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Bone Loss in HCV/HIV Coinfection
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Don't take this study wrong, and I'm not at all convinced their findings are correct because men suffer seriously with bone loss in HIV, just like women can. In fact some think there is a uniqure aspecxt for men in HIV. In HIV men have unexpectedly more serious bone loss than one would have thought and it's thought this could be associated with testosterone, lipoatrophy, lifestyle and additional unknown causes. In HIV-negatives HCV is associated with bone oss so again I'm not sure of the significance in this study regarding men. Still, this study found coinfected women have more bone loss than HIV+ women, that of itself suggests a good reason to follow closely coinfected women for bone loss
Viral Hepatitis is Associated with Reduced Bone Mineral Density in ...
Background: Viral hepatitis has been associated with reduced bone mineral density (BMD) in HIV-uninfected patients, but the mechanisms for bone loss remain ...
www.natap.org/2009/CROI/croi_143.htm
Viral hepatitis is associated with reduced bone mineral density in HIV-infected women but not men
AIDS Oct 2009
Lo Re, Vincent III; Guaraldi, Giovanni; Leonard, Mary B; Localio, Anthony R; Lin, Jennifer; Orlando, Gabriella; Zirilli, Lucia; Rochira, Vincenzo; Kostman, Jay R; Tebas, Pablo
aDivision of Infectious Diseases, Department of Medicine, USA
bCenter for Clinical Epidemiology and Biostatistics, Department of Biostatistics and Epidemiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA
cInfectious Diseases Clinic, Department of Medicine, University of Modena and Reggio Emilia, Modena, Italy
dDivision of Nephrology, Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA
eUnit of Endocrinology and Metabolism, Department of Medicine, Endocrinology and Metabolism, Geriatrics, University of Modena and Reggio Emilia, Modena, Italy.
This study was published in AIDS after being presented at CROI in Feb 2009. Although the authors claim that in coinfected women:
"Interestingly, we found that men had lower mean BMD Z-scores at the lumbar spine and femoral neck compared with women....This finding has been reported in other studies of HIV-infected patients [31,32], but the reasons for these differences are unclear. Future studies should examine differences in BMD among HIV-infected women and men and evaluate hormonal, anthropometric, virologic, immunologic, and therapeutic factors that may account for these differences."
"Overall, men had lower mean Z-scores at the lumbar spine than women [-0.50 versus +0.06; difference = -0.56 (95% CI -0.42 to -0.69); P < 0.001]. However, viral hepatitis-coinfected women had significantly lower mean lumbar spine Z-scores compared with HIV-monoinfected women....In contrast, coinfected men had similar mean Z-scores at the lumbar spine compared with monoinfected men....When we performed adjusted analyses excluding HBsAg-positive patients, mean lumbar Z-scores remained lower in HCV-coinfected compared with HIV-monoinfected women [-1.31 versus -0.82; difference = -0.49 (95% CI -0.74 to -0.25); P < 0.001] but not between coinfected and monoinfected men.....Men also had lower mean Z-scores at the femoral neck than women"
Conclusion
We found that viral hepatitis increased the risk of low BMD among HIV-infected patients. Future studies should evaluate fracture rates and examine risk factors and potential mechanisms for low bone mass among HIV/viral hepatitis-coinfected patients.
Abstract
Objective: Few studies have examined the impact of viral hepatitis on bone mineral density (BMD), and none have done so among HIV-infected patients. Our objective was to determine whether viral hepatitis was associated with low BMD in HIV.
Design: A cross-sectional study among 1237 HIV-infected patients (625 with viral hepatitis).
Methods: Dual-energy X-ray absorptiometry scans of the lumbar spine and femoral neck were obtained. Clinical data, hepatitis B and C status, and markers of bone metabolism were determined at dual-energy X-ray absorptiometry scanning. Multivariable logistic regression examined the association between hepatitis and low BMD (Z-score ≤-2.0 at the lumbar spine, femoral neck, or both).
