sonnenberg et al. (2007) suggested the D1-D2 region of the nuclear 28S rDNA gene as a taxonomic marker. It could complement DNA barcoding studies based on mitochondrial DNA sequences. In addition, molecular testing for reproductive parasites and crossing experiments using isofemale lines should be standard procedure to delineate biological species.

spider mites
tetranychidae
coi
reproductive parasites
barcoding
tetranychus urticae


The Implications of Maternal Overweight and Obesity on the Course of Pregnancy and Birth Outcomes

The increasing prevalence of overweight and obesity among women of childbearing age is a growing public health concern in the United States. The average body mass index (BMI) is increasing among all age categories and women enter pregnancy at higher weights. Women are also more likely to retain gestational weight with each pregnancy. Women who are overweight (BMI 25–30) and obese (BMI ≥30) are at greater risk of adverse reproductive health outcomes compared to women of normal weight status (BMI 19.8–25). This article provides an overview of the complications associated with maternal overweight and obesity including diabetes, pre-eclampsia, c-sections, and birth defects. We present updated information on the weight trends among women. Finally, we present an overview of the prevention studies aimed at adolescents and women prior to pregnancy.
Introduction
The effect of maternal nutritional status prior to pregnancy on birth outcomes is of great public health importance. Epidemiological studies have shown a clear association between maternal pregravid weight and birth outcomes. As a marker of nutritional status, a woman's pregravid body mass index (BMI = kg/m2), if low (<19.8 BMI), may reflect chronic nutritional deficiency whereas a high BMI (>26.1 BMI) reflects an imbalance between energy intake and expenditure, and thus varying degrees of adiposity [1]. The effects of each on birth outcomes differ, with low BMI being associated with intrauterine growth retardation (IUGR), preterm birth, and iron deficiency anemia [2–5]. Whereas, BMI above the normal range of 19.8 to 26.1 (IOM 1990) is associated with a number of adverse reproductive health outcomes. For example, infertility [6] gestational diabetes [7], pregnancy induced hypertension and pre-eclampsia [8], birth defects [9], large for gestational age (LGA) or macrosomia (>4500 g) [10], cesarean sections [11–13], prolonged labor [14], and recently postpartum anemia [15–17] have all been associated with maternal overweight yet the exact mechanisms have not been identified. The purpose of this paper is to describe an overview of the complications associated with maternal overweight and obesity; present updated information on the weight trends among women; and review prevention studies aimed at adolescents and women prior to pregnancy.
Prevalence of overweight and obesity among women of childbearing age
Until recently, most studies on the topic of maternal pregravid weight focused on the lower end of the weight spectrum; however, with the shift in body weight that has occurred globally, more recent research has examined the effect of overweight and obesity on birth outcomes. Obesity world wide (>30 BMI) now exists at a prevalence of 15–20% and accounts for 2–7% of the total health care costs [18]. In the US, the latest NHANES survey (1999–2002) indicate that 26% of non-pregnant women 20–39 years of age are overweight (25–29.9 BMI), and 29% are obese (>29.9 BMI) [19]. On average, obesity among all women appears to have peaked at 33% with no appreciable increase between 1999–2000 and 2003–2004 [20].
Determinants of overweight among women
A higher proportion of women of child bearing age are overweight or obese compared to men, and women of younger or older ages. In addition, upwards of 80% of African American women are either overweight or obese [19]. Using 2003–2004 data from NHANES, non-Hispanic black women are twice as likely to be obese than their non-Hispanic white counterparts (OR 2.01, 95% CI, 1.76, 2.29) [20]. Among individuals with less than a high school education, the prevalence of obesity was roughly twice that of college graduates [21]. Paradoxically, low-income households appear to have a higher risk of obesity [22], although, at the same time they are more likely to be at risk of food insecurity. Household food insecurity has been associated with increased BMI [23] and an increased risk of overweight [24] in women, but not among men or children [25–27].
Influences of adolescent overweight on reproductive health
Moreover, thirty percent of non-pregnant adolescent girls 12–19 years of age are considered overweight or at risk, based on a BMI for age at the 85th percentile or higher [19]. Excessive weight gain at younger ages is associated with earlier menarche, especially among black and Hispanic girls. Younger and more severely obese girls reaching reproductive capacity perpetuate obesity if left untreated, directly through the influence of maternal weight on fetal origins and indirectly through maternal to child social and developmental interactions [28].
Pregravid BMI is a predictor of gestational weight gain
Among women who become pregnant, the shift towards higher pregravid weight also appears evident [29]. One study using a perinatal data base to ascertain weight before pregnancy for a 19 year period showed that the mean maternal weight of women at their first prenatal visit increased by 20% between 1980 and 1999, and the percentage of women weighing >200 lbs at that visit increased from 7.3 to 24% and for those weighing >300 lbs the percentage increased from 2 to 11% [30]. Another study using a perinatal data system of all live births from 8 contiguous counties in New York, an 11% increase in pregravid overweight and an 8% increase in pregravid obesity between 1999 and 2003 [29]. Not only are more women beginning pregnancy at a higher BMI, but women are also gaining in excess of the 1990 Institute of Medicine recommendation for gestational weight gain [1]. Specifically, and overweight and obese women are more likely to gain excessive gestational weight and keep it on after delivery [31–33]. In affluent countries, women retain some weight with each successive pregnancy, gaining more weight than their non-pregnant counterparts [34, 35]. These observations beg the far larger and more important question of how and when to intervene in order to optimize reproductive and individual health?
