Research Proposal

Title of Research

Assessment of body fat in HIV-positive patients to diagnose HIV-associated lipodystrophy using

multiple frequency bio-impedance analysis

Introduction

The HIV-associated lipodystrophy syndrome (HALS) is a phenomenon rendered by the

rise of fat loss from the periphery and/or centralized fat accumulation in the abdominal,

dorsocervical regions and breasts including hyperlipidemia, insulin resistance and lactic

acidaemia. The syndrome is a common adverse effect of HIV treatment with highly active

antiretroviral therapy (HAART), which comprises morphological and metabolic changes

(Mallewa et al., 2008). The combination of drugs from different families used in HAART, for

example, nucleoside reverse transcriptase inhibitors (NRTIs) with protease inhibitors (P1s),

increase the prevalence and severity of HIV-associated lipodystrophy syndrome (HALS) (John,

Nolan, & Mallal, 2001).

The distribution of adipose tissue accounts for two processes, i.e., lipoatrophy and

lipohypertrophy. Lipoatrophy involves a loss of subcutaneous adipose tissue that typically occurs

in the extremities, buttocks and face, while lipohypertrophy mainly occurs in the visceral

compartment of the abdomen, in breast tissue in women and less commonly among men, and, even

more rarely, in the dorsocervical area (Bedimo, 2008).

1
 Hence, it is highly desirable for accurate and precise determination of body composition to

be developed, factoring its association with the various metabolic changes for HIV-associated

lipodystrophy individuals (HALS). Methods such as computed tomography, magnetic resonance,

DXA and hydrostatic weighing generate precise measurement enough to give useful results, but

their cost becomes limiting factor and they are usually not available in most institutions. However,

skinfold (SF) and bioelectrical impedance (BIA) techniques are well-known for its simplicity,

affordable cost and non-invasive which mark them as highly suitable method for the estimation of

body composition.

Bioelectrical impedance (BIA) technique presents a great potential for the estimation of

body composition. The BIA instrument is portable, safe and non-invasive and yields rapid and

reproducible results (Kushner, 1992; Kyle et al., 2004a). The resistance and reactance values

obtained by BIA can be used to estimate body composition based on predictive equations (Kotler,

Burastero, Wang, & Pierson, 1996). In view of the prediction equation however, the equations

constructed for HIV-negative individuals are deemed inappropriate as well as dangerous for HIV-

positive individuals and therefore, the equation should be specifically built and validated for the

group of HIV-infected individual only.

Study Background

In 1981, five cases of Pneumocystis carinii pneumonia were presented to the United States

Centre for Disease Control in homosexual men who had also presented with decreased

CD4+ T cell count resulting in a deficit in cell-mediated immunity (Gottlieb et al., 1981). The

men were later found to be suffering from an acquired immune deficiency syndrome

2
(AIDS), caused by a retrovirus named human T-lymphotropic virus type III /

lymphadenopathy-associated virus (Sarngadharan, Devico, Bruch, Schüpbach, & Gallo, 1985),

or as it called nowadays, human immunodeficiency virus (HIV).

The retrovirus attacks CD4+ T-helper cells, a type of white blood cell in the immune

system and replicates persistently inside these cells which later weakened the body line of defence

system gradually. Antiretroviral therapy treatment helps combat the infection by the consumption

of antiretroviral drug. The highly active antiretroviral therapy (HAART) usually comprises of a

combination of at least three antiretroviral drugs from at least two different antiretroviral classes

required for the HIV viral load count to be suppressed and avoid immune destruction, hence

prolongs the life expectancy of HIV infected patients.

HIV associated lipodystrophy syndrome (HALS) is a complication of human

immunodeficiency virus (HIV) infection and antiretroviral drugs ( ARVs) (Galescu, Bhangoo,

& Ten, 2013). HALS can be described by peripheral lipoatrophy, localized fat accumulation

(visceral, back of neck and lipomata), hyperlipidaemia, insulin resistance and hyperglycaemia

(Chen, Misra, & Garg, 2002) which could lead to cardiovascular disease and diabetes mellitus. In

general, HALS is characterized by different patterns of body fat distribution which are identified

by clinical and body composition assessment, including bioimpedance analysis (BIA) (P Freitas

et al., 2011).

