Bone loss in elderly men: increased endosteal bone loss and stable periosteal apposition. The prospective MINOS study

Introduction Longitudinal studies on the age-related bone loss in men concerns the decrease in areal bone mineral density (aBMD), which can be qualified as “apparent bone loss” because it does not reflect the change in bone mineral content (BMC). Loss of BMC can be referred to as “net bone loss” because it does not take into account the morphological basis of the bone loss (decreased periosteal apposition; endosteal bone loss, i.e. bone loss on the trabecular, endocortical and intracortical surfaces). The aim of this study was to assess age-related apparent net and endosteal bone loss as well as their morphological basis and age-related changes during a prospective follow-up in a large cohort of elderly men.
Introduction
Osteoporosis in elderly men is a major problem of public health. Fracture incidence increases exponentially with age, thus, total number of fragility fractures in men increases rapidly, mainly due to the increasing life expectancy [1]. Currently, 25–30% of fragility fractures occur in men [1, 2]. Moreover, postfracture morbidity and mortality are higher in men than in women [3, 4].
Bone loss is defined usually as the rate of decrease in areal bone mineral density (aBMD) measured by dual X-ray absorptiometry (DXA) [5–7]. However, aBMD of a given region of interest (ROI) is determined by its bone mineral content (BMC) and by its projected area. For tubular bones, ROI length is kept constant, and the projected area depends on bone width. Bone width increases with ageing due to the periosteal apposition [8, 9]. Thus, if the amount of bone deposited on the periosteal surface and that resorbed on the endosteal surfaces (i.e. trabecular, endocortical and intracortical) are equal, BMC remains constant, but aBMD decreases because bone width and its projected area have increased. Therefore, it is more appropriate to refer the decrease in aBMD assessed longitudinally as “apparent bone loss”.
Changes in BMC of a given ROI are determined not only by the quantity of bone resorbed on the endosteal surfaces but also by the quantity of bone deposited on the periosteal surface. Thus, change in BMC does not reflect the quantity of bone really lost on the endosteal surfaces and, therefore, the decrease in BMC is better qualified as “net bone loss”. Estimation of bone gain due to periosteal apposition would allow assessment of the quantity of bone really lost on the endosteal surfaces. This quantity of bone could be referred as to “endosteal bone loss”.
In elderly men, apparent bone loss accelerates with age [5–7]. However, the morphological basis underlying this acceleration has not been studied, and it is unclear whether it is related to faster outward displacement of a constant amount of bone, to higher endosteal bone loss and/or to a lower periosteal apposition. Moreover, only a few studies have compared the rate of apparent bone loss at different sites of measurement in elderly men [10–12], although it appears that the morphological basis underlying apprent bone loss may vary according to ROI [13].
Therefore, the aim of this study was to assess age-related apparent net and endosteal bone loss as well as their morphological basis and the age-related changes during a long-term prospective follow-up in a large cohort of elderly men (the MINOS study).
Subjects and methods
Cohort
The MINOS study is a prospective study of osteoporosis and its determinants in men, which was initiated in 1995 [14]. It is the result of a collaboration between the National Institute of Health and Medical Research (INSERM) and Société de Secours Minière de Bourgogne (SSBM) in Montceau les Mines, a town located 130 km northwest of Lyon in the Department (District) of Saône et Loire. The town has a population of 21,000 inhabitants, including 7,150 men > 19 years if age. SSMB is one of the largest health insurance companies in this town. The study was performed in accord with the Helsinki Declaration of 1975 as revised in 1983. The MINOS cohort consists of 1,040 men aged 19–85 recruited between 1995 and 1998. All men responded to an epidemiological questionnaire covering demographic and behavioural information as well as detailed medical history. Men aged 19–50 were examined once, whereas 790 men aged 50–85 were followed up prospectively for 90 months. Every 18 months, they were invited to attend the follow-up examination composed of an epidemiological questionnaire and DXA measurement. Radiograph of the spine were performed at baseline, 36 and 90 months.
Measurements
aBMD and BMC were measured at the lumbar spine (L2–L4), hip and whole body using pencil-beam DXA (QDR 1500, Hologic Inc., Waltham, MA, USA) and at the distal nondominant forearm using single energy X-ray absorptiometry (Osteometer, DTX-100, Denmark). The OsteoDyne Hip Positioner System (HPS) was used to minimise hip positioning error. ROI of the femoral neck was positioned perpendicularly to the axis of the femoral neck to cover its narrowest part. When necessary, the femoral neck edges were adjusted manually. The QDR 1500 device was calibrated daily using a lumbar spine phantom, yielding a CV for aBMD of 0.33 %. Twice a month, the Hologic hip phantom was measured, yielding a long-term CV of 0.94 % for femoral neck aBMD and 1.05 % for the femoral neck projected area. Also twice a month, a human lumbar spine embedded in methyl methacrylate was measured. Its long-term CV was 1.07 % for BMC of L2–L4, 1.07 % for the projected area of L2–L4, and 0.62 % for aBMD of L2–L4. At the distal forearm, the distal site includes 20 mm of radius situated proximally to the site where the spacing between the medial edge of the radius and the lateral edge of the ulna is 8 mm. Scans with evident error of positioning were excluded. The densitometer was calibrated daily using a calibration standard for DTX 100; its long-term CV was 0.47 % for aBMD and 0.15 % for the projected area.
Dimensions of the vertebral body of the third lumbar vertebra (L3) were measured on the anteroposterior and lateral radiographs of the lumbar spine performed at baseline and at 90 months. The cross-sectional area of L3 was calculated based on the anteroposterior and frontal diameters measured in the narrowest site of the vertebral body. Osteoarthritis of the lumbar spine was assessed, as described previously [14].
The external diameter of bone was calculated as the projected area of ROI divided by its length. The rate of age-related periosteal expansion was calculated as the average annual increase in the external diameter (femoral neck, distal radius and distal ulna), projected area (total hip) or cross-sectional area (L3). Age-related periosteal apposition (ΔBMCPA) was estimated as the mass of bone deposited on the outer surface of bone since the first measurement (Fig. 1). The volume of the ellipsoid cylinder was calculated assuming that the short axis was 0.75 of the long axis (external diameter). Then, the mass of the deposited bone was calculated as the product of the cylinder volume and the volumetric density of bone mineral (1.15 g/cm3) [15]. We assumed that during the follow-up, BMC was determined by baseline BMC, bone mass deposited on the outer surface (ΔBMCPA) and endosteal bone loss (ΔBMCEBL). Thus, ΔBMCEBL can be calculated using BMC at baseline and during the follow-up and the calculated value of ΔBMCPA. The concept of endosteal bone loss does not make any assumption as to its underlying morphological basis (cortical thinning, increased cortical porosity, trabecular bone loss) nor on the proportion of cortical to trabecular bone. It only reflects the loss of bone mineral “inside” bone, which is represented in Fig. 1 by the change of the colour from black to grey.
Fig. 1Calculation of bone mass deposited by periosteal apposition (ΔBMCPA) and of endosteal bone loss (ΔBMCEBL) between baseline (t0) and follow-up (t1). Long semi-axes of the ellipse at baseline (R0) and during the followup (R1) are equal to half the external diameter of the region of interest (ROI). Bone mass deposited by periosteal apposition (ΔBMCPA) is the product of the ellipsoid volume and the vBMD of cortical bone (d = 1.15 g/cm3). Coefficient 0.75 reflects bone flattening in the anteroposterior projection in comparison with its diameter in the frontal projection. Endosteal bone loss (ΔBMCEBL) is presented graphically as the brighter colour of the entire cross-sectional area of bone (black at baseline and light grey at follow-up) in order to indicate that the “endosteal bone loss” makes no distinction as to the morphological basis (cortical thninning, increase in cortical porosity, trabecular bone loss)
Statistical methods
All calculations were performed by using SAS version 8.2 software (SAS Institute Inc., Cary, NC, USA). Correlation between continuous variables was assessed by Pearson’s simple correlation coefficient. Individual slopes were calculated by using simple linear regression. Comparisons of the individual slopes between age groups were performed by analysis of variance (ANOVA) and adjusted for multiple comparisons by Dunnett-Hsu test. Participants attended two to six exams; those who had few measurements, e.g. two, had them over different periods of follow-up (18–90 months). Individual slopes were calculated by using two to six points distributed over 18–90 months, which could influence accuracy of the calculation. We adjusted for the duration of follow-up or the number of measurements to check whether duration of follow-up and number of measurements influenced the results. Each of them entered significant in the majority of models, although they influenced the results only to a limited degree. We present data adjusted for the duration of follow-up because this variable attained higher level of significance in the models and contributed more to the final model.
Results
Characteristics of the investigated cohort
Sixty-five men who abandoned the study after the first examination were, at baseline, older and had lower physical activity but higher prevalence of diabetes mellitus, rheumatoid arthritis and parkinsonism than the 725 men who participated in the follow-up (Table 1). They had lower aBMD and BMC at all sites of measurement except for the lumbar spine. The difference between the groups became weaker after the adjustment for age; however, it remained significant for certain sites.
