2000 HIV Human Functional Genomics Partnership Program

Study population A cohort with a total of 2000 HIV patients will be built, consisting of a discovery cohort (n=1200) and confirmation cohort (n=800). The investigators estimate a 2-year inclusion and 2-year follow-up period and will strive for the inclusion of several clinical phenotypes such as long-term non-progressors (~2-3%), immunologic non-responders (~3%), and rapid progressors (~4-5%) and classical risk group patients such as men who have sex with men (MSM), females, and subjects from Sub-Sahara Africa. Patients will be recruited from the following Dutch HIV Treatment Centers: Radboudumc (Nijmegen), Erasmus MC (Rotterdam), OLVG (Amsterdam), Elisabeth Twee-Steden Ziekenhuis (Tilburg).

A sample size calculation cannot be provided due to the variable frequencies of the various traits in genome-microbiome interaction, and their effect on cytokine production. Because of the explorative nature of this study, the size calculation will be variable depending on the type of polymorphism analyzed. The frequencies of the various microorganism classes in the colonizing microbiome is not known in our study population, and therefore power calculations are impossible to be performed.

Earlier studies in the Human Functional Genomics Project have assessed microbiome traits in 250-500 individuals. An earlier HFGP study included a uniform cohort of 200 HIV-infected individuals (all MSM). The present study will also include HIV-infected patients with a more extreme phenotype, females and subjects originating from Sub-Sahara Africa. Because of this, the investigators decided to increase the number of individuals tested to 2000, consisting of a discovery cohort of 1200 subjects, and a confirmation cohort of 800 participants.

Study visits and procedures At inclusion

1. The investigators will collect metadata using questionnaires on lifestyle, health and clinical symptoms, including neuropsychiatric symptoms. Relevant data will also be collected from the patients records in the medical centers and the HIV Monitoring Foundation.
2. Co-pathology will be assesed:

• Cardiovascular risk scores (D:A:D risk score and Framingham CVD score) will be collected as well as ECG and intima-media thickness (IMT) measurements.
• Non-Alcoholic Fatty Liver Disease (NAFLD) assessment through liver ultrasound and FibroScan.

1. Blood will be drawn (90 ml):

• DNA will be isolated for genetic and epigenetic analysis.
• The function of the immune system will be analyzed at several levels using circulating white blood cells from venous blood.
• Metabolism and proteomics will be analyzed
• Virological analysis, characterizing the HIV reservoir, HIV resistance and viral sequences in circulating DNA and RNA.

1. Microbiome analysis will be performed on stool and saliva samples.
2. Urine sample will be collected for creatinine measurements and microbiome.

After 2 years follow-up

1. The investigators will collect metadata using questionnaires on lifestyle, health and clinical symptoms, including neuropsychiatric symptoms. Relevant data will also be collected from the patients records in the medical centers and the HIV Monitoring Foundation.
2. Co-pathology will be assesed:

• Cardiovascular risk scores (D:A:D risk score and Framingham CVD score) will be collected as well as ECG.
• Non-Alcoholic Fatty Liver Disease (NAFLD) assessment through liver ultrasound and FibroScan.

1. Blood samples (10ml) will be collected for biomarker and infection/inflammation parameter analysis.

Patient informed consent procedure Patients will always be approached first by a treating physician or specialized nurse. They will be provided with information and will be asked if a physician-researcher may contact the patient. If there is oral consent for this, the researcher contacts the patient in one week to ask if they are interested in participating in the study. the physician-researcher will answer any questions the patient may have and plan the first visit. The informed consent procedure will be conducted during the patient's first visit.

Patient registry procedures All patient data will be recorded only in coded form in a secure database with audit and edit trail (CASTOR EDC). During the informed consent procedure, the patient is given a study code. The identifying patient data and study code is kept in a password-protected key file, only accessible to the local research team. No patient-identifying data will leave the local medical center.

In the questionnaires and eCRFs in CASTOR, the investigators will enter as many data checks as possible

Biomaterials All biomaterial (blood, stool, urine, saliva) will be transported to and processed in the laboratory of Experimental Internal Medicine of the Radboudumc in Nijmegen. Here, all the biomaterial will also be stored collectively. All materials will be handled and stored only under the patient's study code.

Monitoring Monitoring will be performed by an independent monitor according to the latest guidelines of the The Netherlands Federation of University Medical Centers. An initiation and close-out visit will be planned in plenary form with all the centers in Radboudumc. There will be one extra visit per center. During these visits informed consents will be checked, as well as inclusion and exclusion criteria and source data verification in a small subset of total participants.

