To our knowledge, this is the first investigation on calcium intake and its relationship with BMD in adult subjects with HIV-infection. Previous studies in this area focused on calcium intake and its potential metabolic impact in HIV-subjects, or on its link with BMD in the subset of a HIV- pediatric population [21–23]. Despite the typical southern Italian regional habit of drinking a glass of milk with coffee in the morning (about 300 mg of calcium), median daily calcium intake in our HIV-infected subjects was lower than the recommended daily amount, as reported by other Asian and European studies [24–26]. Milk is a good source of both calcium and vitamin D but other calcium-rich foods are needed to create a balanced diet assuring sufficient calcium and vitamin D intake in an HIV-population.
Parmesan cheese could be included in the "Mediterranean diet group" of foods because it is usually added to pasta and pizza. In fact, in our study interview Parmesan (25 g = 300 mg calcium) was regularly consumed in 90% of the HIV-subjects.
Among the various dairy foods available we considered yogurt, which contains live active cultures and a substantial amount of calcium and vitamin D. Interestingly, we found that weekly yogurt intake was a protective independent predictor of BMD in the lumbar spine.
When the patients were divided into two groups the analysis showed a more favorable relationship between yogurt plus milk and BMD than milk alone. No previous study has examined the relationship between yogurt intake and BMD in HIV-infected subjects and only Sahni et al.  showed that milk and high yogurt intake were positively associated with hip and spine BMD in immunocompetent subjects in the Framingham Offspring Study.
Probiotic microorganisms have considerable immunomodulatory effects in the gut-associated lymphoid tissue . The importance of the gut microbiota for the development of the host’s immune system and the suggested connection between gut microbiota and bone mass in mice led the researchers to investigate the impact of probiotic yogurt on bone mass, especially in the HIV- population . Further studies are required to evaluate the gut microbiota and other micronutrients as a novel therapeutic target for osteoporosis .
In the present study the prevalence of osteoporosis was significantly higher in HIV- infected than in uninfected subjects, a result similar to previous meta-analyses .
Currently, more intensive investigations have been made to better understand whether HIV infection per se is a risk factor for low BMD [1, 7]. Since the virus itself may induce hypovitaminosis D through many mechanisms (increased levels of TNF-α, inhibition of renal hydroxylation and induction of 25-hydroxyvitamin D consumption by the macrophages and lymphocytes) [7, 32, 33], the abnormal biochemical features found in our HIV-infected subjects compared with the controls, in particular the lower vitamin D levels, may support the hypothesis that HIV per se might play a role in the pathogenesis of bone loss. In addition, this finding in a sunny country like Italy might suggest the need to develop and recommend new key steps of calcium and vitamin D intake in the prevention of osteoporosis.
Several HIV-related factors were correlated with BMD on univariate analysis in both the lumbar spine and femoral neck in our HIV-infected subjects. The correlation between time of HIV diagnosis and BMD in the femoral neck as well as nadir CD4+ T-cell count < 200 cells/μL and BMD in both the lumbar spine and femoral neck and the finding that nadir CD4+ T-cell count < 200 cells/μL was an independent predictor of low BMD, suggest that HIV infection per se and in particular the more advanced stages of the disease might affect the skeletal system. This finding also suggests that an earlier commencement of ART might have a beneficial effect on BMD.
On the other hand, we found a significant negative correlation between longer duration of ART, especially PI exposure, and both lumbar spine and femoral neck BMD on univariate analysis.
How to strike a balance between the various aspects to be considered when providing ART, (such as when treatment should be commenced or the classes of drugs to be used, its influence on the progression of HIV disease and, in the present case, its effects on BMD) in order to maximize its benefits is an ongoing debate. In this respect, while Grund et al.  showed that BMD continuously declined in a HIV-group receiving continuous ART, Bolland et al.  found no evidence of accelerated bone loss over 6 years in middle-aged HIV-infected men treated with ART . Many studies on the influence of ART have shown that the relative risk of low BMD is greater when PI are used [31, 37–39], in agreement with our results. Unfortunately, we were unable to evaluate which drugs of the PI class were responsible for the low BMD, whereas other authors have shown that indinavir inhibits bone formation and lopinavir/ritonavir and atazanavir/ritonavir are associated with BMD loss [31, 39].
We did not find a significant correlation between tenofovir exposure > 1 year and BMD in HIV-subjects, unlike previous reports [39, 40]. Recent data showed that the introduction of tenofovir or emtricitabine/tenofovir was associated with a 0.8 %–1.1 % decrease in BMD which occurred mainly within the first year in high-risk HIV-infected individuals . In our study the small number of enrolled HIV subjects and the long exposure to different antiretroviral drugs made it difficult to show the real impact of tenofovir exposure on BMD health.
Moreover, no significant correlation was found between the small number of fractures reported in our HIV- subjects and BMD at either of the sites. In a recent Italian study, metabolic factors such as BMI and diabetes mellitus were independently correlated with vertebral fractures in HIV-infected patients . Although low BMD in the setting of HIV-infection is frequently observed, data on its relationship with fracture rate are still under investigation .
To improve the ability to predict subsequent fragility fracture in our patients, we used the WHO FRAX equation . Although fracture risk at ten years and calcium intake showed an inverse relationship this was not statistically significant. However, the role of the FRAX score in the HIV-population is still under debate because the HIV-independent risk factors, HIV-related parameters and ART characteristics were not evaluated by the score itself .
Similar to other studies on enrolled HIV-infected subjects with a long duration of infection, we found that older age and BMI values had a negative or positive correlation with BMD, respectively [45–47]. In this respect, a meta-analysis on ten studies showed that low BMI was the main factor behind the lower BMD values in HIV-infected than in normal uninfected individuals .
The results regarding the relationship between dairy product consumption and BMI are contradictory . However, some cross-sectional data suggest that lower-fat dairy products such as milk and yogurt are associated with lower adiposity . The positive correlation between higher BMI values and BMD in the femoral neck DXA T-score or the increasing quartiles of calcium intake, suggest a protective role for calcium intake in the prevention of BMD loss in our HIV-subjects . This observation may also encourage physicians to recommend an increase in calcium consumption especially in HIV-subjects with low BMD.
However, the ideal daily calcium intake to prevent osteoporosis in HIV-positive subjects is not clear because it depends not only on known general and environmental risk factors but also on the patient’s long-term immuno-virological course.
For most people, a daily calcium intake of between 1000 and 1300 mg is both safe and potent but we hypothesize that the dose should be reviewed in relation to ART and HIV-status.
Recent data have suggested that patients with AIDS and addicted to heroin might be at particular risk for bone loss . The independent negative correlation found between drug addiction and low BMD in our HIV subjects confirms this finding.
Another interesting result emerging from our study is the independent correlation between HIV/HCV co-infection and BMD.
Vitamin D deficiency was found in HIV/HCV co-infected patients . Moreover, higher vitamin D levels were independently associated with a rapid virological response in patients with genotype 1 chronic hepatitis C . Increasing vitamin D intake may positively modulate the response to antiviral treatment in HCV-infected or HIV/HCV co-infected patients. Further research is necessary to better understand the impact of viral hepatitis on BMD in HIV-infected and uninfected subjects and discuss the clinical applications and future direction of this field .