Table 1Comparison of 725 men participating in the prospective study and 65 men lost to follow-up after recruitmentParameterFollow-up (n = 725)No follow-up (n = 65)p value*p value**Age (years)65 ± 770 ± 8< 0.0001Body weight (kg)80 ± 1379 ± 150.29Body height (cm)169 ± 6168 ± 70.21BMI (kg/m2)27.98 ± 3.6427.75 ± 4.510.63Fat mass (kg)22.02 ± 7.4823.23 ± 9.110.28Lean mass (kg)54.54 ± 6.6652.16 ± 7.56< 0.01NSTobacco smoking (%)11.811.60.98Physical activity (h/week)21.8 ± 12.717.2 ± 11.3< 0.005NSPrevalent fractures (%)13.819.70.18Diabetes (%)6.515.7< 0.005Rhumatoid arthritis (%)1.45.7< 0.01Parkinsonism (%)1.55.7< 0.02Lumbar spine BMD (g/cm2)1.031 ± 0.1841.052 ± 0.2130.21Femoral neck BMD (g/cm2)0.845 ± 0.1210.803 ± 0.127< 0.01NSFemoral neck BMC (g)5.111 ± 0.8494.883 ± 0.934< 0.04NSFemoral neck width (cm)4.082 ± 0.3164.123 ± 0.3560.31Trochanter BMD (g/cm2)0.740 ± 0.1090.691 ± 0.121< 0.001< 0.03Total hip BMD (g/cm2)0.966 ± 0.1270.910 ± 0.157< 0.001NSWhole-body BMC (g)2706.6 ± 410.42550.3 ± 472.7< 0.005NSWhole-body BMD (g/cm2)1.210 ± 0.1081.167 ± 0.121< 0.003< 0.05Distal forearm BMD0.524 ± 0.0650.483 ± 0.070< 0.0001< 0.01Ultradistal radius BMD0.430 ± 0.0640.399 ± 0.072< 0.001< 0.05Radius BMD (g/cm2)0.556 ± 0.0680.513 ± 0.075< 0.0001< 0.01Radius BMC (g)2.743 ± 0.4032.527 ± 0.422< 0.0001< 0.01Radius width (cm)2.471 ± 0.2072.468 ± 0.2160.94Ulna BMD0.476 ± 0.0660.438 ± 0.070< 0.0001< 0.01Ulna BMC (g)1.502 ± 0.2441.401 ± 0.246< 0.0001< 0.01Ulna width (cm)1.659 ± 0.1361.684 ± 0.1520.16BMD bone mineral density, BMC bone mineral content, NS not significantp* difference between the groupsp** age-adjusted difference between the groups
Characteristics of bone loss
BMC and aBMD of the lumbar spine increased significantly (Table 2) faster in men with severe arthritis (5.95 ± 16.15 vs 1.74 ± 10.96 mg/cm2/year; p < 0.0001). At the femoral neck, aBMD decreased, whereas BMC increased. For other sites of measurement (hip, whole body, distal forearm), both aBMD and BMC decreased. The fractional apparent and net bone loss (percentage of initial value) were fastest at the distal forearm and slowest at the whole body. All the above absolute and fractional changes in aBMD and BMC were significantly different from 0 for all ROIs.
Table 2Average rate of apparent bone loss [change in areal bone mineral density (aBMD)], net bone loss [change in bone mineral content (BMC)] and of periosteal expansion (increase in bone width or area) as well as the simple correlation coefficients of these variables with age in 725 men aged 50–85 at baseline followed up prospectively for 90 months (the prospective MINOS study)Site of measurementYearly changeCorrelation with ageBone mineral density(mg/cm2/year)(%/year)rp Lumbar spine4.205 ± 14.210.495 ± 2.910−0.0690.07 Femoral neck−2.463 ± 8.305−0.282 ± 1.019−0.154< 0.0001 Trochanter−1.963 ± 8.031−0.276 ± 1.123−0.196< 0.0001 Total hip−4.714 ± 8.451−0.496 ± 0.930−0.213< 0.0001 Whole body−2.081 ± 8.712−0.177 ± 0.723−0.0450.22 Distal forearm−2.937 ± 4.178−0.580 ± 0.870−0.202< 0.0001 Distal radius−2.986 ± 5.340−0.561 ± 1.041−0.180< 0.0001 Distal ulna−3.353 ± 5.131−0.730 ± 1.206−0.119< 0.002 Ultradistal radius−1.823 ± 5.334−0.426 ± 1.285−0.128< 0.001Bone mineral content(mg/year) (%/year) −0.0410.27 L3118.17 ± 552.900.658 ± 2.954 Femoral neck12.67 ± 50.380.263 ± 1.010−0.0510.18 Total hip−226.1 ± 0.687−0.504 ± 1.551−0.120< 0.002 Whole body−7565.9 ± 22500.9−0.294 ± 0.877−0.192< 0.0001 Distal radius−11.26 ± 25.90−0.426 ± 0.978−0.179< 0.0001 Distal ulna−8.64 ± 16.82−0.559 ± 1.164−0.123< 0.001Bone size L3 cross-section (mm2/year)35.64 ± 225.400.167 ± 1.184−0.0560.10 Femoral neck (μm/year)133.1 ± 217.20.321 ± 0.5030.0700.06 Total hip (mm2/year)4.82 ± 42.230.112 ± 0.9150.0070.85 Distal radius (μm/year)60.48 ± 362.660.257 ± 1.4970.0340.36 Distal ulna (μm/year)58.27 ± 156.610.355 ± 0.9370.0130.73
External diameter of the femoral neck, distal radius and distal ulna as well as the cross-sectional area of L3 and the projected area of total hip increased significantly during the follow-up. Fractional increase in bone size varied from 0.17% to 0.36% per year across the sites.
Characteristics of the rate of bone loss according to age at baseline
At the hip and distal forearm, rate of apparent bone loss (change in aBMD) was negatively correlated with age, indicating and age-related acceleration of apparent bone loss (Table 2). After the age of 70, the apparent bone loss was two (for distal forearm) to 23 (for trochanter) times faster than in men aged younger than 60 at baseline (Fig. 2). A similar trend was found when apparent bone loss was expressed as the percentage of the baseline value of aBMD.
Fig. 2Comparison of the absolute values of apparent bone loss [change in areal bone mineral density (aBMD)] according to age group (black bars 50–60 years, pointed bars 61–70 years, white bars > 70 years at baseline). The slopes are significantly different from 0 (p < 0.005–0.0001) for all regions of interest (ROIs) and for all age groups, except for the trochanter in the youngest group
Net bone loss (change in BMC) also accelerated with age at the total hip, whole body and bones of distal forearm. In men aged older than 70, net bone loss was two to four times as fast in comparison with men aged younger than 60 at baseline, both for the absolute values of net bone loss and for data expressed as percentage of baseline BMC (Fig. 3).
Fig. 3Comparison of the absolute values of net bone loss [(change in bone mineral content (BMC)] according to age group (black bars 50–60 years, pointed bars 61–70 years, white bars >70 years old). The slopes are different from 0 (p < 0.005–0.0001) for all regions of interest (ROIs) and for all age groups
In 183 men aged 50–60, rates of apparent and net bone loss were significantly different from 0 for all ROIs, except the trochanter. In 387 men aged 61–70 and in 155 men aged older than 70 at baseline, rates of apparent and net bone loss were significantly different from 0 for all ROIs. The rate of periosteal expansion (expressed as the change in the projected area of bone) was significantly different from 0 for all ROIs and for all three age groups. However, it did not vary across age groups for any site of measurement. Similarly, at distal forearm bones, periosteal apposition estimated as bone mass deposited annually did not change across the age groups (Fig. 4). By contrast, estimated endosteal bone loss was about twice as high after the age of 70 compared with men aged younger than 60 at baseline.
Fig. 4Upper panel: comparison of absolute values of annual rates of increase in external diameter at the distal radius and ulna according to age group. Lower panel: comparison of absolute values of rates of deposition of bone mass by periosteal apposition (positive hatched bars) and rate of endosteal bone loss (negative pointed bars) at the distal radius and ulna according to age group. The slopes are significantly different from 0 (< 0.005–0.0001) for periosteal apposition and for endosteal bone loss for both bones and for all age groups. Please note, in the lower panel, scales are different for radius and ulna
Discussion
In men, aBMD increased at the spine and decreased at the hip, distal forearm and whole body. At these sites, apparent bone loss (change in aBMD) was associated with net bone loss (change in BMC) except for the femoral neck. Apparent and net bone loss accelerated with age, whereas periosteal expansion rate remained stable. At the distal radius and ulna, endosteal bone loss accelerated with age, whereas the rate of periosteal expansion remained stable. Apparent bone loss at the hip and distal forearm was associated with a parallel net bone loss. At these ROIs, endosteal bone loss exceeded the periosteal apposition, and apparent bone loss was determined by outward displacement of lower quantity of bone mineral. Longitudinally assessed age-related apparent bone loss at the hip and distal forearm has been described in several cohorts of Caucasian men [5–7, 10, 16], but their morphological basis has not been investigated. In elderly men, apparent bone loss was associated with net bone loss at the intertrochanter ROI but with a gain in BMC at the total hip and femoral neck (narrow neck ROI) [13]. We found the same trend for the femoral neck but not for the total hip. This discrepancy is surprising because our cohort is younger. As the net bone loss accelerates with age, faster net bone loss (and not bone gain) could be expected in the older British cohort.
We confirm longitudinal and cross-sectional data on the gain in aBMD of lumbar spine in elderly men [7, 12, 14, 16–18]. Increase in the cross-sectional area of L3 at the midheight of the vertebral body is determined mainly by the periosteal expansion. This part is less involved in arthritis. Measurements performed on the X-rays allow for exclusion of the pathological calcifications. We do not speculate on the morphological basis underlying the increase in spine aBMD. Vertebral BMC and aBMD in the posteroanterior projection are determined by changes in the vertebral body and the posterior arch. Posterior arch and its processes are built mainly of cortical bone. As their periosteal surface is relatively high, its age-related gain of BMC due to periosteal apposition may be considerable. At the vertebral body, changes in BMC are determined by loss of trabecular bone inside and by the increase in BMC due to the periosteal apposition and the aggravation of arthritis. At the hip and distal forearm, both aBMD and BMC decreased, in agreement with previous studies [6, 16, 17]. Both apparent and net bone loss accelerated with ageing; by contrast, periosteal apposition remained constant. Thus, age-related acceleration of the apparent and net bone loss is determined by the acceleration of bone loss on endosteal surfaces. Apparent and net bone loss were significant in all age groups.
