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Title Page: Growth Parameters in HIV Vertically Infected Adolescents on

Antiretroviral Therapy in Rio de Janeiro, Brazil.

Short running headline: Growth Parameters in HIV-infected Adolescents, in Brazil.

First and last name of each author: Buonora, Sibelle1; Nogueira, Susie

2; Pone, Marcus

Vinicius3; Aloé, Marisa

4; Oliveira, Ricardo Hugo

1; Hofer, Cristina

2.

Name of each author's department, institution or hospital, location and country:

1. Department of Pediatrics, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

2. Infectious Diseases Service, Department of Preventive Medicine, Universidade Federal do

Rio de Janeiro, Rio de Janeiro, Brazil.

3. Department of Pediatrics, Instituto Fernandes Figueira, Fundação Osvaldo Cruz, Rio de

Janeiro, Brazil.

4. Department of Pediatrics, Hospital Municipal Jesus, Secretaria Municipal de Saúde, Rio de

Janeiro, Brazil.

No support was given to the Project, except for a scholarship from Coordenação de

Aperfeiçoamento de Pessoal de Nível Superior, granted to Sibelle Buonora.

There is no possible conflict of interest by any author of this paper.

This study was reviewed and approved by the Instituto de Puericultura e Pediatria Martagão

Gesteira, Instituto Fernandes Figueira, and Hospital Municipal Jesus Ethical Committees.

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Abstract:

Background: Growth failure was an important factor to initiate or change antiretroviral (ARV)

therapy on HIV-infected children.

Our main goal is to assess the impact the HIV infection has on the growth parameters of

adolescents, in Brazil, who vertically acquired HIV.

Methods: This is a retrospective longitudinal study, involving adolescents, aged 10 to 20 years old,

who vertically acquired HIV and were followed up until December 2003 in one of the three main

referral centers for pediatric HIV/AIDS care in Rio de Janeiro, Brazil. Variables related to

demographic, clinical, and laboratorial issues were studied. Their length and weight were measured

with standard clinic scales in all visits.

Results: 108 subjects were enrolled. The median age was 12.7 years. The median time of follow up

was 97.2 months. 61 (56.5%) were female.

The difference between the baseline and the final weight z-score measures was -0.31 (p=0.02).

Patients with final weight Z-score ≤ -2 used more antiretroviral regimens during their life (average

=4.13) than those with Z-score > -2 (average= 2.90), p<0.01, and they also had a lower final CD4+

cell percentage - average of 19% vs 24% (p<0.01), respectively.

The difference between the baseline and the final height z-score measures was -0.27 (p<0.01).

Several factors were associated with final height Z-score ≤ -2: Clinical stage C during follow up

(RR=1.60, 95%CI=1.11 -2.31), chronic diarrhea during follow-up (RR=2.02, 95%CI=1.04-3.90),

HAART use (RR=1.41, 95%CI=1.16-1.71), Number of ARV regimens (p<0.01) and Final CD4+

cell percentage (p<0.01). In the multivariate analysis, the presentation of Clinical stage C during

follow-up was the significant variable (OR=4.04, 95%CI=1.23-13.28).

Conclusion: Even on HAART, HIV positive adolescents have lower growth parameters than the rest

of the population and it is associated with a worse prognosis.

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Introduction

Highly active antiretroviral therapy (HAART) has transformed the human immunodeficiency virus

(HIV) infection from a near-uniformly fatal disease into a chronic, manageable illness. Before

HAART, 5-10% of HIV-positive children survived longer than 3 years after their serologic

diagnosis. Now, this mortality rate decreased up to 70%, consequently, the number of HIV-positive

adolescents who were vertically infected is increasing (1).

Several studies evaluated the growth retardation in HIV-positive children, however, the follow up

period was limited and they were not targeted at adolescent populations (2,3). Growth failure is a

morbidity/mortality predictor in these children (4-9),

and is considered an important factor to initiate or change antiretroviral therapy (10).

In developing countries, several factors are associated with growth retardation, including

nutritional, socio-economic, cultural, and genetic issues. There is scarce data on the growth

evaluation of HIV-infected children in developing countries and none considering the HAART use.

Our main goal is to assess the impact the HIV infection has in the growth parameters of adolescents

who vertically acquired it.

Methods

This is a retrospective longitudinal study involving adolescents, aged 10 to 20 years old, who

vertically acquired HIV. Inclusion criteria were: being alive until December, 2003 and being

followed-up in one of the three main referral centers for pediatric HIV/AIDS care in Rio de Janeiro,

Brazil. The referral centers were Instituto de Puericultura e Pediatria Martagão Gesteira (IPPMG),

Instituto Fernandes Figueira (IFF), and Hospital Municipal Jesus (HMJ). Patients were excluded if

the vertical transmission of HIV was not certified, if they were transferred/died before December

2003 or if they failed to attend follow-up for over a year.