Results: Mean BMD Z-scores were lower among hepatitis-coinfected women at the lumbar spine {-0.15 versus +0.29; difference = -0.44 [95% confidence Interval (CI) -0.65 to -0.23]; P < 0.001} and femoral neck [-0.64 versus -0.39; difference = -0.25 (95% CI -0.44 to -0.06); P = 0.009] compared with HIV-monoinfected women. No differences in mean BMD Z-scores were observed between coinfected and monoinfected men. After adjustment for age, BMI, duration of HIV, antiretroviral use, physical activity, and smoking, viral hepatitis was associated with low BMD among women (adjusted odds ratio 2.87, 95% CI 1.31-6.29) but not men (adjusted odds ratio 1.19, 95% CI 0.74-1.91). Coinfected women had lower mean parathyroid hormone (60.1 versus 68.1 pg/ml; P = 0.02) but similar mean 25-hydroxyvitamin D (19.1 versus 19.6 ng/ml; P = 0.6) and osteocalcin (3.0 versus 3.2 ng/ml; P = 0.8) concentrations than HIV-monoinfected women.
Conclusion: Viral hepatitis was associated with a higher risk of low BMD among HIV-infected women but not men.
Introduction
Although advances in antiretroviral therapy (ART) have led to a substantial decline in AIDS-related opportunistic infections and increased longevity of HIV-infected patients, complications of chronic viral hepatitis have now emerged as a major cause of morbidity in the HIV population [1]. Existing studies among HIV-infected patients have mainly examined the impact of viral hepatitis coinfection on liver-related complications (e.g., cirrhosis, end-stage liver disease, and hepatocellular carcinoma) [2-4], but the effect of viral hepatitis on nonhepatic outcomes, in particular abnormalities in bone mineral density (BMD) and bone metabolism (termed 'hepatic osteodystrophy'), remains unclear.
Viral hepatitis has been associated with reduced BMD among HIV-uninfected individuals [5,6], and a number of factors related to chronic viral hepatitis have been hypothesized to contribute to low BMD (Fig. 1). Prior studies have suggested that elevated serum levels of cytokines associated with viral hepatitis [e.g., tumor necrosis factor-α, interleukin (IL)-1, and IL-6] could inhibit bone formation and increase bone resorption, leading to low BMD [7,8]. In addition, progressive hepatitis-induced liver dysfunction may be associated with reduced hepatic hydroxylation of vitamin D [9], hypogonadism [10], and impaired hepatic production of insulin-like growth factor-1 and osteoprotegerin, both of which promote bone formation [6], and these conditions can also contribute to low BMD. Moreover, HIV itself is associated with reduced BMD and increased fracture risk [11-13]. However, to date, no study has yet examined whether viral hepatitis is an independent risk factor for low BMD in HIV. Evaluating the association between viral hepatitis and low BMD in HIV-infected patients is important because viral hepatitis is prevalent in HIV [14] and low BMD is a predictor of both fragility fractures [15] and all-cause mortality [16,17]. We therefore examined whether viral hepatitis coinfection was associated with reduced BMD among HIV-infected patients.
Discussion
To our knowledge, this study is the first to examine whether viral hepatitis coinfection was associated with reduced BMD Z-scores in HIV. We unexpectedly identified sex differences in the comparison of BMD Z-scores between coinfected and monoinfected patients. HIV/viral hepatitis-coinfected women had significantly lower BMD Z-scores at both the lumbar spine and femoral neck compared with women with HIV alone. After adjusting for confounding variables, viral hepatitis remained associated with low BMD Z-scores in women. In contrast, we found no differences in lumbar or femoral BMD Z-scores between coinfected and monoinfected men, and viral hepatitis was not associated with reduced BMD Z-scores among HIV-infected men. Regardless of sex, coinfected patients had lower intact PTH concentrations but similar 25-hydroxyvitamin D and octeocalcin levels compared with patients with HIV alone.