Weight intervention studies
Many research studies of dietary, physical activity and weight interventions focus on individuals who are already obese or experiencing co-morbidities such as heart disease, diabetes or cancer. In contrast, only a limited number of interventions have focused on weight loss and/or maintenance of optimal weight in the context of reproductive health. On one end of the spectrum, surgical interventions such as gastric bypass and lap-band surgeries have been successful among obese women of child-bearing age with a decrease of reported rates of gestational diabetes, macrosomia, cesarean section while achieving adequate weight gain [36, 37]. Some interventions have been targeted to address obesity during adolescents, pregnancy, and the postpartum or inter-conceptional periods. These commonly have been school- or clinic-based in nature. School-based randomized intervention trails such as the Trial of Activity in Adolescent Girls (TAAG) are presently underway to determine if increased opportunities for moderate and vigorous activity may decelerate the age related decline in physical activity [38].
Clinic-based interventions
Clinic-based intervention usually begin with obesity management training for physicians because of the existence of a combination of a paucity of nutrition, physical and weight educational materials, lack of knowledge of how to counsel women, and lack of resources and personnel to assist with obesity management. For example, results from a survey of pediatricians found that only 12% of pediatricians reported high self-efficacy in obesity management, although 39% stated that physicians were important health care providers that could be effective with obesity management. Lack of non-MD staff reimbursement, of an on-site dietitian, and of patient educational materials were each highly associated with low self-efficacy in obesity management [39].
Weight loss interventions
Tsai and Wadden [40] conducted a systematic review of major commercial weight loss programs undertaken to provide physicians with information on components, cost, and efficacy of these programs. eDiets.com, Health Management Resources, Take off Pounds Sensibly, OPTIFAST, and Weight Watchers were represented in the academic literature. The review identified Weight Watchers as the least costly with maintenance of 3.2% loss of initial weight at two years. Medically supervised very-low-calorie diet programs had the greatest weight loss (approximately 15 to 25% of initial weight), but were associated with high costs and high attrition rates. Finally, Internet (i.e., eDiets.com) and organized self-help programs (i.e., Overeaters Anonymous) produced minimal weight loss. In a clinical intervention conducted among pregnant women, physicians were trained to chart and monitor gestational weight gain adequacy based on the Institute of Medicine weight gain recommendations. Women received five patient education newsletters by-mail with action-oriented messages of how to gain adequate weight in pregnancy. Findings indicated that low-income women who received the intervention were less likely to gain excessive gestational weight. Overweight low-income women were at a reduced risk to retain more than 2.7 kg of weight at one year [41]. A hospital based randomized control trail with an intensive stepped-care, behavioral intervention found that the intervention significantly decreased the percentage of normal weight women gaining in excess of the IOM recommendations [42].
Significance of weight status during postpartum period
The postpartum period is hypothesized to significantly contribute to overweight and obesity through pregnancy weight retention. Olson et al. [31] found that over 25% of women participating in a cohort study experienced major weight gain, defined as 4.55 kg or more, at one year postpartum. Gestational weight gain, postpartum exercise frequency, and food intake were all significantly associated with weight change from early pregnancy to one year postpartum. Lower income women who gained more than the IOM recommendation were at increased risk for major weight gain at one year postpartum. In a randomized trial of 40 overweight postpartum women receiving either a 12 week structured or self-directed diet and physical activity intervention, the structured intervention group had significant weight loss (7.3 kg), decrease in percent body fat (6%) and no change in fat-free mass, whereas the self-directed group had no significant change at one year postpartum [43]. Randomized control trials are currently underway in low-income WIC populations receiving usual care or a multi-component intervention including home visits, group classes and monthly telephone counseling [44].
Conclusion
Given that the costs associated with treating obesity are so high and their limited success rates, the best strategy is to prevent obesity from occurring in the first place. However, prevention first requires a clear understanding of its etiology. Obesity is a chronic disease thought to develop from a complex interaction of genotype and environmental factors [45]. Our knowledge of how and why this disease develops is far from complete, but currently funded research projects are targeting the integration of social, behavioral, cultural, and biological factors. In addition, best practices at obesity prevention, treatment and optimal weight maintenance must be identified to provide practitioners with an array of strategies to help curb the ensuing epidemic.

obesity pregnancy women nutrition postpartum