Imaging oxygenation of human tumours

Tumour hypoxia represents a significant challenge to the curability of human tumours leading to treatment resistance and enhanced tumour progression. Tumour hypoxia can be detected by non-invasive and invasive techniques but the inter-relationships between these remains largely undefined. 18F-MISO and Cu-ATSM-PET, and BOLD-MRI are the lead contenders for human application based on their non-invasive nature, ease of use and robustness, measurement of hypoxia status, validity, ability to demonstrate heterogeneity and general availability, these techniques are the primary focus of this review. We discuss where developments are required for hypoxia imaging to become clinically useful and explore potential new uses for hypoxia imaging techniques including biological conformal radiotherapy.
Introduction
The suspicion that tumour hypoxia increased resistance to radiotherapy was first considered in the 1930’s but it was not until 1955 that Tomlinson and Gray showed that chronic hypoxia occurred in human bronchial carcinomas with necrosis occurring approximately 150 μm from blood vessels [1], which is a little larger than the currently known diffusion distance of soluble oxygen in tissues (approximately 70 μm). Decades of research in radiation therapy then followed, much of which focused on attempts to circumvent hypoxia-mediated radio-resistance but these efforts were only moderately successful. Over the last decade, it has become evident that hypoxia changes the patterns of gene expression in several ways that alters the malignant potential of tumours, leading to more aggressive survival traits. As a result, hypoxic cancers are difficult to treat, particularly by radiation and photodynamic therapy [2], but also by cytotoxic chemotherapy. Attempts at circumventing the cure-limiting impact of hypoxia have included the use of hyperbaric oxygen and radiation sensitizer drugs but these have, in general, not proved widely advantageous. However, attempts to take advantage of the presence of tumour hypoxia, such as hypoxia-specific cytotoxins, are more promising. As hypoxia-directed therapies enter into clinical trials, it has become important to non-invasively assess for the presence of hypoxia and to be able to follow how it is modulated by new therapies. Hypoxia imaging may help select the most appropriate population that would benefit from novel hypoxia-directed therapies. In this review we describe the causes for and the effects of tumour hypoxia, as well as summarise the lead contenders for human tumour imaging. We also assess where developments are required for them to become clinically useful imaging tests and explore potential new uses for hypoxia imaging techniques including biologically-directed conformal radiotherapy.
Overview of tumour hypoxia & its importance
For the majority of solid tumours hypoxia develops because of the inability of the vascular system to supply the growing tumour mass with adequate amounts of oxygen. Consequently, both low oxygen tensions and nutrient deprivation contribute to impaired tumour growth such that growth beyond 2 mm requires tumour neovascularisation. The major factors that play roles in the development of tumour cell hypoxia are the known abnormalities in structure and functioning of tumour microvessels [3], the increased diffusion distances between blood vessels (many of which may not even carry oxygenated red blood cells), the expanding tumour cell mass competing for oxygen and the reduced oxygen carrying capacity of blood due to disease- or treatment related anaemia. Thus, there are three distinct types of tumour hypoxia [4]: (1) Perfusion related (acute) hypoxia that results from inadequate blood flow in tumours that is generally the consequence of recognised structural and functional abnormalities of the tumour neovasculature. Such acute hypoxia is often transient, caused by temporary occlusions and temporary rises in interstitial pressure and can affect all cells right up to the vessel wall; (2) Diffusion related (chronic) hypoxia is caused by increased oxygen diffusion distances due to tumour expansion and affects cells greater than 70–100 μm from the nearest capillary, depending on where tumour cells lie in relation to the arterial or venous end of a capillary; (3) Anaemic hypoxia, which relates to reduced O2-carrying capacity of the blood and may be tumour associated or treatment related.
The presence of hypoxia within human tumours before starting treatment has been observed in a variety of tumour types including squamous cell carcinomas, gliomas, adenocarcinomas (breast & pancreas) and in sarcomas. For example, in the normal cervix the pO2 is a median of 42 mmHg compared to a median of 10 mmHg in squamous carcinomas, and for cervix cancer the oxygenation status is independent of size, stage, histopathological type, and grade of malignancy [5]. Oxygen probes, that is, electrodes implanted directly into tumours to measure oxygen concentration by a polarographic technique [6–8] have shown (1) heterogeneity within and between the same tumour types of oxygen concentration and, (2) that hypoxia contributes to poor prognosis; pO2 < 10 mmHg results in poor local tumour control, disease-free survival and overall survival in squamous carcinomas of the head and neck and of the cervix [9, 10].
A large body of clinical evidence suggests that the hypoxia-mediated aggressive behavior of cancer cells and their resistance to therapy is orchestrated by the heterodimeric transcription factor, hypoxia inducible factor-1 alpha (HIF-1α), via a number of molecular events required for the adaptation of tumour cells to hypoxia (including unregulated glycolysis, angiogenesis and mutant p53) [11]. It is also important to realise that in some tumours including uterine lieomyomas, HIF expression is not always correlated with the presence of hypoxia suggesting that other factors including genetic events also contribute to activation of HIF, the most significant one of which is the loss of function of the Von Hippel-Lindau (VHL) tumour suppressor protein which results in constitutive activation of the HIF pathway. HIF-1 controls the expression of a variety of genes, the protein products of which play crucial roles in the acute and chronic adaptation of tumour cells to oxygen deficiency, including enhanced erythropoiesis & glycolysis, promotion of cell survival, inhibition of apoptosis, inhibition of cell differentiation, and angiogenesis. Thus, adaptive changes in the proteome and genome of neoplastic cells result in the emergence of more aggressive clones which are of cells that are more able to overcome nutrient deprivation or escape their hostile environments. Selection pressures by hypoxia and clonal expansion of the more aggressive cell types can result in exacerbations of regional hypoxia, further promoting the development of cell phenotypes that are treatment resistant (Fig. 1). Given the central role of HIF-1 in hypoxia mediated aggressive behaviour of cancer cells and their resistance to therapy, HIF-1 has become a target for the development of anti-cancer drugs [12].
Fig. 1Stylised diagram showing how hypoxia leads to therapy resistance and the development of an aggressive tumour cell phenotype. Figure adapted from [4]
There is debate about whether there is a critical intratumoural pO2 below which detrimental changes begin to occur that is common across cell types. This occurs because experiments performed in cell cultures may not be applicable to in-vivo environments and some of the literature variation can be attributed to the tumour cell type chosen for experiments and the demands of host tissues. With these caveats in mind, the critical pO2 tensions below which cellular functions progressively cease or anticancer treatments are impaired are approximately as follows [13]: Effectiveness of immunotherapy becomes impaired (30–35 mmHg); Photodynamic therapy (15–35 mmHg); Cell death on exposure to radiation (25–30 mmHg); Binding of hypoxia immunohistochemical markers (10–20 mmHg); Proteome changes (1–15 mmHg) and Genome changes (0.2–1 mmHg). The differences in these numbers are smaller than the similarities so that, from a practical perspective, for solid tissue tumors in vivo, a value of between 5–15 mmHg is a good number to remember because of its impact on therapy. This number is in contrast to ischaemic hypoxia in the myocardium or stroke where detrimental effects are experienced at higher O2 [14]. In all these instances the critical oxygen level in tissues reflects the drive to match delivery with metabolic demand.
As noted above, the presence of tumour hypoxia appears to impair the effectiveness of radiotherapy and radiosensitivity is progressively limited as tumour pO2 levels fall. Hypoxia-induced radioresistance is multifactorial with the presence of oxygen mediating DNA damage through the formation of oxygen free radicals which occurs after the interaction of radiation with intracellular water. The ratio of doses administered under well-oxygenated to hypoxic conditions needed to achieve the same biological effect (i.e., cell kill) is called the oxygen enhancement ratio (OER). For sparsely ionising radiations such as x- and gamma rays, the OER at therapeutic doses is between 2.5–3.5 [15]. That is, well oxygenated cells are about three times more sensitive to x- and gamma radiation than the same cells when they are hypoxic. Half maximal sensitivity to x- and gamma rays occurs at oxygen tensions of approximately 2–5 mmHg; above pO2 values of approximately 10–15 mmHg near maximal oxygen effects are seen. However, it should be recognised that sensitivity of cells to radiation is dependent on the phase of the cell cycle, with cells in the G1 phase having a lower OER (i.e., more radiosensitive) than cells in S-phase. As noted above, the oxygen effect is not the only mechanism for radioresistance in hypoxic tumour cells. Evidence is accumulating that the hypoxia-mediated proteomic and genomic changes may also contribute to radioresistance by increasing the levels of heat shock proteins (heat shock proteins (HSPs), are induced in response to environmental stresses like heat, cold and oxygen deprivation [16]) or by increasing the number of tumour cells that can resist apoptosis by mutating p53 (the slowing of cell division is dependent on a protein brake known as p53; the disruption of the functioning of this protein is associated with approximately 50–55% of human cancers).
Clinical imaging of hypoxia
As tumour hypoxia is an important biological characteristic and there is no good or easy clinical way to predict its presence, it has been suggested that imaging may be a good way of non-invasively selecting cancer patients who would benefit from treatments that overcome, circumvent or take advantage of the presence of hypoxia. Since tumour hypoxia is a key mechanism that leads to radioresistance, it has been repeatedly suggested that a hypoxia mapping technique could be integrated with conformal radiotherapy techniques to improve target delineation and dose delivery; this is discussed in more detail below. Imaging could also be used to document whether or not and the extent to which reoxygenation of tumours occurs during radiotherapy. Key requirements of any method that evaluates tumour hypoxia include non-invasive assessments that allow serial changes during treatment to be monitored and evaluation of heterogeneity between and within tumours.
There are a number of ways in which tissue oxygenation status can be assessed in vivo (both invasive and non-invasive) or in vitro using material from biopsy. Non-imaging methods of assessing for the presence of hypoxia in tissues include histological appearance, immunohistochemical staining for intrinsic markers of hypoxia (e.g., carbonic anhydrase IX (CA-IX) and hypoxia inducible factor-1 (HIF-1)) and for the binding of externally administered nitroimidazoles [17, 18].