Few published studies have exclusively examined the impact of viral hepatitis on BMD, and none have done so among HIV-infected patients. Among viral hepatitis-monoinfected patients, the prevalence of reduced lumbar or femoral BMD, or both has been reported to range from 10 to 56% [8,24-28]. However, these studies are difficult to interpret given their small sample sizes, inclusion of predominantly male patients, and use of different BMD outcomes. Importantly, these studies primarily reported low T-scores as their main endpoint. As T-scores represent the comparison of an individual's BMD with a young adult at the time of peak bone mass, T-scores are significantly lower in older patients due to the expected age-related declines in BMD. Therefore, studies reporting T-scores in patients of varied age are likely confounded by age. Rather, in comparing patients across the entire adult age range, Z-scores are the preferred outcome in which an individual's BMD is compared with a reference population of the same age and sex. Using this outcome, we found that 16% of coinfected patients had low lumbar or femoral bone mass, or both. Had we used T-scores, the proportion would have been much higher among older adults and would not have captured the independent effect of coinfection.
We examined several biochemical parameters of bone metabolism to examine potential mechanisms for low BMD due to viral hepatitis in HIV-infected patients. Serum levels of osteocalcin, a protein synthesized by osteoblasts that reflects bone formation, have been reported to be decreased among patients with viral hepatitis [28] and viral cirrhosis [8,24,25,27] compared with healthy viral hepatitis-uninfected individuals, suggesting impaired osteogenesis. However, after adjusting for eGFR, we found no difference in serum levels of osteocalcin according to hepatitis status. Moreover, the mean osteocalcin concentrations observed in coinfected and monoinfected patients in our sample were similar to results found in HIV-uninfected men and women [29]. In addition, serum 25-hydroxyvitamin D concentrations have been reported to be lower among viral hepatitis-monoinfected patients compared with healthy controls [8,24,25,27]. However, after controlling for eGFR, we found that 25-hydroxyvitamin D concentrations were similar between coinfected and monoinfected women and between coinfected and monoinfected men. We did not observe a compensatory increase in intact PTH concentration among the coinfected patients that would have been expected given the generally low 25-hydroxyvitamin D concentrations observed. Reduced values of both serum PTH and 25-hydroxyvitamin D have been reported in studies of viral hepatitis-monoinfected patients [8,23,24,26]. Cytokines associated with viral hepatitis infection (particularly, IL-1 and IL-6) have been shown in vitro to inhibit PTH secretion [30], which might explain this observation.
The precise mechanisms for the association between viral hepatitis and low BMD in HIV-infected women but not men remain unclear. It is unknown whether there are clinically significant differences by sex in serum levels of hepatitis-associated cytokines, markers of bones resorption (e.g., serum pyridinoline and type 1 collagen cross-linked C-telopeptide), or other factors that maintain bone balance (e.g., insulin-like growth factor-1 and osteoprotegerin) among HIV-infected patients, but such variables are not routinely measured by the Modena HIV Metabolic Clinic Cohort. Moreover, the effect of both HIV and viral hepatitis coinfection, particularly in the setting of hepatic dysfunction, on ovarian function and estrogen production in women is unknown. To elucidate the mechanisms for low BMD in coinfected women, future studies should evaluate serum levels of cytokines, markers of bone resorption, insulin-like growth factor-1, osteoprotegerin, estradiol, testosterone, follicle-stimulating hormone, and sex hormone-binding globulin.
The increased risk of low BMD among HIV/viral hepatitis-coinfected women has important clinical implications. As low BMD is a predictor of fragility fractures [15], our data suggest that viral hepatitis-coinfected women are at even greater risk for vertebral and hip fractures compared with those with HIV alone. Moreover, low BMD is also associated with increased mortality [16,17]. As liver disease due to viral hepatitis currently accounts for the second leading cause of death in HIV [1], low BMD in coinfected women might contribute further to mortality, particularly as this population ages. The impact of therapies to improve bone mass (e.g., calcium, vitamin D, and bisphosphonates) as well as the effect of chronic HBV and HCV treatments on BMD should also be evaluated.