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The charts of all the adolescents were reviewed using a structured questionnaire. Variables related

to demographic, clinical, and laboratorial issues were collected. In the charts, their length

(recumbent length for subjects < 2 years or standing height for subjects ≥ 2 years) and weight were

measured with standard clinic scales in all visits. Clinical Stage (11) was evaluated in every visit.

Nutritional data were not collected.

Antiretroviral (ARV) use was also evaluated. If a patient used one or more ARV for over a month,

this would qualify as an ARV regimen.

HAART was defined as the use of triple ARV therapy (any class), but the use of three-nucleoside

transcriptase reverse inhibitor, when these patients started on HAART, they were maintained on it

for the rest of their lives.

Statistics analysis

First and last visit weight and height Z-scores, adjusted for gender and age, were calculated using

Nutchildren, Epinfo-2000, CDC, USA.

In order to compare baseline and last height and weight Z-score, paired T-test was used. The null

hypothesis was that the difference between last and baseline height and weight Z-scores was zero,

such as the general population. The Z-score measures were also categorized as -2 or lower versus

higher than –2 (our main endpoint). We evaluated possible risk factors associated with lower Z-

scores. The Pearsons Chi-square test was used in the bivariate analysis of all categorical variables.

If any cell presented with an expected value equal or below 5, the Fisher exact test was used. The

Student T-test was used in the bivariate analysis of the continuous variables, and if any variable

presented a non-normal distribution, the Mann Whitney test was used.

The correlation between baseline and final height and weight Z-scores were evaluated by the

Spearman correlation test.

Further logistic regression was used to evaluate possible co-variables and p-value 0.10 was used

to keep a variable in the model.

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Epi-info 2000, CDC, USA and SPSS v 11.0,USA were used on this analysis.

Ethical Considerations

This study was reviewed and approved by the IPPMG, IFF, and HMJ Ethical Committees.

Results

In December 2003, 138 adolescents were being followed-up in these Units. Thirty individuals were

excluded; 17 patients acquired HIV through blood transfusions and for 13 patients, the mode of

transmission was not identified. We enrolled 108 subjects, although we did not have all the

anthropometric data for two patients.

The patients median age was 12.7 years (10.5-19.5 years old). The median time of follow-up was

97.2 months (16-168 months). Of the 61 (56.5%) women enrolled in the study, we were able to

retrieve the information of age at menarche from 23 of them (38%). It ranged from 11-14 years and

the average was 12.3 years.

Most of these patients came from lower social economic classes and the average monthly income

per family was of US$200 (1.2 minimum salaries, in Brazil) (12).

Initial height and weight Z-scores were highly correlated; rho=0.79 (p<0.01), as well as final height

and weight Z-scores; rho=0.82 (p<0.01).

Weight

The average baseline weight Z-score was -0.84 (SD=1.42) and the average final weight Z-score

was-1.15 (SD=1.32). The difference between the baseline and the final measures was -0.31

(p=0.02).

At the first evaluation, 19 adolescents had weight Z-score -2 or lower and by the end of the follow-

up, this number had increased to 30 patients. Among the 19 patients who originally presented lower

Z-scores, 10 continued in this category by the end of the follow-up.

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Patients with lower final weight Z-scores used more antiretroviral regimens throughout life and also

had a lower final CD4+ cell count average (Table 1).

Height

The average baseline height Z-score was -0.94 (SD=1.23) and the average final height Z-score was-

1.20 (SD=1.18). The difference between the baseline and the final measures was -0.27 (p<0.01).

At the first evaluation, 17 adolescents had height Z-score -2 or lower and by the end of follow-up,

this number increased to 30 patients. Among the 17 patients who originally presented lower Z-

scores, 10 continued in this category by the end of the follow up. Individuals classified as C

category during the follow up had a greater chance of being shorter by the end of the observation.

They also had a lower final CD4+ cell count average and more chronic diarrhea, which is defined

as at least two loose stools per day for >30 days (11), (Table 2). In the multivariate analysis (Table

3), after adjusting for being in the clinical stage C during follow-up (OR=4.04, 95%CI=1.23-13.28),

chronic diarrhea was not associated with a lower final height Z-score.

HAART use was more common among adolescents with lower final height Z-scores, who were also

more antiretroviral-experienced. However, they were being followed-up longer than adolescents

with higher final height Z-scores and once adjustments regarding follow-up lengths were made, it

was seen that individuals with lower height Z-score did not use more regimens.

Discussion:

In this study, we demonstrated that even on HAART, vertically HIV-infected children tend to have

decreased growth parameters than the rest of the population and that they maintain lower

parameters once adolescence is reached. Children who presented lower height Z-scores tend to have

lower weight Z-scores, which are highly correlated.

Adolescents with lower final height and weight z-scores were more severely ill (stage C) and had

worse immunological parameters (lower final CD4+ cell count). As in other studies, in developed

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countries (12-16), height z-scores were more efficient in predicting worse prognosis than weight z-

scores, probably due to the fact that it is a more precise measure of chronic progression.