From an imaging perspective, an ideal test would: (1) distinguish normoxia / hypoxia /anoxia/necrosis, (2) distinguish between perfusion-related (acute) and diffusion-related (chronic) hypoxia if possible, (3) reflect cellular in preference to vascular pO2, (4) be applicable to any tumour site with complete loco-regional evaluation, (5) be simple to perform, non-toxic and allow repeated measurements, and (6) be sensitive at pO2 levels relevant to tumour therapy. Therefore, the challenge for hypoxia imaging is to measure low levels of tissue pO2 on a spatial scale similar to the O2-diffusion distance (70–100 μm); a much smaller dimension than can be achieved with human imaging techniques. Currently available MRI and PET methods were compared at a National Institute of Health/National Cancer Institute of the USA sponsored workshop in April 2004 and it was noted that only a few techniques have potential for in vivo assessment in humans particularly for repeated, sequential measurements. 18F-MISO and 60/64Cu-ATSM PET, and BOLD-MRI are the lead contenders for human application based on their non-invasive nature, ease of use and robustness, measurement of hypoxia status, validity, ability to demonstrate heterogeneity and general availability (Table 1).
Table 1Comparison of techniques for evaluating human tumour hypoxiaTechnique and key referencesInvasive investigationRequires injectionMeasuresClinically Validated in RTGeneral availability (1–5; poor-wide)Monitors changes in pO218F-MISO PET [19, 24, 51–53]NoYesHypoxia±3No18F-AZA PET [54]NoYesHypoxiaNo2No18F-EF5 PET [55]NoYesHypoxia±2No64Cu ATSM [25, 26, 28, 34, 35]NoYesHypoxia±3NoBOLD-MRI [36]NoNo[dHb] in RBCsYes4YesPolorographic electrode [6–8]YesNopO2Yes2Yes
18F-MISO
[18F]Fluoromisonidazole, 3-fluoro-1-(2′-nitro-1′-imidazolyl)-2-propanol or (18F-MISO), is the prototype hypoxia imaging agent whose uptake is homogeneous in most normal tissues, reflecting its high partition coefficient that nears unity, and whose delivery to tumours is not limited by perfusion [19]. The initial distribution of 18F-MISO is flow dependent, as with any freely diffusible tracer, but local oxygen tension is the major determinant of its retention above normal background in tissues after 2 hours (Fig. 2). 18F-MISO accumulates in tissues by binding to intracellular macromolecules when pO2 < 10 mmHg. Retention within tissues is dependent on nitroreductase activity (that is, on reduction status of a NO2 group on the imidazole ring) (Fig. 3) and accumulation in hypoxic tissues over a range of blood flows has been noted, including within the intestinal lumen where it is retained in anaerobes!
Fig. 2The 18FDG-PET image (bottom left panel) shows increase uptake in both the oropharyngeal tumour (arrow) and in the left neck nodal metastasis (asterix). The 18F-MISO images (bottom right panel) were acquired in a dynamic mode and representative images after 1 minute, 30 minutes and 240 minutes are shown together with time-activity curves from the two regions of interest indicated in the FDG-PET image. The early distribution (1 minute) shows hyperperfusion in the region of the primary tumour and metastasis because of the high partition coefficient of 18F-MISO. After 2 hours, only the left neck nodal metastasis is shown to be hypoxicFig. 3The structure of [18F]-fluoromisonidazole, 18F-MISO, and its mechanism of retention in hypoxic tissues. The partition coefficient of 18F-MISO is near unity so the molecule diffuses freely into all cells. Once 18F-MISO is in an environment where electron transport is occurring (viable tissues), the –NO2 substituent (which has a high electron affinity) takes on an electron to form the radical anion reduction product. If O2 is also present, that electron is rapidly transferred to oxygen and 18F-MISO changes back to its original structure and can leave the cell. However, if a second electron from cellular metabolism reacts with the nitroimidazole to form the 2-electron reduction product, the molecule reacts non-discriminately with peptides and RNA within the cell and becomes trapped. Thus, retention of FMISO is inversely related to the intracellular partial pressure of O2 as shown in the lower left panel [45]. This mechanism is confirmed by the autoradiograph of a tumour spheroid (bottom right panel) with a radius of approximately 0.5 mm that shows no retention in the necrotic core or in the well-oxygenated outer sphere but intense uptake (white spots) in a donut like ring where cells are hypoxic
Hypoxia can be imaged with 18F-MISO PET in a procedure that is well-tolerated by the patients. Imaging requires 20–30 min and starts anywhere from 75 to 150 min after injection, making it similar to the bone scan with which most cancer patients are familiar. Useful and well-validated images can be achieved with a modest dose of radiation, typically 250 MBq. No arterial sampling or metabolite analysis is required and synthesis is achieved through relatively simple modifications of nucleophilic displacement / deprotection synthesis boxes such as are used for fluoro-deoxyglucose (18F-FDG). In the USA, F-MISO has investigational new drug (IND) authorisation from the Food and Drug Administration (FDA) as an investigational product for use in humans. Unlike Eppendorf pO2 histography, 18F-MISO is only sensitive to the presence of hypoxia in viable cells; 18F-MISO is not retained in necrosis because the electron transport chain that reduces the nitroimidazole to a bioreductive alkylating agent is no longer active (Fig. 3). Limitations of 18F-MISO PET include the modest signal-to-noise ratio of raw 18F-MISO PET images but if a venous blood sample is acquired during the mid-course of the imaging procedure and used to calculate a Tumour:Blood (T/B) ratio image, then normoxic uptake (T/B < 1) can be electronically subtracted to increase image contrast. Several studies in a range of hypoxic tumours, stroke and hypoxic myocardium [14] have shown that a T/B of >1.2 reliably identifies the presence of hypoxia. The presence of high normal liver uptake impairs complete assessment of liver lesions and urinary excretion interferes with imaging near the bladder.
18F-MISO PET is able to monitor the changing hypoxia status of lung tumours during radiotherapy [20]. Studies in sarcoma [21] and head and neck cancer [22–24] have demonstrated a correlation of 18F-FMISO uptake with poor outcome to radiation and chemotherapy.
Cu-ATSM
Cu-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM) holds exceptional promise as an agent for delineating the extent of hypoxia within tumours with PET. Numerous pre-clinical studies have evaluated and validated its use for imaging of hypoxia in tumours and other tissues [25–32]. The mechanism of retention of the reagent in hypoxic tissues is largely attributed to the low oxygen tensions and the subsequent altered redox environment of hypoxic tumours (increased NADH levels) (Fig. 4). Clinical studies, well-tolerated by patients, involved 60Cu-ATSM imaging sessions of about 60 minutes with analysis of 30–60 minute summed-images. This time frame not only yields excellent data with good image quality (Fig. 4) in a very short time frame which opens up the opportunity with the shorter-lived 60Cu to perform multiple imaging sessions. A number of radioactive copper isotopes with longer half lives are available, e.g. 64Cu (t1/2 = 12.74 h) [33], enabling wide geographic distribution and the United States FDA recently approved an IND application for the study of 64Cu-ATSM for the imaging of hypoxia in human tumours.
Fig. 4Retention mechanism of Cu(II)ATSM in hypoxic tissues. (a) Cu(II)ATSM is bioreduced (Cu(II) to Cu(I)) once entering the cell. The reduced intermediate species (likely to be [Cu-ATSM]-) is trapped within the cell because of its charge. This transient complex can then go through one of two competing pathways: reoxidation to the uncharged Cu(II) species (which can escape by diffusion), or proton-induced dissociation (which releases copper to be irreversibly sequestered by intracellular proteins). [Cu-ATSM]- favours the reoxidation route because it is easily oxidised but chemically more resistant to protonation. Copper from Cu(II) ATSM is trapped reversibly as [Cu-ATSM]- (if oxygen is absent), with the possibility of irreversible trapping by dissociation over a longer period. Cu(II)ATSM is thus hypoxia-selective. (b) High quality axial 60Cu-ATSM-PET image through the mid-upper thorax demonstrates heterogeneously increased uptake (arrow) within a known lung cancer in the aorto-pulmonary window and left suprahilar region. PET images representing summed data were obtained from 30 to 60 minutes after injection of 60Cu-ATSM
In human studies of lung [34] and cervix cervical cancers [35], encouraging evidence has emerged that 60Cu-ATSM can act as a prognostic indicator for response to therapy. In the prospective study of 14 humans with non-small cell lung cancer, a semi-quantitative analysis of the 60Cu-ATSM muscle-to-tumour ratio was able to discriminate those likely to respond to therapy from non-responders [34]. A similar study in 14 women with cervical cancer demonstrated a similar predictive value in the tumour response to therapy [35].
BOLD-MRI
Blood oxygenation level dependent (BOLD) and intrinsic susceptibility weighted MRI are interchangeable terms. As in any MR image, tissue contrast in BOLD images is affected by intrinsic tissue properties including spin-lattice and spin-spin relaxations. Additionally, BOLD MRI contrast is affected by blood flow and paramagnetic deoxyhaemoglobin within red blood cells (oxyhaemoglobin is not paramagnetic). Deoxyhaemoglobin increases the MR transverse relaxation rate (R2*) of water in blood and surrounding tissues thus BOLD-MRI is sensitive to pO2 within, and in tissues adjacent to perfused vessels [36]. Static tissue components include iron content (e.g. myoglobin found in muscle) and presence of fibrosis or ligamentous structures (e.g. in benign prostatic hyperplasia and the suspensory ligaments of the breasts) also affect the appearances of intrinsic susceptibility weighted images. In order to decouple the effects of flow from deoxyhaemoglobin and static components it is necessary to measure the T2* relaxation rate (R2* = 1/ T2*) which can be done by using a multi-echo GRE sequence (Fig. 5). Decoupling of flow from static effects on R2* images occurs because the flow component can be thought of as affecting individual T2* images of a multi-gradient echo sequence equally. It is important to remember that, although synthetic R2* images are free of the contribution of blood flow (that is, they mainly reflect deoxyhaemoglobin content and static tissue components), improving blood flow and vascular functioning will also increase tissue oxygenation, which can be seen by changes in R2* images.
