The European Collaborative Study

Data were available from the prospective ECS, on children bom from 1984. Children born to

HIV infected mothers are followed according to the standard ECS protocol (Appendix 2.1), with

detailed clinical and laboratory information collected regularly by clinicians in paediatric referral

centres in major hospitals in Padua, Berlin, Edinburgh, Madrid, Valencia, Amsterdam,

Stockholm, Bmssels, Genoa and Barcelona (Figure 2.1 and Appendix 2.2). Children are seen at

birth, around three and six weeks, 3, 4.5 and six months and then at 3-monthly intervals until 24

months. Subsequently, infected children are seen at least twice a year, uninfected children once a

year, and clinical and laboratory information and current treatment recorded on standard forms.

Data are also recorded when children make visits, because of symptoms, outwith the protocol

schedule. Parental consent is obtained before enrolment in the ECS, and the study is approved by

the local ethics committees.

Figure 2.1 Location of Paediatric centres of the ECS

Results presented in this thesis are based on ECS data available at the time of each analysis. The

last of the four analyses used data collected before September 2002. By this time, 190 (10.2%)

infected and 1666 (89.8%) uninfected children had been enrolled in ECS paediatric centres

(Figure 2.2).

Figure 2.2: ECS children by year of birth and infection status □ Infected Children ■ Uninfected Children

160

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0 ) 0 > 0 > 0 > 0 > 0 > 0 ) a > 0 ) 0 > 0 > 0 > 0 ) 0 > o o o

2.2.1 Role of the researcher

As this work was carried out as part of the ongoing study, which I joined in 1999 as the ECS

statistician, it is appropriate to clarify my role in the research presented in this thesis. The data

collection was the responsibility of local clinicians, and data verification, entry and database

management was carried out by epidemiologists at the ECS coordinating centre at the Institute of

Child Health, London. I was given the responsibility for the analysis of the data relating to

infected children in particular, with the aim of clarifying the patterns of disease progression in vertically-infected children in Europe.

The direction and nature of the analyses were agreed in consultation with my supervisor. Professor Marie-Louise Newell, and co-supervisor Mario Cortina-Borja. The strategic and technical development of all analyses presented here were entirely my responsibility. The work has resulted in five manuscripts for publication in peer-reviewed journals (Appendix 2.3). I was involved in the preparation of all five manuscripts, as part of the ECS team.

2.2.2 Diagnosis of HIV

Until the development of polymerase chain reaction (PGR) and virus culture tests, detection of HIV-infection in newborn children of infected mothers was based on anti-HIV immunoglobulin-G antibodies. As maternal antibodies cross the placenta and remaining in the newborn system for up to 18 months of hfe or more, antibody testing is not specific in these infants, and diagnosis by PCR and virus culture is the informative test (72). A child is regarded as HTV-infected if it has at least two positive tests or meets the criteria for AIDS. Children are considered uninfected with HIV if they test negative for HIV-antibodies at least twice at 6 to 18 months of age, there is no other laboratory indication of infection and they do not meet the ADDS diagnosis criteria (127).

2.2.3 Paediatric classification system

The CDC developed the Public Health Service classification system for HIV infection for the monitoring of health service needs. The system for children less than 13 years of age was revised in 1994 (72) in response to knowledge gained on the development of the clinical symptoms and

immunological status of HIV in children. The refinement of the classification provides an easy to use system comprised of mutually exclusive categories which reflects the stage of the disease for HIV-infected children. In clinical practice, the classification is progressive in that once a child is classified they are not reclassified into less severe categories even if there is a subsequent clinical or immunological improvement.

The CDC classification system for paediatric HIV infection has proved a useful framework for analysis of clinical information on infected children. There are four clinical categories based on signs and symptoms and three immunological categories which classify children according to the severity of their immunosuppression attributable to HIV infection (72). The two category structures represent the progressive staging of the disease (Appendix 2.4 A and B). The four categories for clinical manifestations in infected children are: N (asymptomatic), A (mildly symptomatic), B (moderately severe symptoms, including lymphoid interstitial pneumonitis (LIP), and C (severe symptoms) according to the CDC (72) (Appendix 2.4 A). Children who died with HIV-related disease were classified in a separate category. Normal (category 1), and moderate (2), and severe (3) immune suppression was assessed by CD4^ cell counts or percentages appropriate for age at the time of assessment (Appendix 2.4 B).