In women, menopause results in the acceleration of endocortical resorption and deceleration of periosteal apposition [19]. Thus, after menopause, periosteal apposition continues, as reported previously by Ahlborg et al. [20]. However, it does not increase sufficiently to offset endocortical bone loss. On the contrary, it decreases, which may contribute to the deterioration of bone strength and increased fracture risk. We postulated that postmenopausal osteoporosis is a disease of failed adaptation of bone [19]. However, in men also, age-related increase in endosteal bone loss is not accompanied by an increase in periosteal apposition that would offset the possible loss of strength. Periosteal apposition does not adapt to the endosteal bone loss and remains stable. This imbalance may result in a decrease of bone strength. Thus, in men also, osteoporosis seems to be a disease of failed adaptation.
Our data are consistent with the cross-sectional data obtained in elderly men by using the high-resolution peripheral quantitative computerised tomography (hr-pQCT) showing the increase in bone size at the axial and peripheral skeleton, as well as endosteal bone loss characterised by decrease in the trabecular volumetric BMD (vBMD) determined mainly by the decrease in the trabecular number, increase in total marrow area and cortical thinning, indicating endocortical resorption, and decrease in cortical vBMD [21, 22]. The decrease in cortical vBMD is determined mainly by the increase in the midcortical and subendocortical porosity, whereas subperiosteal porosity and real volumetric density of the mineral of cortical bone remain relatively stable during ageing [15, 23].
Our data help in understanding the difference in the age-related increase in fracture incidence. Young men have bigger bones than women, even after adjustment for age, weight and height [24, 25]. Then, aBMD and BMC decrease. Age-related acceleration of apparent and net bone loss is consistent with the exponential increase in fracture incidence. However, in contrast to women, periosteal apposition rate remains stable in men and may partly offset the endosteal bone loss and associated loss of bone strength. In elderly men, bones are larger than in women (because they were larger in youth and age-related periosteal apposition was greater), net bone loss is less (because periosteal apposition continues to deposit bone on the outer surface) and loss of bone strength starts later (because continuing periosteal expansion compensates partly for the loss of bone strength due to endosteal bone loss). These data are consistent with epidemiological observations that in men, fracture incidence increases later and, for a given age, is lower than in women [1, 26, 27].
Our study has limitations. Montceau les Mines is a small town, and its inhabitants may not be representative of the French population. The response rate for the invitation was 23%. Men who abandoned the study after the first examination were older and sicker, although they represent only 8% of the initial cohort. Men who were followed up may have been healthier than the general population, especially in the oldest group. However, this difference would have underestimated the age-related bone loss and its age-related acceleration. A number of men had lower number of DXA scans because they did not attend examinations regularly or abandoned the study before the end of the follow-up. A low number of DXA values and shorter follow-up could influence the accuracy of estimation of slopes. However, adjustment for the follow-up duration or the number of scans did not influence the results.
DXA presents limitations in the evaluation of bone width. In very old men, subperiosteal bone mass can be low and not recognised by the edge-detection system. This artefact can underestimate the bone width in elderly men and the age-related increase in bone width mainly in the cross-sectional studies (where the age range is large) but less so in the longitudinal study (where the follow-up period is shorter). The projected area of femoral neck may be overestimated because of calcifications in fibrous tissue. The measured radius site is established by the device. According to the individual anatomy, this site may be more distal (larger and more trabecular) or more proximal (narrower and cortical). Again, this artefact may introduce a bias mainly in cross-sectional studies.
Calculation of endosteal bone loss is indirect and based on the assumptions such as uniform bone flattening, constant subperiosteal bone vBMD and proportional periosteal expansion in all axes. By contrast, the advantage of this concept is that we do not make any assumption on the morphological basis underlying the endosteal bone loss (cortical thinning or trabecular bone loss, proportion of cortical to trabecular bone, similar or different rates of trabecular and cortical bone loss, etc.). Finally, our calculation of endosteal bone loss was carried out for the predominantly cortical sites and, although globally consistent with the cross-sectional data obtained by hr-pQCT, may not necessarily apply for the predominantly trabecular sites.
In conclusion, in a large cohort of elderly men, age-related apparent bone loss (aBMD) at the hip, distal forearm and whole body was determined by the net bone loss (BMC), except for the femoral neck. Apparent and net bone loss accelerated with age, whereas the periosteal expansion rate (widening of ROI) remained constant. At the distal forearm, age-related acceleration of the apparent bone loss was determined by the higher endosteal bone loss, whereas the periosteal apposition rate (estimated mass of deposited bone) remained constant.

bone loss
men
periosteal apposition
osteoporosis


Symptomatic asymmetry in the first six months of life: differential diagnosis

Asymmetry in infancy is a clinical condition with a wide variation in appearances (shape, posture, and movement), etiology, localization, and severity. The prevalence of an asymmetric positional preference is 12% of all newborns during the first six months of life. The asymmetry is either idiopathic or symptomatic. Pediatricians and physiotherapists have to distinguish symptomatic asymmetry (SA) from idiopathic asymmetry (IA) when examining young infants with a positional preference to determine the prognosis and the intervention strategy. The majority of cases will be idiopathic, but the initial presentation of a positional preference might be a symptom of a more serious underlying disorder. The purpose of this review is to synthesize the current information on the incidence of SA, as well as the possible causes and the accompanying signs that differentiate SA from IA. This review presents an overview of the nine most prevalent disorders in infants in their first six months of life leading to SA. We have discovered that the literature does not provide a comprehensive analysis of the incidence, characteristics, signs, and symptoms of SA. Knowledge of the presented clues is important in the clinical decision making with regard to young infants with asymmetry. We recommend to design a valid and useful screening instrument.
Introduction
The objective of this descriptive review is to determine and classify the possible causes of asymmetry seen in young infants who have an asymmetric head and/or body posture, as well as to present an overview of the nine most prevalent disorders in infants in the first six months of life leading to the diagnosis of symptomatic asymmetry (SA). Asymmetric infants form an increasing and complicated group of children seen by professionals from various clinical specialties, such as well baby clinic physicians, pediatricians, pediatric physiotherapists, orthopedic surgeons, and plastic surgeons [4, 6, 19, 37, 50, 61]. Asymmetry in infancy is a mostly benign symptom, but in this early phase of life, the differential diagnostics are extensive. The background of the professional influences the way in which associated clinical problems are evaluated. A screening instrument would be helpful. The first step in this process is to synthesize the current information in the literature about differential diagnostics.
Twelve percent of all Dutch newborns develop a positional preference in the first few months of life, different from the physiological asymmetry [6]. A “positional preference” is defined as a condition in which the infant’s head is turned toward one side most of the time and active movement to the other side is restricted [4, 6]. About 25% of these infants (approximately 5,000 a year in the Netherlands) are referred to pediatric physical therapists [6]. Asymmetry in infancy is a clinical condition with a wide variation in appearances (shape, posture, and movement), etiology, localization, and severity. From the referred infants, the asymmetry is either idiopathic or symptomatic, and originates ante- and/or postpartum [37, 48, 50, 51, 61]. In case of an idiopathic asymmetry (IA), the etiology is uncertain; environmental factors play a major role in the development of the asymmetry [6, 34, 35, 48, 62]. In SA, an underlying disorder, disease, or dysfunction causes the asymmetry. The majority of cases will be idiopathic, but an initial presentation of positional preference might be a symptom of a serious underlying problem. In the last decade, many studies on the appearances of IA have been published. If the focus in diagnostics and pattern recognition is on IA, there is a chance that an SA will be missed [3, 20]. When examining young infants with a positional preference, differentiating SA from IA is necessary to determine the prognosis and to choose appropriate intervention strategies.
This review will address the following question: which diagnoses, incidence rates, signs, and symptoms are described in the literature and are thought to cause a symptomatic asymmetrical posture or movement pattern in infants during their first six months of life?
Methods
Search strategy
This review is based on a comprehensive literature search on SA. The following strategy is used: peer-reviewed literature on this topic in journals with a science citation index was searched, as well as clinical textbooks from the various clinical specialties. Computerized bibliographic databases were searched (PubMed, Pedro, Cinahl, and Cochrane Controlled Trials Register), and related papers and their references. General keywords used were: asymmetry, plagiocephaly, torticollis, posture, scoliosis, (differential) diagnosis, and screening. The search then focused on specific diagnoses that might cause asymmetry in infancy condition with the keywords: etiology, tumors, disorders (related to) vision, hearing, central nervous, or musculoskeletal system, obstetric complications, brachial plexus palsy/lesion, clinical syndromes (Grisel, Sandifer), congenital anomalies and syndromes, gastroesophageal reflux, developmental dysplasia of the hip, paroxysmal torticollis, (birth) trauma, and clavicle fracture. Finally, we focused on the incidence and prevalence. The search was limited to citations that included: “all infants, birth–23 months,” had an abstract, were written in English, and the search terms were in the title or abstract. The year of publication was not restricted. When more papers on the same subject were found, the most current studies were chosen. Only diagnoses that could be observed in infants in the first six months of life were included. Unique case reports and innocuous abnormalities that require no specific treatment were not included.
Results
The prevalence and/or incidence of the various medical diagnoses leading to SA was not always documented. Some disorders had no consistency in their reported incidence rates. The majority of children with a positional preference or asymmetry during the first six months of life are diagnosed with an IA [4, 6, 34, 35, 37, 48, 50, 51]. Table 1 shows a selection of the most frequently detected disorders causing an SA.