Fig. 5Data acquisition and quantification of BOLD-MRI in the prostate gland. Gradient recalled-echo MR images acquired at 1.5 T with a fixed repetition time and flip angle (TR ~100 msec; alpha 40 degrees) with lengthening echo-time (TE) are acquired through the prostate gland (bottom row of images). These images show increased susceptibility (T2*) effects with increasing TE. The rate of signal intensity decay in the dorsal aspect of the prostate is dependent on intrinsic T2* relaxation (local structure), deoxyhaemoglobin concentration [dHb] and local blood flow. Synthetic R2* (=1/T2*) images are created by plotting the natural logarithm of the signal intensity against the TE (top left panel). R2* map (top right panel) reflects on the structure of tissues and local [dHb] but inflow effects are minimised; however, R2* maps retain sensitivity to pO2 changes caused by alterations in blood flow
The observations made in the previous paragraph imply two intuitive inferences. (1) BOLD-MRI images are more likely to reflect on acute (perfusion-related) tissue hypoxia which, as stated above, occurs because of transient occlusions of vessels, simply because hypoxic areas extend to the level of the blood vessels. In contradistinction, chronic hypoxia is less likely to be reflected by BOLD-MRI because the red blood cells in vessels are too distant from the area of hypoxia. (2) For BOLD-MRI to be able to inform on tissue oxygenation status, it is important for red blood cells to be delivered to the tissue in question. Human and xenograft studies have shown that tumour perfusion varies widely and that red blood cell perfusion is not simply related to the absence/presence of vessels; plenty of tumour vessels maybe present but perfusion by red blood cells may not occur [37]. This observation probably explains in part why no direct correlations between baseline R2* and tissue pO2 have been observed (that is, R2* does NOT measure tissue pO2). So it is necessary to know or to determine the distribution of blood volume in tissue in order to be able to correctly interpret R2* images in order to infer oxygenation status. Thus, if a tissue is perfused but has a high baseline R2* in one area/region compared to another area/region in the same tissue (i.e. the statistic components are the same), then one can infer that the high R2* region is relatively more hypoxic; this hypothesis is supported by recent preclinical and clinical data [38, 39].
As stated above, the use of BOLD-MRI for assessment of tissue hypoxia is predicated on the assumption that the oxygenation of haemoglobin is proportional to blood arterial pO2 which is in equilibrium with oxygenation of surrounding tissues. Many studies have shown that changes in R2* in response to vasomodulation with Carbogen (95% CO2:5% O2) inhalation, for example, are temporally correlated with changes in tissue pO2. Tumours differ in their responses to carbogen inhalation with only 50–60% of human tumours showing changes in R2* [40, 41]. The reasons for these limited and heterogeneous responses are complex but undoubtedly include the fact that tumours have adapted to widely different perfusion and that, even when vessels are present, red blood cell transport along these vessels may not be effective as demonstrated by Robinson et al. [37]. Thus, hypoxic tumours with high blood volume (due to high microvessel density coupled with large vessels) will not only have raised baseline R2* values but are more likely to respond to Carbogen. This will be reflected by large changes in R2*; and it is these hypoxic tumours that show positive radiosensitisation with Carbogen. On the other hand, hypoxic tumours with low blood volume (due to lower microvessel density, or due to small vessels) will have lower baseline R2* values and are thus less likely to respond to Carbogen. In this situation, there will be negligible changes in R2* and such hypoxic tumours do not show radiosensitisation with Carbogen [42]. Readers should also note that the BOLD response to Carbogen is also dependent on the ability of the underlying maturity of the vasculature with mature vessels able to respond actively to vasoconstrictory and vasodilatory stimuli [43].
The primary advantages of BOLD-MRI are that there is no need to administer exogenous radioactive contrast material and images at high temporal and with high spatial resolution can be obtained and repeated as needed. It is possible to decouple the effects of flow and deoxyhaemoglobin which are seen in native BOLD images and so to demonstrate changes in oxygenation independent of changes in blood flow. Major limitations of BOLD-MRI include the fact that they do not measure tissue pO2 directly (either in blood or tissues because of a non-linear relationship of R2* and tissue pO2), the images obtained have low signal to noise ratio and clinical studies with Carbogen vasomodulation are technically challenging (approximately 25–35% of patient examinations fail due to respiratory distress caused by an increased respiratory drive induced by Carbogen [40, 41]. BOLD-MRI appears most sensitive to oxygen levels adjacent to perfused vessels (that is, perfusion related or acute hypoxia) and BOLD-MRI sensitivity to more distant diffusion related or chronic hypoxia is an unknown.
Hypoxia guided radiotherapy
Recently introduced, technological improvements in radiotherapy delivery systems, including intensity-modulated radiotherapy (IMRT), have provided a means for shaping the dose distribution not only to the geometry of target volumes, and also to the differences in radiobiology across tumours [44]. Thus, it is now possible to define an additional “target within the target” as 3D pixel maps of the prescribed dose incorporating biological information derived from functional images; sometimes called dose painting by numbers [45, 46]. This allows treatment to the desired dose with escalation based on biologically relevant data, such as hypoxia which was discussed above, that is mechanistically related to therapeutic outcome. As an example of this, the spatial distribution of 60/64Cu-ATSM uptake on PET was successfully fused with CT radiotherapy planning images to show a proof of concept. This theoretical treatment planning would deliver higher doses of radiation via intensity modulated radiotherapy techniques (IMRT) to the most hypoxic regions of head and neck tumours [47].
In the case of hypoxia, it is important to remember that if the tumour stays hypoxic throughout the course of a treatment, more (escalating) radiation alone may not be sufficient to overcome the hypoxia-induced resistance. Hence, from a therapeutic perspective it is additionally important to know when and if reoxygenation occurs after initial radiotherapy (either by a reduction of oxygen consumption by the arrest of proliferation or by death of well oxygenated cells [15] (Fig. 6). However, data correlating treatment success with hypoxia and reoxygenation changes occurring during treatment are sparse; the extent and rapidity of reoxygenation is impossible to predict for individual tumours. The complexity of the processes leading to reoxygenation suggests the need for repeated imaging during the initial phase of treatment to determine the best time for dose adaptation. Koh et al. used 18F-MISO PET imaging to detect reoxygenation of some lung tumours after just a few treatments, whereas other patients exhibited no reduction in hypoxia even over the full course of radiotherapy [20]. Thus, dose escalation to target hypoxic areas in all patients at the beginning of the treatment could be a wasted effort because reoxygenation would change its distribution. Dose escalation is possibly best undertaken towards the end of treatment, at a time when hypoxic radioresistant cells are predominate.
Fig. 6The re-oxygenation phenomenon. Tumours contain mixtures of aerated and hypoxic cells. Radiation is effective at eliminating well oxygenated cells because they are radiosensitive. Reoxygenation cause the preradiation pattern to return which can be eliminated by further radiation fractions but a progressive decrease in the tumour mass occurs after a series of fractions. Figure adapted from [15]
Recently, Thorwarth et al. presented a study where dynamic (mapping of perfusion) and static (mapping of hypoxia) 18F-MISO scans were obtained in 15 patients with head-and-neck cancers who were subsequently treated with chemoradiation [48]. Their data suggested that subsequent treatment failure was related to both pretherapy hypoxia and concurrent poor perfusion. This result hints that reoxygenation did not occur as a consequence of the deficient vasculature of tumours [49]. Thus, it may be necessary not only to target hypoxic regions but also areas of poor blood flow. Dose escalation maps can be derived from dynamic 18F-MISO PET scans for targeting poor perfusion, while functional planning target volumes could be obtained from late static scans (that reflect hypoxia); both concepts lend themselves equally well to IMRT (Fig. 7). As a caution, simulated treatment plans also suggest that some patients may require such high radiation doses that will lead to a high risk of late complications in the vicinity of the tumour bed, such as necrosis, rupture of blood vessels or excessive fibrosis.
Fig. 7Dose escalation map superimposed on CT with Planning Target Volume (red). Isodose lines show conformality to hypoxic lymph node with a maximum dose increase by 20%. This is the same patient illustrated in Fig. 2
Challenges for hypoxia imaging techniques
As hypoxia imaging techniques move from academic research environments to routine clinical usage, it becomes important to recognise the unique challenges of clinical translation. For example, it is important that patient examination times are short to improve patient compliance particularly for repeated examinations. Thus, the need for doing dynamic scanning followed by several static scans over a prolonged period of time with PET techniques could prove a disincentive for patients. However, as noted above, not all patients undergoing hypoxia studies by PET need dynamic imaging although it may be useful in selected patients where the aim is to demonstrate both perfusion and hypoxia (for example to demonstrate reperfusion-reoxygenation). In-contradistinction, the interpretation of BOLD-MRI does require that the distribution of blood flow/volume is known and this can be done in clinical studies using dynamic contrast enhanced MRI (DCE-MRI) [50]. Whatever the chosen technique for clinical translation, there needs to be standardisation of imaging procedures and analysis methods in order to allow techniques to become more completely validated, for use in clinical trials. Amongst other issues that require addressing when clinical trials are being designed include the need for quantification, test-retest variability and data collection in body parts where there is a large degree of physiological movement such as the lungs and liver.
A practical question often asked is whether it is necessary to quantify imaging data to answer important clinical questions. Subjective assessments work well enough in the clinic; however it is important to realise that subjective criteria cannot be applied simply from one centre to another particularly, when different equipment and imaging routines and human observers are used. Quantification techniques aim to minimise errors that can result from the use of different equipment and imaging protocols. Quantification techniques also enable the derivation of parameters that are based on some understanding of physiological processes and so can provide insights into tumour biology, for example the simple T/B and T/M ratios described for the two PET procedures. Quantification techniques are preferred when serial imaging studies are anticipated, for example when evaluating response to novel anticancer therapeutics.
The reproducibility of the imaging technique should also be known in order to estimate the sample size required to evaluate therapy efficacy. Variation between measurements of the same quantity on the same individual can be caused either by measurement error or by physiological changes between measurements. Whilst it is possible (in theory) to reduce measurement error, physiological variation is inherent, and can cause difficulty in attempts to characterise disease or to monitor the effects of therapy. An estimate of measurement error enables us to decide whether a change in observation represents a real change. Data addressing the precision and measurement variability of hypoxia imaging techniques are urgently needed and should be an integral part of any prospective study that evaluates functional response to therapy to allow assessments of individual patients and group changes.