Interestingly, we found that men had lower mean BMD Z-scores at the lumbar spine and femoral neck compared with women. This finding has been reported in other studies of HIV-infected patients [31,32], but the reasons for these differences are unclear. Future studies should examine differences in BMD among HIV-infected women and men and evaluate hormonal, anthropometric, virologic, immunologic, and therapeutic factors that may account for these differences.
Our study had several limitations. First, as cross-sectional studies evaluate exposure and disease status at the same point in time, this study design is limited in its ability to determine whether exposure preceded or resulted from disease. However, with regard to this study, low BMD does not lead to HBV or HCV infections. As such, a cross-sectional study design remains appropriate to determine the association between viral hepatitis and low BMD.
Second, identification of HCV status was based on anti-HCV testing alone, as the cohort does not routinely test for HCV RNA, but not all anti-HCV-positive patients are HCV viremic. However, in the case of HIV/HCV coinfection, 95% of HIV-infected patients who are anti-HCV positive have HCV viremia [33,34], minimizing the likelihood for misclassification. Similarly, HBsAg-positive patients may not have had active HBV replication, especially if they were treated with antiretroviral medications that are also active against HBV. To eliminate the possibility of this affecting our results, we performed subanalyses excluding HBsAg-positive patients, given the small number of patients with HBV infection in the sample.
Third, a number of potential confounding variables were evaluated and controlled for analytically, but multivariable analyses may not entirely eliminate residual confounding from additional unmeasured factors, as is always true for all observational studies. We did not have information on the duration of viral hepatitis infection, stage of hepatic fibrosis, or hepatitis genotype, and therefore could not examine the relationship between these variables and reduced BMD. In particular, advanced liver histologic disease stage could negatively impact BMD in viral hepatitis, but liver biopsy results were not collected by the cohort.
Fourth, patients were only from Italy, a homogeneous population, potentially limiting the generalizability of our results.
Finally, this study was limited by the absence of concurrent, local non-HIV-infected controls for the generation of Z-scores. Rather, this study relied on the DXA manufacturer's reference data in ambulatory white volunteers from the United States, Europe, and Australia [35]. However, potential differences in BMD between the manufacturer's reference data and the Modena non-HIV-infected population would not be expected to explain the sex differences in the effects of viral hepatitis coinfection observed here.
Results
Patient characteristics
A total of 1237 patients (625 HIV/viral hepatitis coinfected; 612 HIV monoinfected) were included in the study. Demographic and clinical characteristics of the study groups are summarized in Table 1. Eighty-five (9%) patients were HBsAg positive, and 572 (47%) were anti-HCV positive. Coinfected patients had lower BMIs, a longer median duration of HIV infection, lower nadir as well as total CD4 cell counts at the time of DXA, more commonly were physically inactive, less commonly drank alcohol, more commonly smoked cigarettes, and had lower serum 25-hydroxyvitamin D concentrations.
Differences in bone mineral density Z-scores by viral hepatitis status
A statistically significant interaction between viral hepatitis and sex was identified at the lumbar spine (P = 0.002) and femoral neck (P = 0.04). As a result, all subsequent analyses were stratified by sex.
Differences in lumbar spine bone mineral density Z-scores
Overall, men had lower mean Z-scores at the lumbar spine than women [-0.50 versus +0.06; difference = -0.56 (95% CI -0.42 to -0.69); P < 0.001]. However, viral hepatitis-coinfected women had significantly lower mean lumbar spine Z-scores compared with HIV-monoinfected women [-0.15 versus +0.29; difference = -0.44 (95% CI -0.65 to -0.23); P < 0.001]. After adjustment for age, BMI, duration of HIV infection, use of ART, physical activity, and smoking, deficits in lumbar BMD remained greater among coinfected women [difference = -0.51 (95% CI -0.75 to -0.27); P < 0.001; Table 2]. In contrast, coinfected men had similar mean Z-scores at the lumbar spine compared with monoinfected men [-0.52 versus -0.47; difference = -0.05 (95% CI -0.22 to +0.12); P = 0.5]. Adjustment for confounders (Table 2) yielded little change in the difference in lumbar BMD between these two groups [difference = -0.12 (95% CI -0.32 to +0.08); P = 0.2]. When we performed adjusted analyses excluding HBsAg-positive patients, mean lumbar Z-scores remained lower in HCV-coinfected compared with HIV-monoinfected women [-1.31 versus -0.82; difference = -0.49 (95% CI -0.74 to -0.25); P < 0.001] but not between coinfected and monoinfected men [-1.24 versus -1.14; difference = -0.10 (95% CI -0.32 to -0.12); P = 0.4].