2.2.4 Treatment

Practice of ART administration for the prevention of HIV-related disease progression in the ECS has varied between centres and among children, and has not always been consistent with policy (58). Policies on initiation and change of therapy have varied fi'om routine administration upon

confirmation of infection, to decisions being determined by the specific clinical, immunological and virological circumstances of the individual child.

As of the last analysis, 67 (35.3%) remained ART-naiVe, 28 (14.7%) had been administered monotherapy only, 14 (7.4%) double therapy and 81 (42.6%) had been treated with a combination of three or more ART drugs, at some point during follow-up. The proportion of children treated is different for all analyses presented, and breakdown is given in the corresponding results chapters. Different categorisations of ART have been used in different analyses, depending on the context and data constraints, and are outlined in the relevant methods sections. Generally, combinations of two or more therapies have been categorised together, owing to the restricted numbers of observations. These treatment categorisations always excluded ART given neonatally as prophylaxis for prevention of vertical transmission.

In some analyses, children were also categorised according to receipt, or not, of PCP prophylaxis and IVIG treatment.

2.2.5 HIV RNA viral load

HIV RNA viral load is quantified by the number of copies per millilitre (copies/ml) of blood plasma. In the ECS, laboratory tests including HIV RNA viral load, are carried out locally according to standard procedures. HIV RNA viral load is measured by quantitative reverse transcriptase PCR assay (Amplicor Monitor, Roche Diagnostic Systems, Branchburg, New Jersey, USA) or Nucleic Acid Sequence-Based Amplification (NASBA) (bioMérieux, Durham, USA). Values are conventionally logio transformed to resolve non-Normality and heteroscedasticity.

An aspect of assay systems used in the quantification of RNA viral load is that they measure values above particular cut-off values for detection, below which the assays used are not sufficiently sensitive; in this way measurements are potentially left-censored. This problem is more serious for data ascertained using older generations of assay systems which are less accurate due to higher detection cut-off points in the region of 2000-4000 copies/ml. Assays in use in laboratories in the developed world today have lower limits in the region of just 50-100 copies/ml.

2.2.6 Immunological markers

In the ECS, standard, locally carried out laboratory tests for CD4^, CD8^ cell count and absolute lymphocyte measurements are based on flow cytometry (FAC Scan) with Becton-Dickinson antibodies. Venous blood specimens are anti-coagulated using ethylene diamine tetra acid or heparin and processed and an automated haemocytometer is used to obtain total, or absolute, lymphocyte counts and the subtypes o f white blood cell. Counts are expressed in units of 10^ cells per litre (x 10® cells/I). As for HIV RNA viral load, analyses of CD4^, CD8^ cell counts and absolute lymphocyte measurements were logio transformed throughout as it was successful in Normalising residuals and resolving heteroscedasticity, and was thus appropriate for the apphcation of linear models. CD4^ cell counts measurements were also expressed as a percentage of absolute lymphocyte count.

2.2.7 General definitions

Black children in the ECS cohort are almost all bom to mothers fi'om sub-Saharan Africa. As most of the enrolled children are bom to Caucasian or black Afiican women, investigation of differences by race was restricted to comparison of data on children bom to white mothers to

data on children bom to black mothers. With the exception of the clinical patterns investigation, stratification by gender was carried out in the analyses. Children were classified as severely premature if bom at or before 34 weeks of gestation, as moderately premature between 34 and 37 weeks and as fiill-term beyond 37 weeks. Children were categorised according to receipt, or not, of zidovudine (or other ART) neonatally as prophylaxis for the prevention of vertical transmission. Maternal CD4^ cell count measurement were categorised above or below 500 x 10^ cells/1; the dichotomy maximised the power of the assessment of the effect.