It is intuitive that analysis and presentation of imaging data needs to take into account the heterogeneity of tumour hypoxia. The presence of motion can invalidate functional parameter estimates particularly for pixel-by-pixel analyses and this is especially true for high spatial resolution techniques such as BOLD-MRI. Motion is averaged in PET imaging because imaging times are long. In that case, pixel-analyses of the data and the issues of heterogeneity assessment can become less meaningful. The first step in heterogeneity analysis includes ROI definition which should be performed independent on the hypoxia imaging being assessed. For BOLD-MRI this could be done by anatomic MRI images and for PET studies could include the CT component of CT-PET studies although some groups have used ROIs defined in 18FDG-PET images. However, ROIs defined on 18FDG-PET images are know to be prone to error as far as tumour boundaries are concerned and are highly dependent on the level of threshold chosen. Whole tumour ROIs yield outputs with good signal-to-noise ratio, but lack spatial resolution and are prone to partial volume averaging errors and thus are unable to evaluate tumour heterogeneity. Pixel mapping has the advantages of an improved appreciation of heterogeneity of hypoxia and the risk of missing important diagnostic information and of creating ROIs that contain more than one tissue type is reduced. An important advantage of pixel mapping is being able to spatially map tumour characteristics such as hypoxia, glucose metabolism and blood flow and to be able to probe the spatial correlations between different kinetic parameters, providing unique insights into tumour structure, function and response to treatment.
Conclusions
To summarise, tumour hypoxia is common and its effects represents a significant challenge to the curability of human tumours, leading to treatment resistance and enhanced tumour progression. Tumour hypoxia can be detected by non-invasive and invasive techniques but the inter-relationship between these techniques needs to be better defined; human validation of the utility of hypoxia imaging is sparse at best. Anti-hypoxia therapies exist in the clinic and more are on their way. Either they don’t work very well or we don’t know how to use them optimally. Hypoxia imaging may allow better definition of a sub-population of cancer patients that would benefit for novel anti-hypoxia directed therapies.

hypoxia
bold-mri
radiotherapy
cancer
cu-atsm-pet
f-miso-pet
tumour resistance


Comparison of three perioperative fluid regimes for laparoscopic donor nephrectomy

Background Pneumoperitoneum (PP), as used for laparoscopic procedures, impairs stroke volume, renal blood flow, glomerular filtration rate and urine output. This study investigated whether perioperative fluid management can abolish these negative effects of PP on hemodynamics.
Laparoscopic donor nephrectomy (LDN) has become the method of choice to procure kidneys from living donors, mainly because of the reduced procedure-related morbidity and faster convalescence period [1–3]. Despite the benefits to the donor, there are concerns over the transient deterioration of renal function in the recipient of the kidney procured by the laparoscopic technique, compared with open donor nephrectomy (ODN) [2, 4–6]. The exact mechanism of delayed graft function after LDN is not fully understood.
Pneumoperitoneum (PP) elevates intra-abdominal pressure (IAP), causing a decrease in renal blood flow (RBF) and glomerular filtration rate (GFR) resulting in oliguria [7–10]. In an animal model, London et al. have shown that PP resulted in a decrease in RBF during normal saline infusion, whereas RBF did not decrease if volume expansion was given [11]. From these results vigorous hydration up to 2 l/h of crystalloids during LDN in patients has nowadays been advocated [1, 12–15].
In 52 patients, Bergman et al. [16] found, however, no difference in graft function after LDN between aggressive (>10ml/kg/h) and conservative (<10 ml/kg/h) intraoperative fluid management. Volume loading after establishment of PP is perhaps too late to counterbalance the collapsed venal system. Biancofiore et al. [17] studied the effect of volume loading on graft function with a crystalloid infusion starting the night before surgery. Early graft function did not differ between ODN and LDN, although the serum creatinine declined earlier, but not significantly, in those receiving kidneys from ODN procedure.
Fasting before operation and induction of anesthesia leads to relative hypovolemia and the goal is therefore to compensate this before PP is started. In this study, we compared three different fluid regimes in LDN patients, in which the effect of pre-hydration together with a bolus of colloids given just before induction of anesthesia and a second one just before inflation of PP on hemodynamics was of special interest.
Methods
Patients undergoing LDN from June 2001 to November 2001 (N = 21) were included in the study. The anesthetic procedure was performed according to a strict protocol for medication, ventilation and fluid regimen. In our hospital the donor patients are admitted the day before the operation, they are fasted during the night from 00:00 and operated on at 08.00 the next morning. Patients were randomized the day before operation by sealed envelopes by the responsible anesthetist to three different fluid regimens (Table 1): in group 1 fluid administration was started at 22:00 the day before operation with 3 ml/ideal body weight (IBW)/h Ringers lactate (RL) until operation. Before induction of anesthesia, the patients received 6 ml/IBW of colloid (6% HES 130/0.4); thereafter 13 ml/IBW/h RL was started until nephrectomy, before installation of PP another bolus of 6 ml/IBW colloid was given. Group 2 received overnight infusion in the same way as in group 1 with a bolus of 6 ml/IBW colloid just before induction. During operation, an infusion was started with 13 ml/IBW/h RL and 2 ml/IBW/h of colloid was given for three hours. Group 3 was fasted from 00:00 on the day of operation and received only an infusion during operation with 13 ml/IBW/h RL and 4 ml/IBW/h of colloid for three hours. After nephrectomy the infusion protocol was adjusted, so that exactly six hours after start of operation all the patients had received in total 9 ml/IBW/h RL. Patients were fitted with anti-thrombosis stockings.
Table 1.Infusion protocols used in the three groupsGroupnml RL/IBW/h pre-hydrationaml RL/IBW/h preoperativebml HES/IBW before inductioncml HES/IBW before PPdml HES/IBW/h after PPeml HES/ IBW total1731366122721363 × 21237163 × 412IBW = ideal body weight n = number of patientsa Prehydration from 22:00 the day before laparoscopic donor nephrectomy, until operationb The amount of Ringer’s lactate given during operation, until nephrectomyc Amount of 6% HES 130/0.4 given before induction of anesthesiad Amount of 6% HES 130/0.4 given before installation of pneumoperitoneume Amount of 6% HES 130/0.4 given per hour after installation of pneumoperitoneumHES = 6% HES 130/0.4
Induction of anesthesia was performed with propofol (2 mg/kg) after a bolus of sufentanil (0.3 μg/kg). Muscle relaxation was achieved with rocuronium (0.8 mg/kg) and monitored by train-of-four (TOF) guard, a bolus of rocuronium (0.3 mg/kg) was given for three or more twitches. Anesthesia was maintained with propofol by continuous infusion (4–11 mg/kg/h), aiming at a bispectral index between 45 and 55 (BIS monitor; Aspect Medical Systems, Newton, MA, USA), and analgesia was achieved by continuous infusion of sufentanil (0.4 μg/kg/h) until nephrectomy. One hour after the start of operation 20 mg mannitol was given intravenously.
After intubation all patients were ventilated in a pressure-controlled mode using a closed-loop ventilator (Physioflex®, Dräger, Lübeck, Germany) with the following initial settings: FiO2 of 0.4, positive end-expiratory pressure (PEEP) of 7 cm H2O and peak inspiratory pressure (PIP) of 22 cm H2O. Ventilation frequency was adjusted to keep PetCO2 between 4 and 5.5 kPa. After induction of anesthesia and before positioning of the patient, an esophageal Doppler probe (HemoSonicTM 100, Arrow International Inc., Reading, PA, USA) was positioned for measuring stroke volume (SV) and left ventricular ejection time, corrected for heart rate (LVETc) [18–20].
After positioning the patient in full lateral nephrectomy position, PP was installed with an IAP of 12 mmHg, which was constantly maintained at this level. All operations were done by the same team of anesthesiologists and surgeons. The surgical techniques have been described in detail elsewhere [21].
Mean arterial pressure (MAP) and SV (available after induction of anesthesia) were monitored noninvasively every five minutes. Urine output was measured from 22:00 the day before until the introduction of PP (T0), and was then measured every hour up to six hours thereafter (T1–6). Blood samples of the donors were collected to determine creatinine levels the day before operation, after induction of anesthesia, six hours after installation of PP, two days, one month, and one year after operation. Creatinine clearance (CrCl) was determined using the Cockcroft-Gault formula [22].
Statistical analysis
Data analysis was performed using SPSS for Windows (version 14.0, SPSS Inc., Chicago, USA). Data are presented as means with standard deviation (SD). Differences between the groups were analyzed using the independent t-test, depending on Levene’s test, pooled or unpooled. Repeated measures with a general linear model from SPSS were used to assess significance for CrCl. A (two-sided) p-value < 0.05 was considered statistically significant.
Results
Baseline characteristics are shown in Table 2 and were comparable between the three groups. After induction of anesthesia, SV was significantly higher in both pre-hydrated groups compared to the control group (Fig. 1). After repositioning from supine to lateral, SV decreased significantly in the control group but not in groups 1 and 2 (Fig. 1). After installation of PP, SV remained stable in group 1 but not in groups 2 and 3 (Fig. 1).
Table 2.Demographic data on the three groups, mean (SD)Group 1Group 2Group 3Age (yrs)56 (11)53 (9)55 (12)Weight (kg)72 (9)82 (8)74 (7)IBW (kg)70 (8)78 (8)71 (6)Male/female 2/54/33/4Operation time (min)237 (37)251 (46)226 (31)IBW = ideal body weightFig. 1.Stroke volume changes during laparoscopic donor nephrectomy, comparing three different fluid regimens: # symbol p < 0.05 for groups 1 and 2 versus control group 3; o symbol p < 0.05 for group 1 versus group 2; × symbol p < 0.05 versus supine position. Data are mean ± standard deviation.
After induction, LVETc was higher in group 1 compared to the control group during the whole procedure and remained stable (Table 3). In all groups MAP decreased after induction of anesthesia; in the control group MAP decreased significantly more compared to group 1 (p = 0.03). HR was comparable between the three groups (Table 3).