Table 1Disorders related to symptomatic asymmetry (SA) from the literature searchDisorders with known incidenceIncidence/1,0001. Developmental dysplasia of the hip (DDH)402. Perinatal fracture of the clavicle353. Congenital muscular torticollis (CMT)204. Obstetric brachial plexus palsy45. Central nervous system disorders26. Craniosynostosis/lambdoid suture0.03Remaining groups of disorders7. Congenital abnormalities or malformations Musculoskeletal Chromosomal8. Sensory systems Ocular disorders Hearing disorders9. Acquired asymmetry postpartum in one of the remaining systems (non musculoskeletal)
In the last decade, discussion on positional preference leading to deformational plagiocephaly (DP) has increased substantially. A relatively high number of hits found during a search in January 2008 within PubMed resulted in the following: asymmetry (811), plagiocephaly (206), torticollis (225), posture (405), and scoliosis (623). However, when combined with “differential diagnosis” or “screening,” the result decreased to less than 20 each. Differential diagnosis from craniosynostosis was often described [24, 33, 36, 51]. The main designs were retrospective or prospective descriptive studies and reviews. In diagnoses with a low incidence, the studies were predominantly case reports.
All diagnoses were classified according to the International Classification of Diseases [1]. The results of the literature search are presented below, starting with the diagnosis with the highest incidence rate.
Developmental dysplasia of the hip
Developmental dysplasia of the hip (DDH) has a high rate of co-morbidity with congenital muscular torticollis (8–20%) [11] and, to a lesser extent, with postural torticollis or scoliosis [50]. The reported incidence rate in the Netherlands ranged from 3 to 4% of all newborns [4, 11, 48], with 80% being unilateral [5, 56]. The clinical signs which are described include asymmetry in hip abduction and leg length and/or asymmetrical skin folds in the inguinal and upper thigh region. The strong association with other asymmetries warrants a thorough screening on the signs of developmental dysplasia of the hip in infants with an asymmetry.
Perinatal fracture of the clavicle
A fracture of the clavicle during birth may induce a positional preference in the first weeks of life and, as such, may cause an asymmetry. A perinatal fracture can be an option in the differential diagnostics of asymmetry during the first weeks of life. A co-incidence with other perinatal injuries (like brachial plexus injury) was described by Perlow et al. [49]. The obstetric brachial plexus lesion is described separately. The incidence varies between 0.1 and 3.5% [28, 40, 49, 54], and these fractures usually consolidate within 3 weeks without complications. The clinical presentation can be asymptomatic. When symptomatic, signs include: decreased or absent movement and pain, or tenderness on movement of the arm on the affected side and palpable irregularity along the clavicle [54].
Congenital muscular torticollis
Congenital muscular torticollis (CMT) is frequently described in the literature. Unilateral fibrosis or thickening of and tightness in the sternocleidomastoid muscle can cause a characteristic posture of the head and restricted neck movements. The etiology of the pseudo-tumor or mass is unclear [10, 11, 19, 58]. A compartment syndrome due to intra-uterine malposition is the most frequently mentioned etiological hypothesis [10, 12, 19, 39]. An association with birth trauma and breech presentation is mentioned, but the evidence is weak, since CMT is also seen in infants born via a cesarean section [11, 32]. A pseudo-tumor can be palpated in the second or third week after birth. Incidence rates of CMT vary between 0.3 and 2% [10–12, 19, 58]. Ultrasound screening soon after birth has indicated that the incidence rate could be 3.9%. This screening method tends to be especially sensitive in detecting occult cases of fibrosis [10].
Fibroids of the uterus and other intrauterine tumors are described as a possible etiology to an atrophy of the sternocleidomastoid muscle of the child [18]. This phenomenon can cause a unilateral muscular dysfunction and a strong imbalance between both muscles. The distinction between CMT and postural torticollis is not always clear [15]. A CMT is primarily a condition with a structural fibroid shortening of the sternocleidomastoid muscle, visible and palpable in the first weeks of life, as opposed to a postural torticollis that occurs secondary to a positional preference and a DP [6, 37, 48, 51, 61].
Obstetric brachial plexus palsy
Palsy of the brachial plexus during delivery is caused by traction or compression of the plexus during labor. In most cases, the upper brachial plexus is affected; in 15% of the patients, hand function is also impaired. The described incidence is 0.1–0.4% [31, 49, 52]. The extent of the neural damage becomes evident during the first six months of life [52], although in severe cases, the inactivity of the extremity is observed from birth onwards. Timely recognition of severe cases is important, since neurosurgical intervention can enhance future capacities. Between 20 and 25% of the infants experience persistent functional impairments [31].
Central nervous system disorders
Cerebral palsy (CP) syndromes, in particular, spastic unilateral CP, are neurological disorders that can cause asymmetry [3, 7]. Serious disorders of the central nervous system are generally easy to recognize, but a CP may also be discrete with subtle features. Early diagnosis, before the age of six months, might be difficult [45, 57]. The neurodevelopmental (motor) behavior is an important issue in early recognition: persistent infantile reflexes and abnormal muscle tone, motor delay, abnormal spontaneous movement patterns (especially “general movements”), and poor postural control are more or less predictors of CP [25, 45, 46]. In a review on the epidemiology of CP, the world-wide prevalence of all types of CP is estimated at 0.2% [43].
Hypotonia and developmental delay were also mentioned as causes for developing positional preference and DP. A neurological disorder might be the underlying problem, but it is not always diagnosed at this young age [3, 7, 51].
Craniosynostosis
Craniosynostosis, the premature fusion of one or more cranial sutures, is most frequently described in relation to asymmetry and plagiocephaly, possibly as a result of the over-referral of infants with deformational non-synostotic DP to craniofacial or plastic surgery clinics. Primary craniosynostosis is either simple or compound and part of a genetic syndrome [36]. Premature closing of one lambdoid suture is the most frequently mentioned differential diagnosis of DP [24, 33, 36]. The incidence of this single suture craniosynostosis is rare (1–3 cases to 100,000 newborns) [37, 42] and can be clinically differentiated from DP by four major signs: from the vertex view, a trapezoid head shape can be observed, a palpable unilateral ridge, bulging of the unilateral mastoid, and an asymmetric skull base with tilt to the ipsilateral side [24, 33, 42]. The impact of the premature closure of cranial sutures in complex craniosynostosis is impressive: strong progressive deformation of the skull, risk of increasing intracranial pressure, and developmental problems. When evident, timely surgical intervention is warranted [24, 33, 36, 50, 51].
The following three categories are groups of disorders. Clear incidence rates could not be found.
Congenital abnormalities or malformations
Musculoskeletal congenital malformations must be considered in an asymmetry that is present immediately after birth [3, 7]. Well-known malformations are those of the spine, such as a Klippel-Feil syndrome, hemi-vertebrae, and a hemi-atlas [22, 38, 64]. Exceptional phenomena are hypoplasia or aplasia of the face, neck, or trunk muscles [2]. Patients may show defects in other systems as well, such as syndactyly, deafness, or a congenital heart disease. The co-incidence of defects may be an important sign of a syndrome. An asymmetric development or posture can be an associated finding in a variety of syndromes and abnormalities. These features are often present immediately after birth, but will not always be discovered until a second stage [64]. Local abnormalities, such as a vascular ring (around the trachea) or tracheomalacia, are occasionally an indirect cause of an asymmetric posture [60].
Disorders in sensory systems
In the screening of infants with asymmetry, eye movement and/or vision and hearing disorders must be considered. Infants with congenital nystagmus and restrictive or paralytic strabismus may use anomalous head positions to maximize visual function [26, 47, 63]. No clear incidence rates were found. A predictive factor for an ocular origin of torticollis is the family history of ocular problems, in particular, congenital nystagmus. The ocular pathology may be subtle. In case of doubt, infants must be referred to an ophthalmologist [63].
Theoretically, a unilateral hearing disorder can induce a positional preference in young infants. In the literature search, no match was found for hearing loss and torticollis, except in syndromes such as Klippel-Feil or Moebius. A connection between ear malformation and hearing loss is mentioned [23].
Acquired asymmetry, non-musculoskeletal
A number of disorders in systems other than the musculoskeletal system can cause a postpartum asymmetry, but the asymmetry is not the only symptom. The disorders have in common that their symptoms are not stable and occur some time after birth. The signs and symptoms can be seen as so-called “red flags” and require immediate medical evaluation. It may be secondary to a trauma [27] or to inflammatory conditions, such as pharyngitis [8, 13, 29, 55]. Grisel syndrome (a non-traumatic atlanto-axial rotatory subluxation following infections of the upper respiratory tract) is often described, but never under the age of six months [7, 19, 29]. Another cause can be related to the cardio-respiratory or the digestive system, such as Sandifer syndrome (fluctuating asymmetry with abnormal body movements and contortions of the neck, associated with gastroesophageal reflux) [9, 17, 21]. The most alarming causes of asymmetry are related to neurological syndromes, such as syringomyelia, epilepsy, high intracranial pressure, postencephalitic syndromes, or life-threatening tumors of the central nervous system [3, 7, 12, 19, 20, 30]. These disorders are mainly described in case studies, without proven incidence rates. The signs and symptoms of these non-musculoskeletal causes are described in Table 2.