Differences in femoral neck bone mineral density Z-scores
Men also had lower mean Z-scores at the femoral neck than women [-0.68 versus -0.52; difference = -0.16 (95% CI -0.05 to -0.28); P = 0.006]. Mean femoral neck Z-scores were lower for coinfected versus monoinfected women [-0.64 versus -0.39; difference = -0.25 (95% CI -0.44 to -0.06); P = 0.009]. After adjustment for age, BMI, duration of HIV, ART use, physical activity, and smoking, deficits in femoral BMD were again greater among coinfected women [difference = -0.26 (95% CI -0.47 to -0.04); P = 0.02; Table 3]. Coinfected men had similar mean Z-scores at the femoral neck compared with monoinfected men [-0.72 versus -0.64; difference = -0.08 (95% CI -0.22 to +0.06); P = 0.2]. After adjustment for confounders (Table 3), deficits in femoral neck BMD remained similar between coinfected and monoinfected men [difference = -0.02 (95% CI -0.18 to +0.14); P = 0.8]. When we again excluded HBsAg-positive patients, mean femoral Z-scores were lower in HCV-coinfected versus HIV-monoinfected women [-1.12 versus -0.84; difference = -0.28 (95% CI -0.50 to -0.06); P = 0.01] but not between coinfected and monoinfected men [-2.28 versus -2.27; difference = -0.01 (95% CI -0.18 to +0.17); P = 0.9]. Analyses using imputed data produced similar results.
Risk of low bone mineral density associated with viral hepatitis coinfection
Risk of low lumbar spine bone mineral density
Overall, 97 patients [8%; 17 women (4%) versus 80 men (10%); P < 0.001] had lumbar spine BMD Z-scores of -2.0 or less. Low lumbar BMD was more prevalent in coinfected patients [58 (9%) versus 39 (6%); OR 1.50, 95% CI 0.99-2.29]. Adjustment for age, BMI, duration of HIV infection, ART use, physical activity, and smoking increased the odds of low lumbar BMD in viral hepatitis-coinfected versus monoinfected women (adjusted OR 3.36, 95% CI 0.96-11.75) and coinfected versus monoinfected men (adjusted OR 1.34, 95% CI 0.77-2.36).
Risk of low femoral neck bone mineral density
One hundred patients [8%; 32 women (7%) versus 68 men (9%); P = 0.2] had femoral Z-scores of -2.0 or less. Coinfected patients more commonly had low femoral BMD Z-scores compared with monoinfected ones [56 (9%) versus 44 (7%); OR 1.27, 95% CI 0.84-1.92], but this difference was not statistically significant. After controlling for age, BMI, duration of HIV infection, ART use, physical activity, and smoking, the risk of low femoral BMD was greater but nonsignificant for viral hepatitis-coinfected versus HIV-monoinfected women (adjusted OR 1.87, 95% CI 0.81-4.34) and coinfected versus monoinfected men (adjusted OR 1.03, 95% CI 0.54-1.94).
Risk of low lumbar spine, femoral neck bone mineral density, or both
A total of 170 patients [14%; 43 women (9%) versus 127 men (17%); P < 0.001] were identified with low BMD, defined as either a lumbar or femoral Z-score of -2.0 or less. This outcome was more common among coinfected patients [100 (16%) versus 70 (11%); P = 0.02]. After adjustment for age, BMI, duration of HIV infection, ART use, physical activity, and smoking, viral hepatitis was associated with low BMD in HIV-infected women (adjusted OR 2.87, 95% CI 1.31-6.29) but not men (adjusted OR 1.19, 95% CI 0.74-1.91). When we excluded HBsAg-positive patients, the risk of low BMD remained greater in HCV-coinfected women versus women with HIV alone (adjusted OR 2.99, 95% CI 1.33-6.74), but no association was found between HCV coinfection and low BMD in men (adjusted OR 1.26, 95% CI 0.75-2.10). Results are summarized in Table 4. Analyses using imputed data produced similar results.