Table 3.Data on hemodynamic parameters: heart rate (HR), mean arterial pressure (MAP) and left ventricular ejection time (LVETc), mean (SD). Columns correspond to seven times: before anesthesia (preop); after induction of anesthesia, supine position (supine); full lateral position (lateral); and mean measurement for the first, second, third and fourth 30 minutes after installation of pneumoperitoneum (PP30, PP60, PP90 and PP120, respectively)PreopSupineLateralPP30PP60PP90PP120HR  Group 165 (6)65 (13)63 (20)60 (9)61 (8)60 (10)61 (9)  Group 263 (11)60 (9)61 (14)62 (11)61 (9)63 (6)63 (8)  Group 3 (control) 73 (9)69 (14)61 (6)65 (10)63 (6)60 (7)61 (6)MAP  Group 1102 (11)85 (13)* #81 (19)*93 (14)96 (15)94 (17)98 (20)  Group 2102 (14)73 (10)*81 (15)*107 (10)107 (18)104 (8)102 (12)  Group 3 (control) 105 (12)70 (11)*79 (7)*103 (9)98 (11)95 (12)95 (15)LVETc  Group 1336 (19)#339 (20)#355 (26)#345 (31)335 (30)336 (33)  Group 2313 (29)308 (39)295 (58)°311 (20)°308 (27)300 (28)  Group 3 (control) 294 (26)284 (31)292 (26)307 (18)309 (18)311 (25)* p < 0.05 versus preoperative values# p < 0.05 groups 1 and 2 versus control group° p < 0.05 group 1 versus group 2
Urine output, measured from the start of operation until the moment of kidney extraction, was 1.9 ml/kg/h (range 1.2–3.2) for group 1, 1.4 ml/kg/h (range 0.8–2.3) for group 2, and 1.1 ml/kg/h (range 0.6–1.6) for group 3. In controls, the urine production was significantly lower compared to group 1 (p = 0.01). CrCl decreased in the control group directly after PP, but not in the other groups (Table 4). From two days postoperative, CrCl was comparable between the three study groups (Table 4).
Table 4.Creatinine clearance [mean (SD)] in the three groups at six measurement points: one day before operation; just after induction of anesthesia; at 14:30, six hours after installation of pneumoperitoneum T6; two days after operation; one month after operation; and one year after operationGroup 1Group 2Group 3 (control)CrCl preop (ml/min)89 (19)101 (25)102 (34)CrCl after induction (ml/min)104 (18)109 (20)96 (31)CrCl after the operation (ml/min)87 (17)94 (14)73 (23)ªCrCl D2 (ml/min)63 (10)*65 (11)*64 (19)*CrCl after 1 month (ml/min)63 (12)*67 (11)*63 (24)*CrCl after 1 year (ml/min)71 (13)71 (13)*66 (25)** p < 0.05 versus preoperative valuesª p < 0.05 difference between CrCl preop and CrCl after operation, control group versus groups 1 and 2
Discussion
This study showed that during LDN preoperative hydration together with a bolus of colloid given before induction of anesthesia and before installation of PP resulted in higher SV and higher urine output compared to a fluid regimen with only an intraoperative aggressive fluid infusion. The second group, which received no bolus of colloid before PP in contrast to group 1, showed a significant reduction in SV after installation of PP. In the control group, LVETc and urine output at the moment of kidney extraction showed significantly lower values compared to both pre-hydration groups. CrCl values six hours after the start of the operation was significantly reduced in the control group compared to preoperative values but not in the two pre-hydrated groups; this difference was reduced two days postoperatively.
Clinical studies yield conflicting data concerning the effect of LDN on recipient graft function compared to ODN. The largest study to date compared more than 5,000 kidney transplants from a database and found that LDN was associated with slower early graft function compared to ODN. However, renal function and graft survival at one year was similar between both groups. This was confirmed by retrospective analysis of 120 LDN and 100 ODN in our own institution in which serum creatinine in the recipients was significantly higher in the LDN group only in the first week after transplantation [4]. One very important discriminating factor between ODN and LDN is the pneumoperitoneum. From experimental studies it has become clear that PP decreases RBF and that the magnitude of this decrease is affected by the IAP used, the volume status, and positioning. To counterbalance the increased IAP, vigorous intravenous hydration during LDN is nowadays recommended in an attempt to optimize preload and promote diuresis, but randomized clinical data are missing. In a porcine model, Demyttenaere et al. [23] showed that the decrease in SV and renal cortical perfusion could be prevented by a simple hydration of 15 ml/kg/h saline combined with a bolus 20 ml/kg saline, in accordance with the findings of London et al. [11]. This was also seen in the present study in which pre-hydration with a normal infusion with crystalloids during operation combined with a bolus of colloids just before PP did not decrease SV but improved diuresis. Besides PP, the kidney lateral decubitus position, which is an anti-Trendelenburg position, contributes to hemodynamic alterations by decreasing preload through the effect of gravity on venous return [24]. Yokoyama et al. [25] found no significant change in hemodynamic values after postural change of their patients from supine to lateral but a significant reduction in SV after postural change to kidney position; these patients received a fluid regime of 20 ml/kg/h of crystalloids. This was confirmed by our study in which the control group showed a significant reduction in SV after postural change from supine to kidney position whereas there was no reduction in the two pre-hydrated groups, which received a bolus of colloid just before induction (Fig. 1).
After pre-hydration with crystalloids we infused colloids to achieve optimal plasma expansion just before installation of PP [26]. In our hospital we use 6% HES 130/0.4 for fluid expansion, because the rate for anaphylactic reactions is considerably lower than for gelatin products [27]. However, there are concerns that infusion of certain HES types may influence kidney function [28]. As long as adequate hydration using sufficient amounts of crystalloids are used, the latest generation of HES products (6% HES 130/0.4) do not increase the risk for renal dysfunction even when used in large amounts [29, 30]. Lang et al. [31] even demonstrated that 6% HES 130/0.4 improved tissue oxygenation during and after major surgical procedures compared with a crystalloid-based volume strategy.
In this study, we used the HemoSonicTM, a transoesophageal Doppler ultrasonography (TOD) device, to measure blood flow in the descending aorta. Several studies have confirmed good correlation with cardiac output measured by the thermodilution technique [18, 32]. It has been shown that the accuracy of the device is somewhat operator-dependent [20] and therefore the same two people did all the measurements with this device in the present study. Feldman et al. [33] used LVET to guide their fluid management in LDN patients. In the present study it was shown that LVETc was significantly lower in the control group that did not received pre-hydration and increased over time (Table 2). It should, however, be taken into account that the blood flow with this device is measured in the descending aorta, which is around 70% of the total cardiac output. This could influence our measurements if redistribution of flow away from the descending aorta occurs because of elevated IAP and this is more pronounced in hypovolemic patients.
Some other limitations of this study should be noted. In only four patients a MAG3 scan was performed, which provides the distribution of the function from the two kidneys of the donor. In the four measured patients the harvested kidney contributed 43–48% of the total kidney function, these four patients were divided over all three study groups. However because we do not have the data on the other patients, this could have biased our data on postoperative CrCl. Prehydration of the donor patients conform our protocol, started the night before operation, which contradicts fast-track surgery where kidney donor patients are admitted to hospital on the day of surgery. Also these patients can receive adequate pre-hydration, but further research should be done.
In this study we focused on intraoperative hemodynamic changes. Our data show that preoperative hydration together with a colloid bolus given before induction of anesthesia and before installation of PP resulted in higher SV and higher urine output during LDN, compared to controls that received only an aggressive intraoperative infusion. While under-hydration may contribute to renal dysfunction, perioperative fluid excess can also cause problems, such as pulmonary edema, ileus and increased risk of cardiopulmonary and wound healing complications, which might result in longer hospital stay [34]. However, there is a need to ensure adequate hydration status during PP without being overaggressive. First, our fluid regime will be tested in a large prospective study in order to prevent the negative effect of PP on early graft function in the recipient, and to study possible side-effects in the donor.

pneumoperitoneum
transplantation
renal (kidneys)
anaesthesia
urbanization


Loss of Productivity Due to Neck/Shoulder Symptoms and Hand/Arm Symptoms: Results from the PROMO-Study

Introduction The objective of the present study is to describe the extent of productivity loss among computer workers with neck/shoulder symptoms and hand/arm symptoms, and to examine associations between pain intensity, various physical and psychosocial factors and productivity loss in computer workers with neck/shoulder and hand/arm symptoms.
Introduction
Neck/shoulder and hand/arm symptoms are a common problem in society, in particular among the working population. In the European Union, in 2000 and 2001, 23% of the working population reported work-related pain in neck or shoulders. The percentage reporting work-related muscular pain in the upper limbs was about 13% in the old member states and 20% in the new member states [1]. Although it is not clear to what extent work-related factors contribute to their origin, their impact on working life is huge. Neck/shoulder and hand/arm symptoms can interfere with activities at work, and can cause sickness absence and chronic occupational disability. In the Netherlands in 2001, incident cases of chronic disability for work due to neck and upper limb symptoms added up to 0.1% of the working population, and 6% of the total number of new disability benefits [2].
Besides sickness absence and chronic disability, neck/shoulder and hand/arm symptoms could also lead to reduced work effectiveness. Many workers still go to work despite the feeling that, in the light of their health, they should have taken sick leave. This phenomenon is known as sickness presenteeism [3]. Although they are present at work, their productivity could be reduced due to functional limitations. The extent of productivity loss while present at work is uncertain, but it has been suggested that it accounts for the majority of lost productivity costs associated with chronic pain [4, 5]. Therefore, more knowledge is needed to estimate the magnitude of productivity loss associated with neck/shoulder symptoms and hand/arm symptoms.
Not all musculoskeletal symptoms involve sickness absence or productivity loss. It would be interesting to know more about factors that might influence productivity loss in symptomatic workers. We are inclined to think that these factors might be similar to risk factors for the occurrence of symptoms or for sickness absence due to musculoskeletal symptoms. However, this is not necessarily true and these factors could easily diverge. Knowledge on both types of risk factors is important for primary, secondary and tertiary prevention. While knowledge on risk factors for the occurrence of symptoms is required to prevent them, knowledge on factors associated with productivity loss is needed in the process of retention, reintegration and rehabilitation of workers with symptoms.
Previous studies have shown that pain characteristics, like pain intensity or severity, were predictive factors for a poor prognosis of musculoskeletal symptoms [6–9]. Therefore, it seems plausible that high pain intensity will also have an impairing effect on productivity. Besides pain characteristics, physical, psychosocial and personal factors might affect productivity. In previous studies these factors were already identified as risk factors for the occurrence of symptoms [10–14]. However, as mentioned earlier, this does not necessarily mean they also affect productivity loss in symptomatic workers. The present study is different from studies examining risk factors for sickness absence, as productivity is a broader outcome measure. Moreover, the study population of this study consists of symptomatic workers, and distinguishes between symptomatic workers with and without productivity loss. Studies examining risk factors for sickness absence are usually held in a mixed study population and usually distinguish healthy and symptomatic workers from workers with sickness absence.