Table 2Signs and symptoms of acquired symptomatic asymmetry disorders with a low incidenceSigns and symptomsHints for disordersGeneral historyHeavy painRetro-pharyngeal abscess [13]Vomiting/drowsinessIncreased intracranial pressure [30]Lethargy/irritabilityTumor [12, 30], intracranial injury [27]TraumaIntracranial injury [27]Seizures/convulsionsEpilepsy; increased intracranial pressure; Sandifer syndrome [17]Acute onsetInfection, abscess [13]; Grisel syndrome (>6 months) [29, 55]Stridor, dyspneaVascular ring [60]RefluxSandifer syndrome; pathological gastroesophageal reflux [17]FeverInfection, abscess [13]Specific examinationSunset phenomenonIncreased intracranial pressureBulging anterior fontanelIncreased intracranial pressure, intracranial injury [27]Abnormal courseIncreasing head tiltInfection [13]; tumor [12, 30]Recurrent episodesBenign paroxysmal torticollis [9, 21]
Discussion
Asymmetry in infancy is a condition with a high prevalence in infants in the first six months of life. In the majority of cases, the origin is idiopathic and is often related to environmental factors [6, 15, 34, 35, 37, 62]. This review addresses the possible causes, incidence rates, and symptoms of symptomatic asymmetries due to an underlying disorder, dysfunction, or disease.
Not all of the incidence rates could be found, while some inconsistencies were observed in the current literature. The disorders with a high prevalence are well described in epidemiologic studies. The rarer diseases were, most of the time, documented in case reports without incidence rates. The incidence rates mentioned in the studies are inconsistent because of different opinions regarding the operationalization and assessment of the SA. Frequently, psychometric properties of instruments and concepts have not been described or evaluated. Variations in incidence rates (e.g. CMT) are inevitable, considering the variety in inclusion criteria and diagnostic tests used in the studies. The sequence in estimated incidence rates, as proposed in Table 1, is open to debate.
A clear description of signs and symptoms was not always presented in the literature. The variety in the etiology of asymmetry is considerable. The level of evidence of the included studies varies. Literature of more than 10 years ago mainly described underlying causes of SA, in particular, non-muscular torticollis [3, 7]. They still turned out to be useful in establishing criteria for differential diagnostic screening and are widely cited in current studies. However, an update regarding new developments in studies on infant asymmetry is needed. The exponential increase of plagiocephaly in the last decade, related to the recommendations to put babies on their back to sleep, is reflected in the objectives of recent studies [4, 6, 37, 41]. They mainly focused on IA and its predispositions, with little attention to SA. A number of recent papers described features to distinguish craniosynostosis from DP. Although craniosynostosis has a very low incidence, craniofacial clinics are deluged with infants with DP [14, 15, 37]. One of the positive effects of this situation is that authors from this background described useful clinical diagnostic criteria for craniosynostosis.
van Vlimmeren et al. [61] stated in their review on diagnostic strategies, that asymmetry in infancy is a diagnosis with a large spectrum of features and a multifactorial etiology without consensus on definition, nomenclature, or classification. In the present review, a classification by virtue of etiology is proposed. The dichotomy, symptomatic versus idiopathic, is often used in medicine [16, 44, 53, 59] and fits well with this health problem, since a large number of children have unexplained asymmetry.
Although flow diagrams for diagnostic strategies are presented in some reviews [3, 7, 15, 19, 61], clear clinical diagnostic criteria that could be used were not mentioned. The criteria found in the present review might be considered in a future study. An expert validation, such as a Delphi study with clinical experts, could be a next step towards establishing clinical diagnostic criteria as warning flags in young infants with IA or SA.
This review presents an overview of the most common disorders underlying SA in infants less than six months of age. We have discovered that the literature does not provide a comprehensive analysis of the incidence, characteristics, signs, and symptoms of SA. Knowledge of the presented clues is important in the clinical decision making with regard to young infants with asymmetry. The endpoint of this review may be a starting document for the creation of a protocol, but it needs additional studies in order for it to become a valid and useful screening instrument.

symptomatic asymmetry
differential diagnosis
etiology
infant
screening
plagiocephaly
torticollis


Assessment of Respondent Driven Sampling for Recruiting Female Sex Workers in Two Vietnamese Cities: Reaching the Unseen Sex Worker

Respondent driven sampling (RDS) is a relatively new method to sample hard-to-reach populations. Until this study, female sex workers (FSWs) in Vietnam were sampled using a variety of methods, including time location sampling (TLS), which may not access the more hidden types of FSWs. This paper presents an analysis from an HIV biological and behavioral surveillance survey to assess the feasibility and effectiveness of RDS to sample FSWs, to determine if RDS can reach otherwise inaccessible FSWs in Vietnam and to compare RDS findings of HIV risk factors with a theoretical TLS. Through face-to-face interviews with FSWs in Ho Chi Minh City (HCMC) and Hai Phong (HP), data were collected about the venues where they most often solicit their clients. These data were used to create three variables to assess whether FSWs solicit their clients in locations that are visible, semi-visible and non-visible. For this analysis, the visible group simulates a sample captured using TLS. Survey results in HIV prevalence and related risk factors and service utilization, adjusted for sampling methodology, were compared across each of the three FSW visibility groups to assess potential bias in TLS relative to RDS. The number of self-reported visible FSWs (HCMC: n=311; HP: n=162) was much larger than those of the semi-visible (HCMC: n=65; HP: n=43) and non-visible (HCMC: n=37; HP: n=10) FSWs in HCMC and HP. Non-visible FSWs in both cities were just as likely as visible and semi-visible FSWs to be HIV positive (HCMC: visible 14.5%, semi-visible 13.8%, non-visible 13.5%, p value = 0.982; HP: visible 35.2%, semi-visible 30.2%, non-visible 30.0%, p value = 0.801), to practice behaviors that put them at risk for contracting and transmitting HIV (injecting drug use—HCMC: visible 13.8%, semi-visible 12.3%, non-visible 5.4%, p value = 0.347; HP: visible 38.9%, semi-visible 23.3%, non-visible 30.0%, p value = 0.378, to have no condom use in the past month —HCMC only: visible 52.7%, semi-visible 63.1%, non-visible 48.6%, p value = 0.249) and to have symptoms of a sexually transmitted infection (STI) in the past year (HCMC: visible 16.1%, semi-visible 12.3%, non-visible 16.2%, p value = 0.742; HP: visible 13.6%, semi-visible 18.6%, non-visible 20.0%, p value = 0.640). There was a difference found among the visible, semi-visible and non-visible groups in HP for no past month condom use (visible 53.1%, semi-visible 79.1%, non-visible 60.0%, p value = 0.009). This study found that RDS was successful at recruiting hidden types of FSWs in Vietnam. Past reports of FSWs in Vietnam have assessed the more visible FSWs as being the most vulnerable and at risk for HIV. Although the number of visible FSWs is much higher than those of the semi and non-visible groups, this study found that the non-visible FSWs are very vulnerable to HIV infection. If prevention programs are targeting and responding to those who are most likely to be assessed (e.g., more visible types of FSWs) then this analysis indicates that a significant proportion of the FSW population at risk for HIV may not be receiving optimal HIV information and services.
Introduction and Background
Vietnam is in the midst of an HIV epidemic concentrated among injection drug users (IDUs) and, increasingly, female sex workers (FSWs). In recent years, the epidemic in Vietnam appears to be moving rapidly from IDU populations to FSWs through sexual contact with injecting male clients and the adoption of injection drug use behaviors by FSWs. Sexual transmission of HIV from FSWs to their non-injecting clients, who in turn have non-commercial sex partners, greatly increases the probability that HIV will spread to the general population. In 2002, the Department of Social Evils in the Ministry of Labor, Invalids, and Social Affairs estimated that 130,000 women work as sex workers in Vietnam (other estimates put the number much higher).1–3
To understand and respond to HIV transmission patterns in Vietnam, numerous surveys have measured HIV prevalence and related risk factors among FSWs. However, the lack of sampling frames, relative rarity of this population, and their stigmatized and/or illegal behaviors, hinder investigators from obtaining representative samples of FSWs. Further, it is important to recognize the heterogeneity of the sex worker population. Broadly, FSW can be dichotomized into direct and indirect sex workers. Indirect sex workers typically have a primary job or source of income other than sex work, though that job may lead to contacts with potential clients (e.g., bar maids or masseuses). Direct sex workers typically do not have another job. Direct sex workers can be further broken down into street-based, brothel-based, or gatekeeper-based. The latter may involve a formal “pimp”/agent relationship or informal referrals from hotel concierges or taxi drivers. Use of the Internet to find clients is increasing also.
There is a concerted effort by the Government of Vietnam to reduce prostitution by placing FSWs in administrative detention in government rehabilitation centers where they can spend anywhere from 1 month to several years. Fear of detention leads FSWs to exercise greater caution and more subtlety on the streets. Recently, FSWs have become even more difficult to reach because the Vietnamese government is imposing penalties (fines, jail sentences and possible closure) on the owners of entertainment establishments where sex is allegedly sold.4 As a result, FSWs will now wait off-site for an establishment owner or some other gatekeeper to contact them at a client’s request.
FSWs are also hard to reach because they are highly mobile and move regularly from bar to bar, between districts and towns, and within and to other provinces.5–9 Some FSWs are difficult to locate because they have primary jobs and engage in sex work part-time. For instance, women from rural areas may travel to urban areas to sell agricultural goods and only sell sex as a way to earn extra income.5 Higher paid FSWs, such as those who solicit clients from scooters, the Internet, through agents and over the telephone, have the financial capacity to remain hidden.5,10
Past efforts to survey FSWs in Vietnam, including annual sentinel HIV surveillance, have relied on convenience samples from rehabilitation centers and street-based FSWs. These samples comprise the type of FSWs who are reportedly the most vulnerable, and therefore most at risk, and visible, (i.e., visible enough to be captured by the police or found by public health staff) and do not represent the entire sex work community.5 Further efforts to assess HIV related risk factors among FSWs have used other non-probability sampling methods, such as institutional sampling, targeted and chain referral sampling.11–16 FSWs in Vietnam have also been sampled using a probability sampling method called time location sampling (TLS), also known as time–space or venue–day–time sampling.17,18 However, TLS can only be representative of those FSWs who are easily located at visible sites, thereby missing potentially important information from other types of FSWs.