Biochemical parameters of bone metabolism by hepatitis status
After adjusting for eGFR, HIV/viral hepatitis-coinfected women had lower mean intact PTH concentrations (60.1 versus 68.1 pg/ml; P = 0.02) but similar mean 25-hydroxyvitamin D (19.1 versus 19.6 ng/ml; P = 0.6) and osteocalcin (3.0 versus 3.2 ng/ml; P = 0.8) concentrations compared with HIV-monoinfected women. Coinfected men also had lower mean intact PTH concentrations (66.1 versus 73.7 pg/ml; P = 0.001) but similar mean 25-hydroxyvitamin D (21.4 versus 22.3 ng/ml; P = 0.3) and osteocalcin (3.4 versus 4.0 ng/ml; P = 0.13) concentrations compared with HIV-monoinfected men after adjusting for eGFR.
Methods
Study design and patients
We performed a cross-sectional study among patients enrolled in the Modena HIV Metabolic Clinic Cohort, which was initiated in September 2004 to examine metabolic alterations among HIV-infected patients seen at the metabolic clinic of the University of Modena and Reggio Emilia School of Medicine (Modena, Italy) [18]. Patients in the cohort have laboratory-confirmed HIV, provide informed consent, and complete a standardized questionnaire at enrollment that collects demographic, clinical, and HIV data. Dual-energy X-ray absorptiometry (DXA) scans are performed to assess BMD, and blood is drawn for hepatitis status, HIV RNA, immune function, and metabolic parameters.
For this study, all patients enrolled in the Modena HIV Metabolic Clinic Cohort between 1 September 2004 and 31 December 2007 were eligible for inclusion. Approval for the study was obtained from the Institutional Review Boards of the University of Pennsylvania and University of Modena and Reggio Emilia.
Main outcome measures
The primary study outcome was the age and sex-specific standard deviation score (Z-score) of BMD of the lumbar spine and femoral neck. This is the appropriate method to assess disease effects across a population of varied age and sex [19]. In addition, Z-scores of -2.0 or less identify patients with BMD below the expected range for age and sex [19]. Thus, we also examined as study outcomes: low lumbar spine BMD, defined as a spine BMD Z-score of -2.0 or less, low femoral neck BMD, defined as a femoral neck BMD Z-score of -2.0 or less, and low BMD, defined as a lumbar, femoral neck BMD Z-score, or both of -2.0 or less.
Data collection
All data were collected from the Modena HIV Metabolic Clinic Cohort's electronic database. Data on age; sex; smoking (number of cigarettes/day); alcohol consumption (grams of alcohol/day); physical activity [none, mild (<4 h weekly), and intensive (≥4 h weekly)]; diabetes mellitus (based on self-reported physician's diagnosis, use of antidiabetic medications, or both); use of corticosteroids, oral estrogens, and calcium and vitamin D supplements; amenorrhea (defined as the absence of menstrual periods for more than 6 months in women who previously had menses); HIV diagnosis date; risk factors for HIV transmission; nadir CD4 T-lymphocyte count; and antiretroviral medications were collected from patients at enrollment using a structured questionnaire. ART use was defined as receipt of three antiretroviral agents.
Body weight was measured using a digital scale to the nearest 0.1 kg with patients wearing light clothes without shoes. Height was measured using a wall-mounted stadiometer to the nearest 0.1 cm. Measurements for body weight and height were completed in triplicate and the mean recorded.