The objective of the present study is to describe the extent of productivity loss among computer workers with neck/shoulder symptoms and hand/arm symptoms, and to examine associations between pain intensity, various physical and psychosocial factors and productivity loss in computer workers with neck/shoulder and hand/arm symptoms.
Methods
Study Population
Data were used from the baseline measurement of the PROMO-study: Prospective Research On Musculoskeletal Disorders in Office Workers [15]. The main purpose of this study was to determine risk factors for neck/shoulder symptoms and hand/arm symptoms among computer workers. The study design was approved by the Medical Ethics Committee of the VU University Medical Center.
The study included workers from five different companies. The five participating companies comprised an insurance company, a department of a university, a public transport company, a brewery, and a financial consultancy firm. Employees from these companies had administrative, professional or management jobs. Altogether approximately 9,000 employees were working in these companies. All employees were invited to participate in the study. To maximize the participation rate, various activities were arranged, varying from the dissemination of brochures to visits at the worksite. Almost 2,500 employees subscribed to the project and signed informed consent. They were requested by e-mail to fill out an electronic questionnaire, accessible via the Internet. Participants who did not want to fill out an electronic questionnaire could fill out a paper version. Out of these employees, 1,951 filled out the questionnaire at baseline, resulting in a response rate of 79% of the subscribed employees.
In the PROMO-study, productivity loss due to neck/shoulder and hand/arm symptoms was self-reported and only assessed in workers reporting symptoms. Therefore, analyses concerning associations between various factors and productivity were limited to respondents reporting work-related neck/shoulder symptoms or hand/arm symptoms during the past three months. This selection contained 654 computer workers.
Productivity Loss
Productivity loss was only assessed for those respondents that reported regular or prolonged neck/shoulder symptoms or hand/arm symptoms in the past three months. A dichotomous variable was constructed, based on the answers on the following questions: (1) ‘Have your symptoms slowed down your work pace?’; (2) ‘Have your symptoms decreased your working hours?’; or (3) ‘Have your symptoms caused disability to work for one or more days?’ These questions were adapted versions of items used in the Swedish questionnaire used in the epi-mouse study. In the epi-mouse study, questions on productivity loss were validated through interviews [16]. They were asked for neck/shoulder symptoms and for hand/arm symptoms separately and referred to the past three months. If one or more of these questions were answered affirmative, it was defined as productivity loss. It was assumed that respondents answering that their symptoms decreased their working hours (2), but did not cause disability for work (3), had not been on sick leave. Only if the question concerning disability for work was answered affirmatively, it was defined as sickness absence.
Neck/Shoulder Symptoms and Arm/Hand Symptoms
In the questionnaire subjects were separately asked about symptoms in the neck/shoulder region and in the arm/hand region. The reason to separate these regions is the expected difference in relationship between symptoms and computer usage; computer usage seems to have more effect on arm/hand symptoms than on neck/shoulder symptoms [17]. In the present study the distinction between the regions was maintained, because the different relationship with computer usage might result in different effects on productivity loss.
Subjects were asked to rate the occurrence of pain or discomfort in the neck/shoulder region as well as in the hand/arm region in the previous 3 months on a four-point scale: ‘no, never’; ‘yes, sometimes’; ‘yes, regularly’; ‘yes, prolonged.’ Subsequently, subjects were asked to estimate whether these symptoms were related to their work, which they could answer with ‘yes, completely’; ‘yes, partly’; ‘possibly’ or ‘no.’ Also, 8 possible specific causes of these symptoms were summed: sport injuries, accidents, skin diseases, a twist or sprain, a cut or burn, a congenital defect, rheumatic disorders and a slipped disc. Neck/shoulder symptoms and arm/hand symptoms were defined as regular or prolonged pain, completely, partly or possibly related to work, and not caused by any listed specific cause.
Independent Variables
To examine which factors are associated with productivity loss in computer workers with neck/shoulder and hand/arm symptoms, analyses were carried out with the following variables: pain intensity, physical activity in leisure time (with BMI as a possible confounder), working hours, mouse position, psychosocial load and overcommitment.
Intensity of Symptoms
The intensity of symptoms was measured using Von Korff scales [18]. Respondents were asked to indicate the mean intensity of their symptoms in the past three months on a scale ranging from 0 (no pain) to 10 (worst pain ever). If they reported symptoms in both neck/shoulder and hand/arm region, the region with the highest score was used in the analyses.
Physical Activity and BMI
Concerning physical activity, the respondents were asked about the number of days per week they usually performed activities of at least moderate intensity, and about the times per week they usually performed activities of vigorous intensity. One variable with three categories was constructed measuring physical activity. The categories were ‘no significant physical activity,’ ‘moderate intensity physical activity, but no physical activity of vigorous intensity,’ and ‘vigorous intensity physical activity.’ Moderate intensity physical activity was defined as performing physical activity causing increased breathing for at least 30 min per day with a frequency of at least 5 days per week [19, 20].Vigorous intensity physical acitivity was defined as performing physical activity causing sweating for at least 20 min per session with a frequency of at least 3 days per week [21, 22]. BMI was computed by body weight (kg) divided by square of height (m2). Data on weight and height were self-reported.
Working Hours
Respondents were asked how many hours per week they worked according to their contract. A dichotomous variable was constructed that distinguished between full-time workers working 37–40 h and part-time workers working 4–36 h per week.
Mouse Position
Respondents were asked about the position of their mouse while using it. This question was illustrated with five possible positions for respondents to select, and a category ‘other position.’ A dichotomous variable was constructed that distinguished between a mouse position close to the keyboard, and positions further away from the keyboard. This dichotomization corresponds to the dichotomization in previous research of Hagberg et al., who defined nonoptimal computer mouse position as: “Those who marked their computer mouse position outside a rectangle close to the operator in a workplace layout figure in the questionnaire” [16].
Psychosocial Load
To assess psychosocial load, two concepts were used, namely effort-reward imbalance and job satisfaction. For the first concept Siegrist’s Effort-Reward Imbalance model (ERI) was used [23, 24]. The theory of this model is that a combination of high effort and low reward could lead to adverse health effects. Effort and reward were measured with the recommended scales of the ERI-model [25]. A validated Dutch version of the questionnaire was used [26] with scores varying from 1 (‘agree’) to 5 (‘disagree, and I am very distressed’). Many different methods have been used to construct a variable indicating ERI [27]. In this study a variable in four categories was constructed: no high effort and no low reward, high effort (but no low reward), low reward (but no high effort) and both high effort and low reward. High effort was assigned when a respondent reported to be distressed or very distressed about one or more of the 5 effort items. Low reward was assigned when a respondent reported to be distressed or very distressed about one or more of the 11 reward items. Cronbachs α was 0.68 for the effort scale and 0.80 for the reward scale.
To assess job satisfaction respondents were asked how they enjoyed their work. To answer this question four categories were presented: ‘never,’ ‘sometimes,’ ‘often,’ and ‘always.’ This variable was dichotomized, resulting in a positive score on job satisfaction containing the responses ‘often’ or ‘always,’ and a negative score containing the responses ‘never’ or ‘sometimes.’
Personal Factor—Overcommitment
Concerning personal factors, the personality trait overcommitment was assessed. The concept “overcommitment” specifies those cognitive, emotional and motivational components within the global concept of Type A behavior that are important in coping with work demands. Overcommitted workers may expose themselves more often to high demands at work, or they may exaggerate their efforts beyond what is formally needed [25]. Overcommitment was assessed with the short version of a standard questionnaire [23–25]. Subjects were asked if they strongly disagreed, disagreed, agreed or strongly agreed on 6 items (e.g., I get easily overwhelmed by time pressures at work). Scores were dichotomized (agree versus disagree) and the added scores on these items resulted in an overcommitment score ranging from 0 to 6. Cronbachs α of the overcommitment scale was 0.74.
Analysis
First, descriptive statistics were used to examine how often symptoms resulted in productivity loss, and to describe the components of productivity loss in terms of sickness absence and decreased performance at work. The association between productivity loss and several determinants was examined with logistic regression analyses using productivity loss as the dichotomous outcome variable. Univariate and multivariate analyses were carried out. In all multivariate analyses, age, gender, level of education, and intensity of symptoms were included as covariates. For the analysis with physical activity, BMI was checked for confounding and for the analysis with the psychosocial work characteristics, the other psychosocial work characteristics were checked for confounding. If their inclusion in the model did not result in a change of more than 10% in the effect estimate, these covariates were not considered as a confounder and not included in the final model.
Results
Productivity Loss
Figure 1 shows that neck/shoulder symptoms were reported more frequently than hand/arm symptoms. Of the total population 10% reported both symptoms. On average, in 26% of the cases reporting symptoms, productivity loss was involved. If both symptoms were reported, they were more often accompanied by productivity loss (36%).
Fig. 1Distribution of neck/shoulder and hand/arm symptoms in a population of computer workers (n = 1,951), and the fraction involving productivity loss within workers reporting symptoms
Figure 2 shows what part of productivity loss was caused by sickness absence and what part was caused by a decreased performance at work. A decreased performance at work means decreased speed or decreased working hours, but no sickness absence. In 32% of all cases reporting productivity loss, this productivity loss was coming from sickness absence. Sickness absence occurred more frequently in workers reporting both symptoms (43%). Among workers reporting arm/hand symptoms, and no neck/shoulder symptoms, productivity loss was composed mainly of decreased productivity at work. Only 11% of these workers reporting productivity loss have been on sick leave during the last 3 months.
Fig. 2Distribution of productivity loss due to neck/shoulder symptoms, hand/arm symptoms or both in a symptomatic population of computer workers (n = 654) and the fraction involving sickness absence within symptomatic workers reporting productivity loss
Associations with Productivity Loss
Table 1 shows odds ratios resulting from the logistic regression analyses. A higher odds ratio represents a higher probability of productivity loss. Pain intensity and psychosocial load, i.e., high effort and low job satisfaction were associated with productivity loss in computer workers with neck/shoulder or arm/hand symptoms. In the analyses of the psychosocial work characteristics, the other psychosocial work characteristics were identified as confounders, while inclusion of physical activity in the model did not change the odds ratios for more than 10%.
