The interest in children's bone health has been expanding over the last decade, after the evidence for a crucial role of bone accrual in the determination of fracture susceptibility in adult life.1 Low bone mineral density (BMD) is a recently recognized metabolic complication of HIV infection and its treatment, even in children and adolescents.2-4 The decreased morbidity and mortality rates of HIV-infected youths attributable to more effective antiretroviral therapy have contributed to an increased survival well into adulthood. Bone health is therefore becoming an emerging issue in these patients, and a precocious screening of patients at higher risk is mandatory to assure an optimal bone mass accrual.
The majority of studies in children employed x-ray-based techniques, such as dual-energy x-ray absorptiometry (DXA). The quantitative use of ultrasound waves for studying bone has a special appeal for its application in children because of its speed, low cost, and lack of ionizing radiation.5-7 Because the equipment for quantitative ultrasound (QUS) measurements is portable, this methodology may represent a convenient way of evaluating bone health in an office setting. Moreover, for countries with limited access to DXA, peripheral QUS may be a reasonable alternative.
The aim of the current study was to assess the applicability of the QUS technique in HIV-infected youths. For this purpose, we related the QUS measurements with those obtained by DXA at the lumbar spine, the whole skeleton, and at the mid shaft of the tibia in a large group of HIV-infected patients with a wide age range.
MATERIALS AND METHODS
Children and adolescents who were part of an ongoing study on body composition and skeletal mineral density in HIV-infected youths represented the study population. We studied 43 boys and 45 girls (78 of white ancestry and 10 of African ancestry), aged from 4.8 to 22.1 years. Eighty-four patients were vertically infected, whereas 4 were infected in the first year of life. Forty patients showed asymptomatic or mild disease, whereas 48 cases had moderate or severe disease. The mean CD4 number was 795 cells per microliter (ranging from 15 to 1946 cells/μL), and the mean CD4 percentage was 31.5 (ranging from 2% to 72%). The great majority (70%) of the patients included in the study had undetectable HIV viral load, whereas 30% of the patients showed a HIV viral load ranging from 1000 to >10,000 copies per milliliter.
Seventy-six patients were receiving a protease inhibitor-based or nonnucleoside reverse transcriptase inhibitor-based highly active antiretroviral regimen, 6 patients were receiving a double nucleoside reverse transcriptase inhibitor regimen, and 6 patients were naive to antiretroviral treatment.
Informed consent was obtained from all the parents or legal guardians of all patients and from the patients when appropriate. The study was approved by the Ethical Committee of the L. Sacco Hospital.
All subjects underwent physical examination to obtain anthropometric measures and to assess pubertal development. Body weight was measured to the nearest 0.1 kg on a balance beam scale (Seca, Hamburg, Germany), and height was measured to the nearest millimeter using a wall-mounted stadiometer (Holtain, Ltd, Crosswell, United Kingdom). Body mass index was calculated as weight on height2 (kilograms per square meter). Pubertal stage was defined according to Tanner criteria.8
The Sunlight Omnisense 7000 device (BeamMed Ltd., Tel Aviv, Israel) was used. The measurements were made at the midshaft tibia of the nondominant leg and at the distal radius of the nondominant arm. The tibia and the radius were then scanned repeatedly on their inner faces with sweeping movements from the posterior to the anterior aspect of the bone. Speed of sound (SOS) measurements were obtained separately by 2 operators (L.C. and G.P.) to examine the interoperator variability. Moreover, QUS measurements were repeated 3 times with repositioning after erasing the skin mark to calculate intraoperator variability. The QUS measurements were expressed both as absolute values and as SD scores (z scores) from a reference population of age- and sex-matched individuals, included in the scanner's software.
Dual-Energy X-ray Absorptiometry
Bone mineral measurements were made with a DXA (DPX-L; Lunar-GE, Madison, WI). The data were acquired and analyzed with a pediatric software (version 1.5h, DPX-L; Lunar Radiation Corp, Madison, WI). The coefficient of variation (CV) of our instrument is 1.4% for the lumbar spine and 1.5% for the whole skeleton, in vivo. According to published data, the effective radiation dose for each scan is about 0.3 μSv (6 μGy) for the lumbar spine and <0.03 μSv (<0.12 μGy) for the whole body scans.9 All scans were performed and evaluated by the same operator (S.M.).