Blood was drawn from all patients for determination of hepatitis B virus (HBV) surface antigen (HBsAg; Elecsys 2010; Roche Diagnostics, Indianapolis, Indiana, USA), hepatitis C virus (HCV) antibody (anti-HCV; Abbott HCV EIA 3.0 enzyme immunoassay; Abbott Laboratories, North Chicago, Illinois, USA), HIV RNA (Abbott RealTime HIV-1 assay; Abbott Laboratories; lower limit of detection: 50 copies/ml), CD4 cell count, thyroid-stimulating hormone (TSH; Advia Centaur TSH assay; Bayer Healthcare, Tarrytown, New York, USA), serum creatinine, and biochemical parameters of bone metabolism, including intact parathyroid hormone (PTH; Liaison N-Tact PTH Assay; DiaSorin; Stillwater, Minnesota, USA), 25-hydroxyvitamin D (DiaSorin 25-hydroxyvitamin D chemiluminescence immunoassay; DiaSorin), and osteocalcin (Liaison osteocalcin Assay; DiaSorin).
All patients enrolled in the cohort had BMD measured by DXA at the lumbar spine (i.e., L1-L4) and femoral neck. All patients were scanned using the same single densitometer (Lunar DPX-MD; Lunar Corporation, Madison, Wisconsin, USA). Values were expressed as Z-scores (number of SDs relative to age within men and women) compared with the manufacturer's reference population. The instrument was calibrated daily with a hydroxyapatite phantom. The in-vivo coefficients of variation of the DXA measures at the lumbar and femoral sites were less than 2%.
Statistical analysis
Differences between HIV/viral hepatitis-coinfected and HIV-monoinfected patients were assessed using chi-square tests for categorical data and Wilcoxon rank-sum tests for continuous data. We first examined interactions between viral hepatitis, age, and sex. Next, multivariable linear regression was used to determine differences in the mean lumbar and femoral neck Z-scores between coinfected and monoinfected patients, after adjusting for potential confounders. Multivariable logistic regression was then used to determine unadjusted and adjusted odds ratios (ORs) and 95% confidence intervals (CIs) of low femoral neck and lumbar spine BMD between patients with and without viral hepatitis. Potential confounders evaluated included age, sex, BMI (calculated as weight/height2), duration of HIV infection, nadir CD4 cell count, HIV viral load and CD4 cell count at the time of DXA, receipt of ART, smoking status, alcohol consumption, physical activity, amenorrhea, and estimated glomerular filtration rate [eGFR, calculated using the formula developed by the Modification of Diet in Renal Disease Study [20]: 186 x (serum creatinine)-1.154 x (age)-0.203 x (0.742 if woman)]. We assessed the impact of confounding by examining the change in the ß-coefficient for viral hepatitis, as potential confounders were included in the model [21]. Additionally, as HBsAg-positive patients may not have had active HBV replication due to treatment with antiretroviral medications that are also active against HBV, we performed subanalyses excluding HBsAg-positive patients and examined the association between chronic HCV alone and low BMD.
Variables of interest were examined for missing data, and the covariate with the highest proportion of missingness was HIV RNA (4%). To ensure that missing data did not affect the validity of our results, multiple imputations were performed to generate 10 data sets [22]. Multivariable linear and logistic regression analyses were then performed on each data set, and results were combined to arrive at appropriate variances that accounted for missing data [23]. As renal function affects serum levels of bone turnover markers, we adjusted for eGFR when comparing these parameters between coinfected and monoinfected patients.
Assuming a type 1 error of 0.05, 80% power, a 5% prevalence of low BMD in HIV-monoinfected patients, and a 1: 1 ratio of monoinfected: coinfected patients, we estimated that 1030 patients (515 with viral hepatitis coinfection) were needed to detect an OR equal to 2.0 of low BMD. This sample size also provided 80% power to detect a difference in Z-score of 0.15 between patients with and without viral hepatitis. All data were analyzed using STATA 10.0 (Stata Corporation, College Station, Texas, USA). Multiple imputation analysis was done using SAS 9.1 (SAS Incorporated, Cary, North Carolina, USA).
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