HALS further divided to two distinct processes which are lipoatrophy and lipohypertrophy.

It is still not understood whether these two processes occupy the same mechanism, or each is

independent processes even though usually both are regarded as distinguishable processes. It is

also noted that both can happen together and separately (Dinges et al., 2005; Mulligan et al., 2006).

3
 There are several aberrations in body composition that occur resulting from the HIV

infection. Continuous loss of body lean mass may also weaken and the consequence is death due

to wasting in HIV-infected individuals. It is shown that fat-free mass and body fat content of HIV-

infected individuals were lower in comparison with normal individuals (Mutimura et al., 2010).

However, there is no significant differences after age and height adjustment. Moreover, the fat

distribution has high percentage of visceral fat and low subcutaneous fat in HIV-infected

individuals than normal individuals (Kotler, Rosenbaum, Wang, & Pierson, 1999).

Common research methods available for body composition assessment are dual-energy X-

ray absorptiometry (DXA), densitometry and multiple dilution method. These methods boast

definite accuracy of measurement even though their use limited and apply to only certain

conditions. Some of the limiting factors are the long hour of time required to perform the

examination, high cost and the degree of technician skills needed for assessment. Whereas, the

common field methods available for body composition assessment are anthropometry and

bioelectrical impedance analysis (BIA). These methods have the following advantages which are

being rapid, easy to use, convenient and inexpensive but they are limited in their accuracy.

Based on the previous studies, the SF-BIA method is not sufficient in altered hydration

condition but is valid and reasonably accurate in healthy population (C. Earthman et al., 2007).

These studies suggest improvement in using the BIA as prediction tool where recalibration is

recommended and adjusted for different population and sub-population (Achim Schwenk et al.,

1999) provided numerous variables are available to eliminate bias and increase accuracy (Horlick

et al., 2002). It is not advised to be used for individual evaluation but BIA is highly suggested over

anthropometry method (P Freitas et al., 2011). One study analyses the use of phase angle from

4
BIA as a substitute for identifying risk of wasting in HIV-infected individuals and it found that

phase angle may contribute as a prognosis tool for lipodystrophy (Achim Schwenk et al., 2000).

Several studies evaluate lipoatrophy and lipohypertrophy consequences on body

composition of HIV-infected individuals. Body composition evaluated by DXA in untreated HIV-

infected subjects shown fat mass and lean mass decreases even though after adjustment on age,

height, body bone mineral density (BMD). This finding is contributed to the low trunk fat mass

while lower limb fat mass is high. (Delpierre et al., 2007) The result finding is similar with patients

treated with HAART, mainly protease inhibitors (PIs) and nucleoside analogue reverse

transcriptase inhibitors (NRTIs) by measuring regional fat with DXA in a longitudinal study

(Mallal, John, Moore, James, & McKinnon, 2000). However, it should be noted that the result may

deviate in performing analysis due to hindrance in quality assurance (Smith et al., 2003) even

though the reproducibility of DXA as diagnostic tool is high regardless of populations when

estimating body fat in HIV lipodystrophy (Cavalcanti, Cheung, Raboud, & Walmsley, 2005)

Objective

The objective of the present study was to develop equations for the estimate of total body fat by

BIA methods in HIV-infected patients on HAART.

Methodology Planning

Measurements of total body fat have already been performed in 300 HIV infected and 250

community dwelling uninfected individuals from the Malaysian HIV and Aging study. In addition,

5
whole body scans using DEXA which are the gold standard in assessment of body composition is

also available in a subset of 200 participants in this cohort which will allow validation of the BIA

assessment in both HIV infected and uninfected individuals. The specific analyses which will be

performed include;

- Calculate and estimate the main body composition parameters (FM, FFM) using most

common and available MF-BIA prediction equations.

- Conduct a comparison study to explore the significance differences in body

composition between HIV+ and HIV- subjects utilizing different prediction modules.

- Conduct a correlation and regression study to explore the main bioimpedance

predictors of HIV-related lipodystrophy syndrome.

- Develop a prediction module to classify the severity zones of HIV-related

lipodystrophy syndrome.