Table 1Associations with productivity loss in a symptomatic population of computer workers (n = 654)% (n)Crude OR (95%CI)Adjusted* OR (95% CI)R2N if item deletedFull model**.140Pain intensity    Continuous measure (0–10)1.24 (1.13–1.37) 1.26 (1.12–1.41) .104Physical activity in leisure time    No significant physical activity63 (408)1.001.00.131    Moderate intensity (5 × 30 min/week)22 (141)0.73 (0.46–1.17)0.74 (0.43–1.27)    Vigorous intensity (3 × 20 min/week)16 (99)1.48 (0.92–2.39)1.27 (0.70–2.27)Working hours    Part-time (4–36 h)61 (399)1.001.00.135    Full-time (37–40 h)39 (255)1.37 (0.96–1.96) 1.36 (0.87–2.11) Mouse position    Close to the keyboard35 (229)1.001.00.134    Other position65 (425)0.93 (0.64–1.34) 0.70 (0.45–1.08) Psychosocial loadEffort-Reward Imbalance    No high effort, no low reward40 (258)1.001.00.115    No high effort, low reward20 (130)1.81 (1.10–2.99)1.43 (0.76–2.67)    High effort, no low reward18 (119)1.89 (1.14–3.16)2.26 (1.24–4.12)    High effort, low reward21 (138)2.69 (1.68–4.32)1.95 (1.09–3.50)Job satisfaction/task enjoyment    Always21 (138)1.001.00.110    Often65 (423)1.29 (0.80–2.08)1.62 (0.89–2.92)    Never/sometimes14 (93)3.10 (1.72–5.58)3.10 (1.44–6.67)Overcommitment    Continuous measure (0–6)1.06 (0.96–1.16) 1.09 (0.97–1.22) .140Abbreviations: OR, odds ratio; CI, confidence interval; R2N: Nagelkerke R2 index of global model fit (0 = lack of fit, 1 = perfect fit)* Adjusted for gender, age, level of education and intensity of symptoms; the analyses with effort-reward imbalance were additionally adjusted for job satisfaction and the analyses with job satisfaction were additionally adjusted for effort-reward imbalance** The full model contained gender, age, level of education and all variables mentioned in the table
Physical activity in leisure time, working hours, mouse position and overcommitment were not associated with productivity loss in computer workers with neck/shoulder symptoms or arm/hand symptoms. Additional adjustment for BMI in the analyses with physical activity did not result in a change in odds ratio for more than 10%.
Discussion
The purpose of this study was to describe the extent of productivity loss among computer workers with neck/shoulder symptoms and hand/arm symptoms and to examine associations between various physical, psychosocial and personal factors and productivity. The results show that in 26% of all cases reporting regular or prolonged symptoms in the past three months, productivity loss was involved. Most productivity loss was found in workers reporting both neck/shoulder symptoms and hand/arm symptoms. Overall, about 32% of the productivity loss was coming from sickness absence. Sickness absence occurred more frequently in workers reporting both symptoms (43%) and considerably less frequent in workers reporting only hand/arm symptoms (11%). Symptomatic workers reporting unfavorable psychosocial work characteristics reported more productivity loss.
Comparison with Previous Research
The study population of the present study consisted of office workers with neck/shoulder or hand/arm symptoms. So far, almost all studies using productivity loss as an outcome measure were studies in a mixed population, containing subjects with and without musculoskeletal symptoms. Moreover, most previous research on productivity loss concerned sickness absence, while in the present study a decreased productivity while working was also included. Therefore, the results of previous studies are hard to compare with the present study. In a mixed population, associations between productivity loss and potential risk factors partly reflect the risks for symptom occurrence. In the present study we wanted to examine which factors are related to decreased productivity once symptoms have occurred. These factors do not have to be similar. Nevertheless, we will mention the results of previous research in this section.
In the present study, physical activity in leisure time was not significantly associated with productivity loss in workers with neck/shoulder or hand/arm symptoms. We did not find other studies in a symptomatic working population. A few studies have examined the relation between physical activity and productivity loss in a general population. These studies often found a favorable effect of physical activity. A favorable effect was found of sporting activity on sick leave in general [28], and in particular sick leave due to musculoskeletal disorders [29, 30]. Also an association was found between higher levels of physical activity and job performance [31]. It seems that physical activity has a preventive effect and is positively associated with productivity, but does not affect productivity loss in workers already having symptoms.
The present study did not examine the association between physical load and productivity loss extensively. Our hypothesis was that computer workers with a high physical load (for example adverse ergonomic working conditions or sustained computer or mouse usage) might experience more productivity loss. Conversely, some studies in a general working population examining the effect of the improvement of ergonomic working conditions have shown beneficial effects on productivity [32, 33]. An observational study found a weak association between an adverse mouse position and productivity loss [16] in workers with musculoskeletal symptoms.
The present study could not confirm the relation between an adverse mouse position and productivity loss. It might be more interesting to examine the relation between productivity loss and duration of computer use, since studies show that duration is more consistently associated with musculoskeletal symptoms than posture [10, 17]. However, the design of the present study is not suitable to examine this relationship. The results of the analyses would produce a biased view, because in a population of computer workers, productivity loss means almost automatically less computer usage. Preliminary analyses confirmed this assumption: in the subpopulation of symptomatic computer workers, those reporting productivity loss also reported less computer usage, in particular mouse usage. Therefore, analyses examining the association between computer usage and productivity loss would result in a negative association, i.e., more computer usage, less productivity loss.
However, we did examine the association with part-time versus full-time work, because this variable is probably not biased. Respondents were asked how many hours they worked according to their contract. The distinction might indicate a form of physical load, assuming that full-time workers are exposed to a higher physical load. Nevertheless, no relation was found between productivity loss and part-time/full-time work. No previous studies were found examining this relationship.
Psychosocial load, in this study defined as effort-reward imbalance and job satisfaction, was strongly associated with productivity loss. No studies were found that examined these psychosocial work characteristics in relation to productivity loss or sickness absence due to musculoskeletal symptoms. Also, no studies were found that examined the relation between psychosocial work characteristics in general and productivity loss, other than sickness absence. Finally, no studies were found that examined these relations in a symptomatic population.
In mixed populations, containing subjects with and without musculoskeletal symptoms, the relation between psychosocial load and sickness absence in general was demonstrated by several studies [34–36]. Few studies have examined the relation with sickness absence due to musculoskeletal symptoms, and their results were conflicting [37, 38].
Limitations of the Study
In the present study, self-reported data were used to assess productivity loss. Productivity loss in this study contains two components: absenteeism and presenteeism. Self-reported data on absenteeism have been found to be reliable and valid when the recall periods are short [39]. However, measuring presenteeism is more complex. For most types of employment, there is no objective account of productivity with which to assess an employee’s performance. In the present study computer usage was measured by a software program. Therefore, in this study population of computer workers it might be considered to use computer usage as a measure for productivity. We decided not to do so, because we assumed that differences in computer usage would be larger between jobs than between workers with and without productivity loss. For this reason, differences in computer usage would probably reflect different kinds of jobs instead of differences in productivity.
The lack of objective instruments to measure productivity also hampers the validation of self-reported measures. One study, using a rather similar productivity measure, compared self-reported productivity loss assessed with a questionnaire with interviews of 50 computer users who had reported musculoskeletal symptoms [16]. The results seem to indicate that productivity loss assessed in the questionnaire could be a slight underestimation.
Another limitation concerning the productivity measure is its dichotomy. No distinction could be made between a minimal and a major loss of productivity. It would be more accurate and it would provide more insight into the magnitude of the problem, if the precise size of the productivity loss was assessed. However, it is very hard for workers to estimate the size of their productivity loss. A recent review concluded that no study has shown that employees can accurately transform their perceived impairments into a quantitative measure [39]. Therefore, since objective instruments to measure productivity are lacking, we are forced to limit ourselves to a subjective dichotomous measure of productivity.
Although in the PROMO-study follow-up measurements were available, we choose to use a cross-sectional design. The main reason is that data on productivity loss were only available for workers with symptoms. Since symptoms generally have an episodic nature the selection of workers with symptoms at baseline is not identical to the selection of workers with symptoms at follow-up. To select workers with data on productivity at baseline as well as at one or more of the follow-up measurements would mean a selection of workers with symptoms at all these measurements. Such a selection would result in a small study population of workers with chronic symptoms.
If we assume that the associations in this study are causal, still the direction of causality cannot be established due to the cross-sectional design. It could be that either psychosocial working conditions caused productivity loss, or productivity loss caused the reporting of adverse psychosocial working conditions, or both. Intervention studies focusing on the prevention of productivity loss among symptomatic workers might shed more light on the direction(s) of causality.
Implications for Practice
The results of this study show that employers should be aware that the consequences of neck/shoulder and hand/arm symptoms are more extensive than the visible sickness absence due to these symptoms. Only one third of workers who experience productivity loss due to their symptoms actually take sick leave. For the other workers productivity loss expresses itself in decreased performance at work. The results concerning factors associated with productivity loss are more difficult to interpret, since there is still a lack of knowledge on how they relate. It seems that symptomatic workers perform better in favorable psychosocial working conditions. An advantageous psychosocial climate might prevent productivity loss in symptomatic workers.
In conclusion, most workers with neck/shoulder symptoms or hand/arm symptoms experience productivity loss from a decreased performance at work and not from sickness absence. Favorable psychosocial work characteristics might prevent productivity loss in symptomatic workers.

productivity
computer workers
psychosocial factors
presenteeism
musculoskeletal symptoms


Mycoplasma hominis deep wound infection after neuromuscular scoliosis surgery: the use of real-time polymerase chain reaction (PCR)

Mycoplasma hominis is a commensal of the genitourinary tract. It mostly causes infections to associated structures of this system; however, occasionally it is a pathogen in nongenitourinary tract infections. Since, M. hominis strains require special growth conditions and cannot be Gram stained, they may be missed or delay diagnosis. This report describes a deep wound infection caused by M. hominis after neuromuscular scoliosis surgery; M. hominis was recovered by real-time polymerase chain reaction (PCR). An awareness of the role of M. hominis as an extragenital pathogen in musculoskeletal infections, especially in neuromuscular scoliosis, being a high-risk group for postoperative wound infection, it is necessary to identify this pathogen. Real-time PCR for postoperative deep wound infection, in patients with a history of genitourinary infections, decreases the delay in diagnosis and treatment. In these cases rapid real-time PCR on deep cultures should be considered.