Bone mineral content (BMC) and BMD were measured at the L2-L4 vertebral level and in the whole skeleton. The BMD measurements were expressed both as absolute values and as SD scores (z scores) from a reference population of age- and sex-matched control subjects, provided by the manufacturer. To explore the relationship between the measurements obtained with QUS in the tibia and the corresponding region measured by DXA, a region of interest was selected manually and placed at the midshaft tibia of the nondominant leg on the total body scan. BMC and BMD values were calculated for each tibial region by the same operator (L.C.).
Descriptive statistics were calculated for all the variables, and data are expressed as the mean (SE). All statistical analyses were conducted at the α = 0.05 level and were 2 tailed. Distribution of the variables was checked using the Shapiro-Wilk W test. The statistical software JMP 7.0 (SAS Institute, Inc, Cary, NC) was used for the analyses.
The CV was calculated for repeated measurements as the SD divided by the mean of the measurements and expressed as percentage.
The differences between the 2 operators were evaluated by Wilcoxon signed rank test for paired samples. The relationships between QUS and DXA measurements were explored by multiple regression analyses to account for sex, race, pubertal development, and anthropometric differences. The differences between z scores obtained with the 2 methods were evaluated by Wilcoxon signed rank test for paired samples. The prevalence of low (z score ≤ −2.0) QUS and DXA measurements was compared by χ2 test.
Study subjects' weight was on average 45.8 (1.7) kg [z score: −0.6 (0.1)], height was 151.5 (2.1) cm [z score: −0.5 (0.1)], and body mass index was 19.2 (0.4) kg/m2 [z score: −0.3 (0.1)]. Twenty patients were prepubertal, whereas 22 were in the early stages of puberty (Tanner stages II and III). The remaining 45 patients were classified in the late stages of pubertal development (Tanner stages IV and V).
To evaluate the SOS measurements' reproducibility, we calculated the CV of at least 3 separate QUS measurements made by one operator on the same subject. The mean CV of operator 1 (OP1) for SOS measurements at the radius was 0.41% (0.05%) and that of operator 2 (OP2) was 0.41% (0.09%). The difference between the 2 operators was not significant (z = −25.4; P = 0.36).
The CVs calculated after repeated QUS measurements of the tibia were 0.34% (0.07%) for OP1 and 0.33% (0.08%) for OP2. The measurements' reproducibility between the 2 operators was not statistically different (z = −4.5; P = 0.88).
We found no correlations between CV values and age or anthropometric measurements, indicating that precision was independent from the subject's size.
We compared the readings obtained on the same subject by the 2 operators. The mean SOS values measured at the tibia were 3836 (26) m/s (OP1) and 3767 (84) m/s (OP2). Paired analysis showed that the difference was statistically significant (z = −60; P = 0.033). Similarly, the absolute readings made at the radius were 3936 (25) m/s (OP1) and 3910 (22) m/s (OP2). The paired test showed a significant difference (z = −79; P = 0.003).
Although the differences were statistically significant, their magnitude was negligible in practice: Measurements of the radius differed by 0.65% (0.21%) between the 2 operators and those of the tibia differed by 0.32% (0.13%). The QUS and DXA measurements of the study population are shown in Table 1.
The final models for multivariate analyses included sex and anthropometric measurements as confounding variables (race and pubertal development did not contribute to the model and were excluded), DXA measurement as independent variable, and SOS as dependent variable. Radius SOS measurements were significantly associated to lumbar spine BMC and BMD measurements (R = 0.59 and R = 0.60, respectively) and to total body BMC and BMD values (R = 0.57 and R = 0.60, respectively). Similarly, SOS values of the tibia showed positive associations with lumbar spine BMC and BMD measurements (R = 0.65 and R = 0.66, respectively) and with total body BMC and BMD measurements (R = 0.62 and R = 0.64, respectively). The multiple regression analyses, considering the SOS of the tibia as the dependent variable, and the corresponding BMC and BMD measurements showed positive associations (R = 0.58 and R = 0.60, respectively).