Following enumeration of lipodystrophy using MF-BIA in the cohort, the result obtained

will be correlated with markers of inflammation, IL-6. The inflammatory marker is involved in the

pathogenesis of cardiovascular disease which is mediated by lipodystrophy in HIV and the

correlation of these markers with MF-BIA will help further validate the utility of this measure in

this population.

So far, the plasma samples required by this study have been collected and stored in

laboratory freeze storage. IL-6 will be measured using conventional ELISA (Quantikine R&D

Systems). The protocols have been previously optimised in the laboratory. These assays will be

performed in the subset of 200 participants who have both MF-BIA and whole body DEXA

performed. Correlations between inflammatory and endothelial damage markers with MF-BIA and

6
DEXA assessments of lipodystrophy will be performed using multivariate linear regression

adjusting for age, gender, ethnicity and CD4 T-cell counts.

The proposed data analysis and statistical methods utilized for evaluation are student-t test

and ANOVA analysis, Correlation analysis, ICC, Multi-Regression analysis, Bland Altman Plot

method and BIVA method.

Expected study outcome

The expected results from this study will be:

 Significance representation of body composition parameters among HIV+ subjects and

differences between healthy subjects.

 MF-BIA body composition prediction module for HIV related lipodystrophy for patient

management purpose.

7
Work Schedule

No 2017 2018
Activities
.
6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12

1 Literatures review
2 Analysis of existing study
3 Submission of research proposal
4 Appointment of supervisor
5 Research proposal defence
6 Data collection and statistical analysis
7 Analysis and evaluation
8 Candidature defence
9 Paper publication
10 Project presentation
11 Writing up
12 Final editing of thesis
13 Final thesis submission