The Vietnamese Ministry of Health, with technical assistance from Centers for Disease Control and Prevention—Global AIDS Program, conducted a survey using a relatively new sampling method to assess HIV prevalence, risk factors and service utilization among FSWs. The goal of the survey was to provide baseline information before increasing numbers of HIV prevention programs targeting these populations were initiated. This sampling method, known as respondent driven sampling (RDS), is a variant of chain-referral sampling that utilizes an incentive for being interviewed and another incentive for recruiting peers (other FSWs) to be interviewed.19–22 Sampling begins by selecting a small number of non-randomly selected members of the target population, known as seeds, to take part in the study. Seeds initiate the chain referral by recruiting a fixed number of peers who, in turn, recruit other peers. The process continues until the target sample size is attained. Links among peers are recorded using anonymous identification numbers.
Although RDS is relatively new to public health researchers, it is starting to be used widely in place of other well known types of sampling methods for hard to reach populations. Researchers are considering the advantages of using RDS in place of TLS, the only other probability method for hard to reach populations, to conduct future HIV biological and behavioral surveillance surveys. TLS has been used to sample FSWs who gather and are accessible at public locations (or venues) that can be listed in a sampling frame.23–25 TLS entails identifying days and times when the target population gathers at specific locations (e.g., brothels, city blocks, bars), constructing a sampling frame of time and location units, randomly selecting these units and systematically intercepting consenting members of the target population for interviews.24–26 When implemented correctly, TLS allows researchers to construct a sample with known probabilities, make statistical inferences to the larger population of venue visitors, and theorize about the introduction of biases that may limit generalization of results to the target population.23
Two TLS surveys of FSWs in Vietnam have been reported. In 2000 Family Health International (FHI) conducted a behavioral survey with karaoke (n=2,302) and street-based (n=1,300) FSWs in five Vietnamese cities using TLS.18 During 2003, the Vietnamese National Institute of Hygiene and Epidemiology collected data from karaoke (n=1,367) and street-based (n=976) FSWs from five provinces.17
Although TLS can produce a probability sample by ensuring that time–location units are enumerated and selected randomly, it allows researchers to reach only those segments of the population who are visible and identifiable. Depending on the population of interest, this may introduce a bias by differential sampling of the portion of the population which is most at-risk. TLS can be expected to miss those segments that are less visible (e.g., FSWs who solicit clients through agents, by telephone or Internet). The “visible” portion of a population does not represent the whole population any more than an institution or clinic-based population does; “hidden” population members have a zero probability of selection, reducing representativeness of the sample.
The objectives of this paper are to present findings from a survey on HIV prevalence, risk behaviors and service utilization to (1) assess the effectiveness of RDS to sample FSWs in Vietnam; and (2) to compare RDS findings with theoretical TLS. This is especially important given that evaluations of RDS used for HIV surveillance have only come from IDUs and males who have sex with males populations in the United States.20,22,27–29 Until this study, RDS has not been evaluated in Asia among FSWs. This methodology warrants investigation to assess its ability to sample hard to reach populations in international settings.
Materials and Methods
Data to evaluate RDS were collected from FSWs in Ho Chi Minh City (HCMC) and Hai Phong (HP), Vietnam. Eligible participants were over the age of 18 and had exchanged sex for money in the previous month. FSWs were recruited by their peers (other FSWs) and provided a small monetary incentive for completing a questionnaire and providing a saliva sample to test for HIV (US \$3 in HP and US \$4 in HCMC). FSWs were provided another incentive for recruiting up to three other FSWs to participate (approximately US \$1 in HP and US \$1.50 in HCMC for each eligible recruit). Data were gathered from FSWs in HP from April to June and in HCMC from May to July, 2004. Recruitment began with 20 FSWs, non-randomly selected by survey staff, in each city. Outreach workers selected these initial recruits, or “seeds,” based on where they most often solicited their clients. A majority of the seeds were from the visible groups, street-based and karaoke-based FSWs. A few seeds from hotels and guesthouses were recruited.
Once seeds consented, they completed a one-hour face-to-face interview with a trained Vietnamese interviewer. The interview instrument consisted of questions about the participant’s demographic background, current and past drug use, sexual behaviors, HIV knowledge, risk perceptions and service utilization, and about where they usually solicit their clients. After the interview, survey staff provided participants with three coupons with which to recruit up to three other FSWs who were co-workers, friends, acquaintances or family members. Each coupon contained a unique identification number and provided basic information about the study and the addresses of the interview sites. Respondent/recruiters were asked to return to collect their incentives for successful recruits. A brief follow-up questionnaire was administered during this visit to ascertain the recruiter’s relationship to their recruits and the number and type of refusals they received. This permitted the assessment of participation bias due to refusals or no-shows.
Oraquick™ HIV antibody rapid test kits for oral fluids were used for HIV testing. No one refused to provide an oral sample. Participants did not learn their test results at the interview site but were provided a voucher to receive a free HIV test with counseling at a nearby voluntary counseling and testing (VCT) center.
For the assessment of bias in a theoretical TLS-drawn sample in comparison to an actual RDS-drawn sample, respondents were classified as accessible, i.e., visible to TLS, if they reached their clients in a public venue. Categories were established from the response to the question, “Where do you usually reach your clients?” Responses were aggregated into three groups: visible (street, park, karaoke bar); semi-visible (brothel, guesthouse, hotel); non-visible (agent, telephone, Internet). Our assumption is that the visible group could potentially be captured by TLS. The semi visible group may or may not be captured with TLS, and the non-visible group would be missed with TLS.1
Statistical Analysis
Adjusted proportions and 95% confidence intervals (ci) for FSWs’ level of visibility were analyzed using the RDS analysis tool (RDSAT).30 RDSAT adjusts proportion estimates for participants’ network sizes and recruitment patterns.20,31 RDSAT adjustments use principles of social network theory to account for biases found in conventional chain referral sampling.31,32
Chi-square proportions and p-values for comparisons of HIV risk factors and service utilization of RDS and theoretical TLS were analyzed using SPSS 11.0. Five of the 20 non-randomly selected seeds in the HP sample, and four seeds in the HCMC sample were not used in the final analysis since they did not recruit any peers. It is not known why these seeds did not recruit anyone. The sample size was calculated based on 15% increase in use and knowledge of CDC sponsored VCT HIV services and outreach. We estimated low baseline knowledge and use of services (no actual data available) with high HIV prevalence. HP sample size was 200 based on higher knowledge from existing health education programs. The sample size in HCMC was 400, assuming lower baseline knowledge. Design effect was set at 2.
This study was reviewed by the Associated Director for Science of the National Center for HIV, STD and TB prevention at CDC who classified this as a surveillance activity. The protocol for this study was reviewed and approved by the LIFE-GAP Office of the Vietnam Ministry of Health.
Findings
A total of 628 individuals, including seeds, were interviewed in fixed interview sites in HCMC (n=413) and HP (n=215). The majority of the FSWs in the HCMC (54.6%) and HP (51.3%) sample were under the age of 29 years old. More than 50% of FSWs in HCMC and HP had no occupation other than selling sex.
During the follow-up visits to collect recruitment incentives, most participants reported giving out all of their coupons. According to the 174 recruiting participants in HCMC and 90 in HP, each of whom received three coupons, a total of 75 (15.3%) individuals in HCMC and 24 (10%) in HP refused to accept a coupon; 147 recruiters in HCMC and 83 in HP reported that they were successful in distributing their coupons and that no one to whom they tried to give a coupon rejected one; so refusals were clustered among a handful of recruiters, 27 in HCMC and 7 in HP. According to recruiters, most refusers did so because of “fear of identification,” (57% in HCMC; 75% in HP), others “felt uncomfortable” (27% in HCMC; 8.3% in HP), still others thought they were “too busy” (10.7% in HCMC), others thought the “incentive not worth the time” (17% HP) and a small number of FSWs in HCMC responded that they were “not interested.”
Overall, the recruited FSWs were diverse with respect to where they solicited their clients. Figure 1 depicts the diversity of sex work types among the links within a single recruitment chain of FSWs in HCMC (n=59 recruits). In this example, the seed recruited three FSWs who were not street-based: two based in guesthouses and one based in karaoke.2 The selected karaoke-based FSW recruited two other karaoke-based and one guesthouse-based FSW. Near the outer waves of this recruitment chain, there are some more hidden types of FSWs such as agent-based and telephone-based FSWs.
Figure 1RDS recruitment network for one seed for type of female sex worker in HCMC, Vietnam.
The distribution by visibility was visible (HCMC, n=311; HP, n=161), semi-visible (HCMC, n=65; HP, n=43) and non-visible (HCMC, n=37; HP, n=12). Figure 2 shows that the largest adjusted proportions of FSWs in both HCMC and HP reached by RDS were visible [HCMC, 73.7%; 95% ci(67.0%, 79.5%) and HP, 75.9%; ci(67.1%, 85.3%)]. RDS reached a small proportion of non-visible [HCMC, 9.3%; ci(6.0%, 13.1%) and HP, 9.7%; ci(1.3%, 18.2%)] and semi-visible FSWs [HCMC, 16.9%; ci(11.5%, 23.4%) and HP, 14.2%; ci(9.1%, 21.2%)], who theoretically could have been missed by TLS.