A total of 3 patients had low SOS measurements (z score ≤ −2.0) at the radius, and 3 had low SOS at the tibia (Fig. 1). In contrast, 9 patients showed low BMD values at the lumbar spine. Finally, 3 patients had total body BMD measurements below −2.0 SD from reference (Fig. 1). The χ2 analysis failed to show difference in distribution of lumbar spine low bone mass measurements and low z scores of the radius (χ2 = 1.2, P = 0.26) and the tibia (χ2 = 2.0, P = 0.14).
The mean z scores for QUS and DXA measurements are shown in Table 1. Paired analyses showed that lumbar spine values differed significantly from those of the radius (z = 1019; P < 0.0001) and the tibia (z = 1128; P < 0.0001). On the contrary, z scores of the total body did not differ from those obtained with QUS.
Ultrasound has been recently introduced as a diagnostic tool for bone diseases, and its use in pediatric patients is expanding.5-7 The ease of use and the lack of radiation exposure are great advantages over more traditional methods. The portability of the QUS devices is also a feature that allows for studies in different locations. QUS measurements can be performed in different skeletal sites, using different approaches. One of the recognized limitations of QUS measurements resides in the paucity of quality assurance procedures.10 Moreover, a specific issue regards intra- and interoperator variability. Too large variations may impair a correct assessment and may lead to unnecessary further examinations or therapeutic attempts. This issue is particularly important when measuring bones that are still growing and changing in shape. For this reason in the current study, 2 different operators measured the SOS of the radius and tibia in a large pediatric sample. The wide age range of the children who underwent QUS measurements assured the presence of diverse bone dimensions. Our results indicate a good reproducibility of the measurements, the CV of both operators being on average below 0.5%. The lack of correlation between the CV and age or anthropometric measurements indicates that precision of SOS measurements is independent of the patient's size and it is constant with age. The precision obtained in the current study is comparable to that of an earlier report that used a similar QUS approach.11
Despite the low variability of measurements, significant differences between the 2 operators have been observed in terms of absolute values. However, although statistically relevant, the differences were below 1% for both skeletal sites. This result is encouraging because consistent results could be obtained with different operators who follow carefully the procedures suggested by the manufacturer.
The applicability of QUS in the study of bone mass of HIV-infected children was assessed by comparing SOS measurements with those obtained with DXA. The latter is the most widely used technique for bone mass measurements in children.5-7 We examined the relationships between measurements after correcting for the confounding effect of sex and anthropometric measurements. Multivariate analyses indicated that the relationships between the 2 techniques were good, and these results suggest that both techniques measure a component of growth and bone mass.
The correlations between QUS and DXA are encouraging, but they do not provide information regarding the ability of QUS to detect low bone mass values and therefore to select patients with increased fracture risk or at risk for developing osteoporosis later in life. The expression of bone mass measurements as SDs (z score) from a reference population matched for sex and age is a recommended method to define people at risk.12 Lumbar spine BMD z scores were lower than −2.0 in about 10% of the patients. A much lower proportion of HIV-infected children had low SOS or total body BMD z score values. The paired analyses comparing QUS and DXA z scores indicated highly significant differences between lumbar spine DXA results and SOS z scores of the radius and tibia. Conversely, no differences were observed between QUS and total body z scores. These results are not surprising. The QUS device we used measures SOS in the cortex of long bones, and the whole skeleton consists of approximately 88% of cortical bone. On the contrary, the lumbar spine measurements include a large portion of trabecular bone, which is constitutively different from cortical bone.
There are some limitations of the current study. First, we studied HIV-infected children 4.8-22.1 years of age with different stages of infection and diverse therapeutic schedules. It is therefore possible that the relative heterogeneity of the cohort studied may have affected the strength of the associations between the 2 techniques. Second, the great majority of the HIV-infected patients showed BMD and SOS values within reference range. Therefore, the association between bone mass measurements we observed may not be representative of the lowest values. Third, our results were obtained from comparisons between 2 specific instruments and may not apply with equipment from other manufacturers.
In summary, our data show that SOS measurements at the radius and tibia in children and adolescents are consistent and reproducible. Moreover, the results of our study show a good agreement between DXA and QUS measurements, particularly evident for total body DXA. Therefore, QUS may be an additional diagnostic tool for the study of cortical bone mass in HIV-infected youths. There is some evidence that SOS measurements are lower in children with fractures compared with nonfractured controls.13 In this respect, QUS could be an alternative instrument in resource-limited settings where more expensive devices for bone mass measurements are not available.
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