8
References
Bedimo, R. J. (2008). Body-fat abnormalities in patients with HIV: progress and
challenges. Journal of the International Association of Physicians in AIDS Care, 7(6),
292–305.
Cavalcanti, R. B., Cheung, A. M., Raboud, J., & Walmsley, S. (2005). Reproducibility of
DXA estimations of body fat in HIV lipodystrophy: implications for clinical research.
Journal of Clinical Densitometry, 8(3), 293–297.
Chen, D., Misra, A., & Garg, A. (2002). Lipodystrophy in human immunodeficiency virus-
infected patients. The Journal of Clinical Endocrinology & Metabolism, 87(11), 4845–
4856.
Delpierre, C., Bonnet, E., Marion-Latard, F., Aquilina, C., Obadia, M., Marchou, B., …
Bernard, J. (2007). Impact of HIV Infection on Total Body Composition in
Treatment—Naive Men Evaluated by Dual-Energy X-ray Absorptiometry Comparison
of 90 Untreated HIV-Infected Men to 241 Controls. Journal of Clinical Densitometry,
10(4), 376–380.
Dinges, W. L., Chen, D., Snell, P. G., Weatherall, P. T., Peterson, D. M., & Garg, A.
(2005). Regional body fat distribution in HIV-infected patients with lipodystrophy.
Journal of Investigative Medicine, 53(1), 15–25.
Earthman, C., Traughber, D., Dobratz, J., & Howell, W. (2007). Bioimpedance
spectroscopy for clinical assessment of fluid distribution and body cell mass. Nutrition
in Clinical Practice, 22(4), 389–405.
Freitas, P., Carvalho, D., Santos, A. C., Mesquita, J., Correia, F., Xerinda, S., … Medina, J.
L. (2011). Assessment of body fat composition disturbances by bioimpedance analysis
in HIV-infected adults. J Endocrinol Invest, 34(10), e321-9.
Galescu, O., Bhangoo, A., & Ten, S. (2013). Insulin resistance, lipodystrophy and
cardiometabolic syndrome in HIV/AIDS. Reviews in Endocrine and Metabolic
Disorders, 14(2), 133–140.
Gottlieb, M. S., Schanker, H. M., Fan, P. T., Saxon, A., Weisman, J. D., & Pozalski, I.
(1981). Pneumocystis pneumonia--Los Angeles. MMWR. Morbidity and Mortality
Weekly Report, 30(21), 250–252.
Horlick, M., Arpadi, S. M., Bethel, J., Wang, J., Moye, J., Cuff, P., … Kotler, D. (2002).
Bioelectrical impedance analysis models for prediction of total body water and fat-free
mass in healthy and HIV-infected children and adolescents. The American Journal of
Clinical Nutrition, 76(5), 991–999.
John, M., Nolan, D., & Mallal, S. (2001). Antiretroviral therapy and the lipodystrophy
syndrome. Antiviral Therapy, 6(1), 9–20.
Kotler, D. P., Burastero, S., Wang, J., & Pierson, R. N. (1996). Prediction of body cell
mass, fat-free mass, and total body water with bioelectrical impedance analysis:
effects of race, sex, and disease. The American Journal of Clinical Nutrition, 64(3),
489S–497S.
Kotler, D. P., Rosenbaum, K., Wang, J., & Pierson, R. N. (1999). Studies of body
9
 composition and fat distribution in HIV-infected and control subjects. JAIDS Journal
of Acquired Immune Deficiency Syndromes, 20(3), 228–237.
Kushner, R. F. (1992). Bioelectrical impedance analysis: a review of principles and
applications. J Am Coll Nutr, 11(2), 199–209.
Kyle, U. G., Bosaeus, I., De Lorenzo, A. D., Deurenberg, P., Elia, M., Gómez, J. M., …
Pirlich, M. (2004a). Bioelectrical impedance analysis—part I: review of principles and
methods. Clinical Nutrition, 23(5), 1226–1243.
Mallal, S. A., John, M., Moore, C. B., James, I. R., & McKinnon, E. J. (2000). Contribution
of nucleoside analogue reverse transcriptase inhibitors to subcutaneous fat wasting in
patients with HIV infection. Aids, 14(10), 1309–1316
Mallewa, J. E., Wilkins, E., Vilar, J., Mallewa, M., Doran, D., Back, D., & Pirmohamed,
M. (2008). HIV-associated lipodystrophy: a review of underlying mechanisms and
therapeutic options. Journal of Antimicrobial Chemotherapy, 62(4), 648–660.
https://doi.org/10.1093/jac/dkn251
Mulligan, K., Parker, R. A., Komarow, L., Grinspoon, S. K., Tebas, P., Robbins, G. K., …
Dubé, M. P. (2006). Mixed patterns of changes in central and peripheral fat following
initiation of antiretroviral therapy in a randomized trial. JAIDS Journal of Acquired
Immune Deficiency Syndromes, 41(5), 590–597.
Mutimura, E., Anastos, K., Lin, Z., Cohen, M., Binagwaho, A., & Kotler, D. P. (2010).
Effect of HIV infection on body composition and fat distribution in Rwandan women.
Journal of the International Association of Physicians in AIDS Care, 9(3), 173–178.
Sarngadharan, M. G., Devico, A. L., Bruch, L., Schüpbach, J., & Gallo, R. C. (1985).
HTLV-III: the etiologic agent of AIDS. In Retroviruses in Human
Lymphoma/leukemia: Proceedings of the 15th International Symposium of the
Princess Takamatsu Cancer Research Fund, Tokyo, 1984 (Vol. 15, p. 301). VSP.
Schwenk, A., Beisenherz, A., Kremer, G., Diehl, V., Salzberger, B., & Fätkenheuer, G.
(1999). Bioelectrical impedance analysis in HIV-infected patients treated with triple
antiretroviral treatment. The American Journal of Clinical Nutrition, 70(5), 867–873.
Schwenk, A., Beisenherz, A., Römer, K., Kremer, G., Salzberger, B., & Elia, M. (2000).
Phase angle from bioelectrical impedance analysis remains an independent predictive
marker in HIV-infected patients in the era of highly active antiretroviral treatment. The
American Journal of Clinical Nutrition, 72(2), 496–501.
Smith, D. E., Hudson, J., Martin, A., Freund, J., Griffiths, M. R., Kalnins, S., …
Investigators, P. D. G. and. (2003). Centralized assessment of dual-energy X-ray
absorptiometry (DEXA) in multicenter studies of HIV-associated lipodystrophy. HIV
Clinical Trials, 4(1), 45–49.

10