Figure 2Distribution of visible, semi-visible and non-visible FSWs in HCMC and Hai Phong, Vietnam; adjusted proportions (number). Visible FSWs are assessed to be the most visible of the FSWs in Vietnam; semi-visible FSWs are assessed as not always being easily identifiable; and non-visible FSWs are assessed as the most hidden types of FSWs in Vietnam.
HIV Risk Factors
There were minor differences among FSW visibility groups with regard to HIV status and related risk behaviors (see Table 1).
Table 1HIV prevalence, risk factors and drug use among visible, semi-visible and non-visible FSWs in Ho Chi Minh City and Hai Phong; unadjusted proportions (number), p-value Ho Chi Minh City (n=413)Hai Phong(n=215)Visible % (n)Semi-visible % (n)Non-visible % (n)p-valueVisible % (n)Semi-visible % (n)Non-visible % (n)p-valueHIV positive status14.5 (45)13.8 (9)13.5 (5)0.98235.2 (57)30.2 (13)30.0 (3)0.801No condom use with a  partner in the past month52.7 (164)63.1 (41)48.6 (18)0.24953.1 (86)79.1 (34)60.0 (6)0.009Ever used non-injection  illicit drugs20.9 (65)18.5 (12)29.7 (11)0.38546.9 (76)37.2 (16)60.0 (6)0.338Ever injected illicit drugs13.8 (43)12.3 (8)5.4 (2)0.34738.9 (63)23.3 (10)30.0 (3)0.378Symptoms of a sexually  transmitted  infection in past year16.1 (50)12.3 (8)16.2 (6)0.74213.6 (22)18.6 (8)20.0 (2)0.640
HIV positive test status did not differ by visibility group in HCMC or HP. In HCMC and HP, a higher proportion of visible FSWs tested positive for HIV (14.5 and 35.2%, respectively) than semi-visible (13.8%, 30.2%) and non-visible (13.5 and 30.0%) FSWs. The only significant difference among the groups for any variable was in HP, for “no past month condom use with a partner” (p value = 0.009).
Visibility groups in both HCMC and HP did not differ with respect to reported use of non-injection illicit drugs ever and injected illicit drugs ever or reporting a sexually transmitted infection (STI) during the previous year.
Service Utilization
Contact with a peer educator (PE), source of HIV information and HIV testing did not vary significantly by visibility group in either city. Among all three visibility groups in both cities, fewer than 50% had ever made contact with a PE. More than 50% of FSWs in both cities (with the exception of semi-visible FSWs in HP) reported receiving their HIV information from television, radio, newspapers or magazines. More HCMC FSWs reported receiving HIV information from friends than from health workers and PEs; this pattern was reversed for HP FSWs (Table 2).
Table 2HIV service utilization among visible, semi-visible and non-visible FSWs in Ho Chi Minh City and Hai Phong; unadjusted proportions (number), p-value Ho Chi Minh City (n = 413)Hai Phong (n=215)Visible % (n)Semi-visible % (n)Non-visible % (n)p-valueVisible % (n)Semi-visible % (n)Non-visible % (n)p-valueEver met with a peer educator33.8 (105)49.2 (32)40.5 (15)0.31031.5 (51)27.9 (12)40.0 (4)0.720Most common source  of HIV information0.0880.106 TV, radio, newspaper  or magazine56.1 (171)70.8 (46)52.9 (18)57.5 (92)45.2 (19)70.0 (7) Health worker/peer educator17.0 (52)9.2 (6)20.6 (7)26.3 (42)38.1 (16)30.0 (3) Friends20.7 (63)13.8 (9)26.5 (9)11.3 (18)16.7 (7)0 Other6.2 (19)6.2 (4)05.0 (8)00Ever had an HIV test45.3 (141)46.2 (30)40.5 (15)0.84134.0 (55)27.9 (12)50.0 (5)0.398Type of facility providing  last HIV test0.9760.285 Public clinic35.6 (51)43.4 (13)33.3 (5)46.3 (25)16.7 (2)40.0 (2) Hospital43.7 (62)36.7 (11)33.3 (5)29.6 (16)25.0 (3)40.0 (2) Private clinic4.2 (6)3.3 (1)6.7 (1)1.9 (2)16.7 (2)0 VCT center6.3 (9)6.7 (2)13.3 (2)9.3 (5)16.7 (2)2.0 (2) Rehab center9.9 (14)10.0 (3)13.3 (2)13.0 (7)25.0 (3)0
Fifty percent or fewer FSWs in each visibility group reported ever having an HIV test. Among those HCMC and HP FSWs who ever had an HIV test, the majority received their test at either a public clinic (district or community public health center or STI clinic) or hospital, with the exception of HP semi-visible FSWs who reported receiving their HIV test from either a hospital or a drug user/sex worker rehabilitation center. Twice as many non-visible (13.3%), in comparison to visible and semi-visible (6.3% each) FSWs in HCMC, reported receiving their HIV test at a VCT center.
Discussion
The analysis of RDS coverage demonstrates that this method may provide modestly better estimates for FSW populations than TLS because it reaches individuals ostensibly inaccessible by TLS. The level of risk and HIV prevalence in the less visible groups of FSWs was similar to the semi-visible and visible FSWs. This would indicate that less visible FSWs merit reasonable efforts to assure that these groups are targeted within all interventions for FSWs. The differences might have differed if two groups emerged rather than three. However, the semi-visible group could not be clearly dichotomized.
The three visibility groups in HCMC and HP did not differ significantly, which may contradict previous reports that higher paid, indirect FSWs, in general, have less HIV risk than direct FSWs.5,11,33 High proportions of respondents among all visibility groups in HCMC and HP reported inconsistent condom use with a sexual partner in the past month, possibly indicating that all three groups of FSWs are not being appropriately educated about using condoms or that they cannot easily access them. Other possible barriers to increased condom use might include poor negotiating skills with their clients or a false perception that a well-dressed client with an occupation may not be HIV-infected. Understanding the perceptions, negotiation skills and the reasons why some FSWs use condoms and others do not is essential to addressing the low proportion of condom use among FSWs in these cities.
The high proportion of non-visible FSWs in HCMC and HP who ever met with a PE indicates that the more hidden FSWs may be receiving more exposure to HIV information than previously believed. HCMC and HP FSWs reported similar sources for HIV information, indicating that PEs may be equally disseminated among the visibility groups. Furthermore the high proportion of non-visible FSWs who ever had an HIV test and their access to HIV testing at public clinics, hospitals and VCT centers indicate that this group has some awareness of and interest in their risk. It would be useful to gather qualitative data to determine how they obtained information and services.
Another key finding was the nature of social networks formed by FSWs in Vietnam. Networks were dense and diverse enough to provide a large sample in a short time that crossed the various types, direct and indirect, of FSWs working in the respective cities. These network properties are essential to the successful use of RDS. RDS had an added advantage over TLS by providing some measure of selection bias by questioning respondents on the characteristics of peers who demurred. TLS cannot determine reasons for not using a venue to solicit clients, only for respondents’ refusal to speak with interviewers.
There were limitations to our study. Categories of visibility were based on the authors’ perceptions of the sex work industry in the respective cities. We did not conduct extensive fieldwork so there was limited knowledge about the types of FSWs in Vietnam. There was too little formal research about characteristics of hotel and guesthouse-based FSWs to confidently dichotomize them as visible or non-visible. With a better understanding of these types of FSWs, reclassification and reanalysis of the data could better demonstrate the capacity of RDS to reach hidden FSWs. Furthermore, the question “Where do you usually reach your clients?” does not signify where a FSW always reaches her clients. The findings here may have non-differentially misclassified some respondents.
Currently RDS data analysis is limited to estimates of proportions. Stratified analysis of the differences in risk factors among visibility groups might yield more information when RDSAT is updated.
The best scenario for testing whether RDS captures more hidden elements of the FSW population would be to conduct nearly contemporaneous surveys using TLS and RDS in the same population. Using RDS data to simulate TLS for comparisons of the two sampling methods is subject to any biases unique to RDS that might not be found in TLS.
Although non-visible FSWs make up a small proportion of the FSW sample in HCMC, their high HIV prevalence and associated risk factors is alarming, especially when past efforts to sample FSWs, using TLS and convenience sampling, and sentinel surveillance in STI clinics, are likely to have missed these groups. Missing the most inaccessible portions of the FSW population could result in a biased understanding of HIV sero-prevalence and risk factors and, consequently, could lead to inefficient planning for HIV prevention and intervention programs for the entire FSW population. Recurrent round-ups of FSWs drive FSWs off the street, making them increasingly difficult to find in public venues. If current prevention programs are targeting and responding to those who are most likely to be assessed (more visible FSWs) then it is possible that an important segment of the FSW population at risk for HIV is not receiving optimal HIV information and services. However, this study found that segments were accessing services at about the same low rates, indicating a need to increase service utilization among all groups of FSWs.
Based on these findings, we recommend using RDS for future surveys of FSWs in Vietnam to prevent the omission of the less accessible members of this population.

sex workers
vietnam
hiv
respondent-driven sampling


Purinergic signaling microenvironments: An introduction

The common theme of this introductory article and the minireviews that follow in this special issue is the concept of microenvironments within tissues and surrounding cells that would be ideal signaling venues for a biologically active purinergic ligand. Collectively, the editors/authors and the other contributing authors agree that nucleotides and nucleosides would be most potent within a confined system. A talented cadre of purinergics has been solicited to discuss purinergic signaling in his or her favorite microenvironment within a given organ or tissue. We are gratified by the large number of original articles that also have successfully navigated the peer review process and are part of this special issue. These concepts are not simply purinergic, but the idea of maximal potency in a tissue microenvironment and surrounding specialized cells within a tissue pertains to any autacoid or paracrine agonist.