These subjects were vertically infected by HIV and followed for a long period, during which,

several ARVs were used, even monotherapy. Therefore, it is important to consider this fact when

observing the lack of effect of HAART on growth parameters. The number of ARV regimens was

almost significant in the multivariate analysis, as this variable reflects the ARV use in a generation

who was exposed to several practices, such as monotherapy, which are inadmissible nowadays. It

probably also reflects the differences in length of follow-up, which is likely to have been an

insignificant variable due to colinearity with the number of ARV regimens.

Another important issue is the hypothesis of whether in this analysis, chronic diarrhea contributed

to this growth deficit or if it was only a marker of disease progression. In the multivariate analysis

we were able to demonstrate that it is probably a marker of illness severity since after adjusting for

final CD4+ cells count and clinical C stage, it was not statistically significant.

Guillén et al demonstrated that virologic control is related to sustained growth (17). Due to

technical problems, most of these adolescents were not able to perform viral load in a routine

manner, therefore, we were not able to evaluate it.

Since this is a retrospective study, its main limitation is that not all information can be retrieved

form charts. Although the lack of nutritional information is an important issue when considering

growth retardation, this is a homogeneous population, of low socioeconomic status from Brazil, and

probably suffered some nutritional privation during their lives.

Pubertal progression is another very important issue when considering growth in adolescents.

Unfortunaly, this information was not retrieved from the charts. As a proxy, we used age of

menarche in a subset of female patients. Interestingly, the average age of menarche in this subset

was not different from the rest of the Brazilian population (18). Consequently, we were not able to

conclude any relation between growth deficit and puberty delay. Other studies demonstrated a

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delayed onset of pubertal development in a more severely ill population (19, 20), and so, we may

conclude that both growth development and puberty maturity are related to illness severity.

In summary, we were able to demonstrate that growth parameters are important surrogate markers

of immunodeficiency even in a developing country, adjusting for number of ARV regimens, and

laboratory parameters (CD4+ cells).

We believe this study approaches a significant challenge regarding the HIV infection: To evaluate

and follow-up HIV-vertically infected adolescents, living in a developing country, and dealing with

poverty, orphanage, decreased growth parameters and the stigma associated with these issues. And

probably, since these patients were very ARV-experienced, they also need to deal with multi-drug-

resistant HIV strains and consequently, antiretroviral regimens with several requirements and more

frequently associated with side effects. We believe that a lot more attention should be paid to this

population in order for them to have the best possible follow-up.

Acknowledgments:

We would like to express our gratitude for the help given by the Coordenação de Aperfeiçoamento

de Pessoal de Nível Superior, and Infectious Diseases Post-Graduation Program – Universidade

Federal do Rio de Janeiro.

We would also like to thank Dr Guilherme Santoro-Lopes for.his important suggestions on this

manuscript, and Mrs Andreia Fiorani for the English correction.

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Table 1: Bivariate Analysis on Final Weight Z-score

Variables Z-score ≤-2 Z-score >-2 RR 95%CI p-value

Baseline CD4+

cell counts – % mean (SD)

18 (14) 19 (11) p=0.71

Clinical stage

C during follow up

19/30 33/76 1.46 1.00-2.12 p=0.09

Chronic

diarrhea during follow up

8/26 15/61 1.24 0.62 – 2.45 p=0.60

HAART use 26/30 49/70 2.17 0.84-5.60 p=0.13

Number ARV

regimens mean (SD)

4.13 (2.18) 2.90 (1.73) p<0.01

Final CD4+

cell count – %

mean (SD)

19 (12) 24 (10) p=0.03

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Table 2: Bivariate Analysis on Final Height Z-score

Variables Z-score ≤-2 Z-score >-2 RR 95%CI p-value

Baseline CD4+

cell counts – %

mean (SD)

17.87 (13.46) 18.98 (11.07) p=0.66

Clinical stage C during

follow up

20/30 32/77 1.60 1.11 -2.31 p=0.02

Chronic diarrhea during

follow up

11/26 13/62 2.02 1.04-3.90 p=0.04

HAART use 28/30 47/71 1.41 1.16-1.71 p<0.01

Number ARV regimens mean

(SD)

4.33 (2.01) 2.80 (1.74) p<0.01

Final CD4+ cell count – %

mean (SD)

17.57 (12.48) 25.10 (9.56) P<0.01

Follow up

duration - mean (years)

(SD)

8.50 (3.00) 7.27 (3.05) P=0.06

Table 3: Multivariate analysis on Final Height Z-score:

Variable OR 95%CI p-value

Clinical stage C during follow up

4.04 1.23-13.28 0.02

Chronic diarrhea during follow up

1.23 0.35-4.20 0.74

Number ARV regimens 1.39 0.97-1.99 0.07

Final CD4+ cell count – % 0.95 0.90-1.01 0.14

Follow up duration 1.16 0.91-1.48 0.24