Introduction
Deep wound infections complicating spinal surgery are sources of major morbidity in neuromuscular scoliotic patients. Enterobacter, Enterococcus, Escherichia coli, Proteus and Staphylococci account for the majority of postoperative spinal wound infections in these patients [28]. Increasingly, previously uncommon pathogens are being identified in surgical infections. These organisms may be difficult to identify and/or resistant to most of the broad-spectrum antibiotics used for preoperative prophylaxis and for the treatment of postoperative wound infections. Among these pathogens, Mycoplasma hominis has been recognized as a cause of postoperative wound infections [25, 27, 31]. M. hominis is a commensal bacterium in humans and is distinguished phenotypically from other bacteria by the minute size and lack of a cell wall. As a result, M. hominis cannot be Gram stained and is resistant to penicillin and other antibiotics that interfere with the cell wall metabolism. In addition, it is a fastidious slow-growing organism, which may not be readily identified by using routine culture protocols. Early diagnosis, however, is of utmost importance for adequate institution of appropriate antimicrobial therapy.
To our knowledge, only two cases of postoperative spinal wound infections due to M. hominis have previously been reported [18]. We describe a case report of a M. hominis postoperative deep wound infection in a patient with a progressive myelomeningocele scoliosis who underwent posterior scoliosis surgery with bone allograft. Real-time polymerase chain reaction (PCR) provided a fast and secure diagnosis, which prevented further complications.
Case presentation
An 11-year-old girl with a complete paraplegia at level L2–L3 caused by a myelomeningocele was admitted to our hospital for surgical correction of a progressive right convex scoliosis. Preoperative physical examination revealed a flexible right convex thoracic scoliosis with a left convex thoracolumbar curve. The unsupported sitting anteroposterior radiograph showed a right convex thoracic scoliosis of 55°and a left convex thoracolumbar curve of 42°with no pelvic obliquity. Her medical history revealed a shunted hydrocephalus with an Arnold Chiari type II malformation, chronic urinary tract infections, and an auto-augmentation of the bladder.
The patient was treated with single stage scoliosis correction involving posterior instrumentation (Xia™ spinal system, Stryker Spine, Cestas, France) from T3 to L5. The spondylodesis was completed by applying allograft bone chips (Netherlands Bone Bank Foundation, Leiden, The Netherlands) over the laminae in the thoracolumbar region. In addition, Collagraft® (Neucoll, Campbell, CA, USA), a synthetic bone graft substitute composed of collagen and a composite mineral (hydroxyapatite and tricalcium phosphate), was applied posterolateral in the lumbosacral region.
Prophylactic antibiotics, cefazolin (cefalosporin, Kefzol®) 1,000 mg IV, were administered at the induction of anaesthesia, and as a second and third dose 8 and 16 h postoperatively, respectively [6]. Because of a chronic urinary tract infection including a positive culture with E. coli, cefradin (cefalosporin, Velosef®) 500 mg orally was continued for 9 days at 8-h intervals.
The postoperative course was uneventful. At day 8 the patient was discharged. At discharge the wound produced negligible clear fluid at the distal end without any signs of infection. However, at day 18 postoperatively, the patient developed fever (39°C) and was readmitted to our centre. On physical examination, an enlarged distal wound dehiscence was seen with increased fluid production. Infection parameters showed an increased C-reactive protein (CRP) (120 mg/l; normal <10 mg/l) and white blood cell (WBC) count (10.9 cells×109/l; normal 4–10 cells×109/l). A surgical intervention was performed including a thorough debridement of necrotic tissue and removal of the bone grafts. The instrumentation was left in place. According to the established guidelines, cultures were taken of various deep areas by fluid aspiration and from the applied bone graft [15, 28, 32]. After extensively irrigating with pulsatile lavage, the wound was closed leaving gentamicin collagen fleeces (Septocoll®, Biomed, Darmstadt, Germany) over the instrumentation. Adjuvant therapy with gentamicin (aminoglycoside, Garamycinl®) 210 mg IV and flucloxacillin (isoxazolylpenicillin, Floxapen®) 2,000 mg IV were initiated immediately after obtaining appropriate intraoperative cultures, and was continued postoperatively at 6-h intervals.
Intraoperative cultures from both the fluid aspiration and the bone graft yielded Mycoplasma after 4 days incubation. Real-time PCR on the peri-operative biopsy material was only positive for Mycoplasma and the species was identified by 16S DNA amplification as M. hominis (Fig. 1). Separately a Collagraft® sample of the same batch was tested for Mycoplasma, however, the culture as well as real-time PCR remained negative. Gentamicin and flucloxacillin were stopped, and doxycycline (tetracycline, Vibra-S®) 100 mg was administered orally at 12-h intervals and continued for 3 weeks. The patient was discharged 5 days after surgery.
Fig. 1Agarose gel analysis of duplo PCR amplification of Mycoplasma DNA. M Molecular weight marker, Lanes 1 and 2 positive PCR amplification of clinical sample, lane 3 negative control, lane 4 positive control. Subsequent sequencing results revealed M. hominis
Wound healing and temperature were monitored at regular intervals at the outpatient clinic. After 3 months, the infection parameters were normalized and the wound was healed (Fig. 2). Four years after surgery there were no signs of infection. Radiographs at final follow-up demonstrated no instrumentation failure and no loss of correction of the scoliotic deformity (Fig. 3).Fig. 2Clinical appearance 3 months after surgical intervention for deep wound infection, located at the distal end of the woundFig. 3AP radiographs of the lumbar spine at the site of the deep wound infection immediately after surgical intervention and debridement of necrotic tissue including removal of the bone grafts (a) and at 4 years of follow-up (b)
Discussion
Mycoplasma hominis is a commensal bacterium in humans. The organism is commonly associated with infections of the genitourinary tract, particularly in females. Rates of colonization in the urogenital tract range from 21 to 54% among women and from 4 to 13% among men [20]. Reports on M. hominis infections outside the genitourinary tract are scarce. Most of them are case reports describing extragenital infections, such as septic arthritis [17, 21], septicemia [11, 12], prosthetic valve endocarditis [3, 10], postoperative wound infections [25, 31], peritonitis [13], neonatal encephalitis [30], meningitis [7, 9], brain abscess [33], thrombophlebitis [27], and mediastinitis [19]. Extragenital infections are mainly related to patients with immunosuppression [3, 7, 9, 17, 19, 21, 25, 27, 30, 33]. Osteomyelitis caused by M. hominis is predominantly reported in combination with hypogammaglobulinemia [5, 14, 16, 24]. In an extensive overview of nongenitourinary M. hominis infections, Madoff and Hooper [18] described only two cases of postoperative deep wound infections caused by M. hominis after orthopedic surgery. Both cases concerned with deep wound infections after scoliosis surgery, one of them with a history of pyelonefritis.
Postoperative deep wound infections after scoliosis surgery are more common in neuromuscular patients than in patients with idiopathic scoliosis [28]. Risk factors associated with increased postoperative wound infection rates include a generalized decline in the immune status of neuromuscular patients, poor personal hygiene, and soiling of the wound. Sponseller et al. [28] reported 25 patients (12%) with a deep wound infection out of a series of 210 surgically treated patients with neuromuscular scoliosis in a 10-year retrospective study. From these 25 patients, 16 had a scoliosis related to myelomeningocele. Two risk factors were found to be significant: the degree of cognitive impairment and use of bone allograft. Furthermore, 52% of the infections were polymicrobial, which could point to contamination during or after surgery. However, the authors did not report any case of M. hominis wound infections in their series.
Antibiotics are used routinely to prevent postoperative wound infection in patients undergoing spinal implant procedures. Currently, first or second-generation cephalosporin (e.g. cefazolin) are recommended as prophylaxis [6]. Prophylactic cefazolin was also routinely given in our case. Since, cephalosporins act on the cell wall of organisms in a manner similar to the penicillins, postoperative wound infection by M. hominis was not prevented. Antimicrobial treatment for M. hominis include protein-synthesis inhibitors such as tetracycline (e.g. doxycycline) [26] and doxycycline was administered accordingly. The postoperative deep wound infection resolved favorably after surgical debridement and appropriate antimicrobial treatment with doxycycline.
The source of M. hominis deep wound infection in our case is unclear. M. hominis is commonly associated with infections of the genitourinary tract [20] and patients with meningomyelocele are known to have a high incidence of urinary tract infections [8]. In addition, it has been shown that children with myelomeningocele have a high incidence of urological complications after surgical treatment of scoliosis [4]. Possibly, in our case the M. hominis could have been present in the urinary tract infection and spread hematogenous or by contamination, either at the time of surgery or secondarily through the wound. Other possible sources are the materials that were used during surgery. Obviously, the instrumentation and the applied Collagraft®, were sterilized. In addition, cultures and PCR on the same batch of the Collagraft® were negative. Bone allograft, used to induce and facilitate spinal fusion, could also have been the source of the infection. The fact that the bone allograft was positive for M. hominis, proved that it was infected before retrieval, however, not that it was the source of the infection itself. Bone allograft has not been reported as a carrier of M. hominis in the literature, however, it must be noted that the standard screening at the Netherlands Bone Bank Foundation does not include screening on M. hominis. Unfortunately, no other specimen of the donor could be retrieved for testing. As a result, haematogenous spread or contamination from a colonized urogenital tract or contamination by the bone allograft as source of the infection could not be excluded.
In the presented case, cultures taken during the reintervention proved to be positive for Mycoplasma. However, the diagnosis of a Mycoplasma infection was delayed due to the fastidious nature of mycoplasmas. To identify the Mycoplasma species, which in our case was cultured, 16S DNA amplification was used. Because isolation of M. hominis is difficult, time consuming, and not routinely done, a rapid specimen processing is required. Real-time PCR on deep cultures could provide a rapid alternative with a higher sensitivity and specificity than culture for the detection of M. hominis [1, 2].
In conclusion, M. hominis infections should be considered in postoperative deep wound infection after neuromuscular spinal surgery, especially in patients with genitourinary tract comorbidity. Since, M. hominis is not covered by routine prophylactic and therapeutic antibiotics, rapid real-time PCR is advised in these patients to initiate appropriate antibiotic treatment.

mycoplasma hominis
wound infection
neuromuscular scoliosis
pcr