Introduction
Adenosine 5′ triphosphate (ATP) and adenosine are extracellular autocrine and paracrine signaling molecules. The most potent effects of ATP and adenosine are likely limited to extracellular microenvironments within tissues or immediately surrounding a cell or group of cells. These extracellular microenvironments may have a limited area of diffusion, have a small volume, have an ideal ionic composition and pH, represent an unstirred layer near a membrane surface, represent a trapped interstitium, or any and all of the above. If P1 or P2 nucleotide receptors are present on a membrane that is exposed to released nucleotide or nucleoside in this microenvironment, these ligands can engage P1 G-protein-coupled receptors, P2Y G-protein-coupled receptors or P2X receptor channels and affect the function of the cell on which it binds. Besides ATP and adenosine, other forms of nucleotides and nucleosides may also be active extracellular signaling ligands and may have unappreciated receptors. Inosine, thymidine, and even cyclic nucleotides are released by and may have effects on mammalian cells. The self-aggregation signal for the slime mold Dictyostelium discoideum, is cyclic adenosine monophosphate (AMP). Receptors for cyclic nucleotides have only been defined until now in the lower biological systems. Receptors for cyclic nucleotides as well as inosine or thymidine may also exist on mammalian cells and receive this autocrine signal within a microenvironment.
Ideal microenvironments for purinergic signaling
For simplicity, we focus on ATP and its metabolites. Many different microenvironments have been studied in great detail and are described and included below. One classical microenvironment that has emerged in importance in the cystic fibrosis (CF) research community is in an open system but is a stirred liquid layer trapped between the luminal membrane of the airways epithelium and the humidified air that fills the lung and airways. The airway surface liquid [ASL, also termed airway surface fluid (ASF), the periciliary liquid layer (PCL), and the epithelial lining fluid (ELF)] bathes motile respiratory cilia [1–4]. The humidified air above this layer and the ciliated apical or luminal membrane beneath this layer help close this microenvironment. Submucosal gland secretions may also contribute importantly to its composition. Ric Boucher and his many coworkers have presented many recent seminal contributions on the purinergic signaling that may occur in the ASL—among its many other properties [3, 4]. There are similar microenvironments of this type that are also discussed in review articles within this special issue. Daniel Marcus and colleagues and Claire Mitchell and coworkers discuss liquid microenvironments in the ear and the eye, respectively [5, 6], which provide a medium for purinergic signaling.
A second classical microenvironment is a closed and open system that is compromised of a secretory gland or acinus and a collecting or draining duct. Iva Novak and colleagues have done seminal work in the pancreas in this regard [7, 8]. Geoff Burnstock has often discussed the rich purinergic signaling that occurs in tubes or ducts [9, 10]. In this microenvironment, glandular and acinar secretions may convey the purinergic ligands so that they may act in an autocrine manner at the acinus or in a paracrine manner within the duct. However, it is an open system in that these secretions eventually exit this tissue environment. The airway submucosal gland beneath the airway surface epithelium may be one such example that conveys purinergic signals to the surface where they may drive ciliary beat and other functions. The pancreatic acinus and duct, the sweat gland and duct, and the bile secretory apparatus and the bile duct are all important examples.
A third microenvironment is a closed system in which the purinergic ligands concentrate within a small volume and within a tissue. Here, the purinergic ligands bind and activate receptors until they are either degraded or reabsorbed by the cells that secreted them or by neighboring cells. The endolymph of the ear and the humors of the eye are ideal examples of such closed systems [11, 12]. Marcus and colleagues and Mitchell and coworkers discuss these systems in minireviews alluded to above [5, 6]. Original articles and reviews in this issue and future issues of Purinergic Signalling speak to such systems in pancreatic islets and in the many renal tubules that comprise the nephron of the kidney [13]. In polycystic kidney and liver disease, tubules remodel into abnormal fluid-filled cysts. Multiple laboratories have argued that the fluid-filled lumen of the encapsulated cyst is a rich yet pathophysiological microenvironment for purinergic signaling [14–20]. The minireviews that follow this introductory article focus on specific microenvironments, cells and tissues that create and/or come in contact with such microenvironments, and all possible modes of purinergic signaling within these microenvironments.
An ideal microenvironment paradigm for extracellular purinergic signaling: the synapse
Many reviews in the past [21–23] and in the initial issues of this journal have documented well the roles of the P2X receptor channel at the synapse between neurons. Indeed, P2Y receptors may play roles in synaptic transmission as well. This is analogous to the interplay between cholinergic and muscarinic receptor systems in cholinergic neurons and synapses in many different nuclei and regions of the brain. In fact, the purinergic field was born from the classical studies of extracellular ATP on the heart by Drury and Szent-Gyorgyi [24], in the definition of purinergic nerves in the enteric nervous system by Burnstock and colleagues [25], and on the release and effects of purinergic ligands such as ATP on skeletal muscle contractility by Tom Forrester (see “Historical Perspective” by T. Forrester in this issue, [26]). The authors lead this series of minireviews with the idea that the synapse is the ideal and, perhaps, the first or founding microenvironment for autocrine and paracrine purinergic biology.
The synapse is designed to have a limited diffusion distance between pre- and postsynaptic membranes where the principal ligand or neurotransmitter is reabsorbed by the presynapse or degraded in the synapse. For the sake of simplicity, our narrative here focused first on a purinergic synapse where released ATP acts as the principal ligand to evoke an action potential in the postsynaptic membrane. ATP as the lead neurotransmitter is released in response to an action potential in the presynaptic bulb that opens voltage-dependent calcium (Ca2+) channels. This voltage-dependent Ca2+ entry signal triggers the fusion or exocytosis of ATP-filled synaptic vesicles so that labile ATP is secreted into the synaptic cleft. Extracellular ATP-gated P2X receptor channels on the postsynaptic membrane have a large extracellular domain poised to bind that ATP immediately upon release within a synaptic microenvironment where there is a short diffusion distance and a small volume. In other words, the synapse is an ideal microenvironment for paracrine purinergic signaling. In this paradigm, the ATP is likely degraded to adenosine diphosphate (ADP), 5′ AMP and adenosine, where adenosine may be recycled back into the presynapse by nucleoside transporters only to be a substrate to synthesize new ATP for metabolism or neurotransmission.
Several additional functions for P2X receptor channels have been proposed in neurotransmission driven by other neurotransmitters. For example, at the glutamate synapse, it is well known that ATP is released as a cotransmitter. A classical example of this is the hair-cell sound transduction process, where glutamate and ATP are coreleased to stimulate auditory afferents [11]. How may ATP affect responses to glutamate at glutamate, N-methyl-D-aspartate (NMDA), and kainate receptor channels in the postsynaptic membrane and/or at the presynapse in terms of release of ligand? Many possible cotransmitter effects have also been entertained and studied. We do not wish to debate the relative roles of P2X receptor channels in neurotransmission. The possible explanations of ATP effects at an excitatory glutamatergic synapse are given in simplistic terms. We only wish to show the synapse as a lead microenvironment to illustrate how and why local ATP signaling may be important.
First, the presence of a postsynaptic P2X receptor channel or channels would potentiate the excitation of glutamate postsynapse. Not only do NMDA, kainate, and glutamate itself potentiate Ca2+ and sodium (Na+) entry as Ca2+ permeable nonselective cation channels, but extracellular ATP-gated P2X receptor channels open a separate pathway for Ca2+ and Na+. Flow of Ca2+ and Na+ through parallel pathways driven by these cotransmitters would lead to a much quicker development of a postsynaptic potential. Moreover, with Ca2+ entering with Na+, it is likely that the action potential may be more long-lived and possibly more robust (i.e., having a shoulder or plateau due to Ca2+ entry beyond the Na+-driven spike). Second, ATP could potentiate glutamate release at the presynaptic neuron by potentiating Ca2+ entry driven by the preceding action potential. A more robust Ca2+ entry would cause more or secondary glutamate release and would keep the postsynaptic neuron firing. As for acetylcholine, ATP also has G-protein-coupled receptors, the P2Y receptors, which could modulate presynaptic and/or postsynaptic neuron function. Third, could metabolites of ATP limit activation of the postsynaptic and presynaptic neuron? One does not wish for the postsynaptic neuron to continue firing ad infinitum. One must have a limiting or negative feedback signal. Could this signal be adenosine? Not only does adenosine need to be recycled back into the neuron as a substrate for new ATP synthesis, but adenosine may also bind to P1 G-protein-coupled receptors to have limiting effects or neuronal function. Of course, ATP release and signaling at an inhibitory synapse driven by γ-aminobutyric acid (GABA) or glycine would have different and modulating effects on inhibitory synapse. We do not discuss or illustrate these possible mechanisms because that is not the intent of this introductory minireview. Synaptic purinergic neurotransmission has been reviewed by Burnstock and others in many seminal reviews [9, 10, 21–23].
Conclusions and future directions
The founding concepts of purinergic signaling within tissues and tissue microenvironments were found in the heart, the enteric system (later to be duplicated in urinary bladder afferents, nociceptive neurons at the dorsal root ganglia of the spinal cord, etc.), and skeletal muscle by Forrester and coworkers [26]. This special issue of Purinergic Signalling focusing on the “Physiology of Nucleotide Release” is dedicated to these and other pioneers who would not give up the fight. All purinergics would agree that we owe the purinergic pioneers a great debt. Through their efforts, establishment of this journal, and the increasing volume of research in purinergic signaling, it is now accepted that ATP and its metabolites have two principal biological roles—as an extracellular signaling ligand and as an intracellular fuel.

signaling nucleotides nucleosides adenosine atp