Data collection for patient registry

Baseline data collected from electronic patient files or the HIV Monitoring Foundation during inclusion visit:

Medical history:

1. Inclusion date
2. Length and weight
3. Last known blood pressure (and date measured)
4. Seroconversion date (if known)
5. Date first HIV-positive test
6. Date start cART
7. HIV transmission risk behavior:

• MSM
• Heterosexual
• Injecting drug use
• Contaminated blood products
• Unknown

1. First CD4 count
2. CD4 nadir
3. CD4/CD8 ratio pre-cART
4. HIV stadium at start cART according to CDC criteria
5. Pre-cART HIV RNA zenith
6. Opportunistic conditions before start cART

• None
• MTB
• Fungal infections
• Parasitic infections,
• Bacterial infections

1. Malignancies before start cART

• None
• If yes, specify what kind, when and treatment

1. Other known medical problems before start cART, which require long-term treatment

• None
• If yes, specify what kind, when and treatment

1. Response cART:

• Period between start cART and viral load < 500 (months)
• Viral loads:
• Undetectable after 24 weeks: yes/no
• Number/frequency of residual viremia (positive test with signal <50 copies): last year and last 5 years
• Number of viral blips (50-200): last year and last 5 years
• Number of viremia: 200-1000: last year and last 5 years
• Number of failing cART (>1000): last year and last 5 years
• Resistance associated mutations pre-cART and following failure
• CD4 reconstitution
• CD4/CD8 ratio and normalization >1 yes/no after cART initiation

1. IRIS, yes/no
2. Opportunistic conditions after start cART (if known)

• None
• MTB
• Fungal infections
• Parasitic infections,
• Bacterial infections

1. Malignancies after start cART (if known)

• None
• If yes, specify what kind, when and treatment

1. Vascular diseases after start cART:

• None
• Stroke/ TIA
• Angina pectoris
• Myocardial infarction
• Claudicatio intermittens
• Venous thrombo-embolism
• Other …

1. SOAs

• None
• HAV: yes/no, if yes: treatment and how often
• HBV:
• Infected: active infection (PCR+) or non-active infection
• Vaccination: No/Yes
• Once with anti-HBs> 100
• Two times vaccination leading to
• anti-HBs> 100
• anti-HBs< 100
• No infection
• HCV: yes/no, if yes: treatment and how often
• Lues: yes/ no: if yes: treatment and how often
• Chlamydia/Go: yes/ no: if yes: treatment and how often
• HPV

1. COVID-19 infection before or after start cART:

• Was a COVID-19 test done and if so type of test if known (PCR or antigen test), date of test and result of laboratory test for COVID-19 (positive or negative)
• COVID-19 Serology results
• Register COVID-19-related hospital admissions
• COVID-19 vaccination: type and date(s)
• Side effects of COVID-19 vaccination:
• Mild: overall malaise, fatigue, muscle ache, pain at injection site, shivers, headache.
• Moderate: mild symptoms to the extend (self)medication/pain relief is required.
• Severe: fever >38.5 degrees Celsius and/or one or more days bedridden

1. cART regimes

• Reasons for switching

1. Prescribed co-medication
2. Full current medication list

Physical examination

• Signs of Kaposi sarcoma
• Lymphadenopathy
• For determination of medication levels: when was the last date and time HIV-medication was taken?
• Other abnormalities: …

Cardiovascular risk profile:

• D:A:D (R) and Framingham scores
• ECG

Laboratory results of date closest to inclusion date (standard care at least once a year according to Dutch guidelines):

• Full blood count
• Liver enzymes (ALAT, AF, bilirubin)
• Creatinine
• HIV-RNA
• CD4 and CD8 counts
• Glucose
• Lipid profile
• TPHA/VDRL
• HCV serology (for all at risk, such as MSM and i.v. drug users)
• If positive, HCV PCR
• HPV diagnostics in females (PAP, PCR)
• Urine diagnostics (if available): α1-microglobulin, protein, albumin, phosphate, creatinin

Baseline data collected from electronic patient files or the HIV Monitoring Foundation during 2-year follow-up visit:

Medical history last 2 years:

1. Date of 2 year follow-up visit
2. Weight
3. Last known blood pressure (and date measured)
4. Response cART last 2 years

• Number of residual viremia: last 2 years
• Number of viral blips (20-200): last 2 years
• Number of viremia (200-1000): last 2 years
• Number of failing cART (>1000): last 2 years
• CD4 count
• CD4/CD8 ratio (if known)

1. Opportunistic conditions last 2 years

• None
• MTB
• Fungal infections
• Parasitic infections,
• Bacterial infections

1. Malignancies in the last 2 years

• None
• If yes, specify what kind, when and treatment

1. Signs/symptoms of cardiovascular diseases in the last 2 years:

• None
• Stroke/ TIA
• Angina pectoris
• Myocardial infarction
• Claudicatio intermittens
• Venous thrombo-embolism
• Other …

1. New SOAs in the last 2 years

• HAV: yes/no, if yes: treatment and how often
• HBV:
• Infected: active infection (PCR+), non-active infection,
• Vaccination: No/Yes
• Once with anti-HBs> 100
• Two times vaccination leading to
• anti-HBs> 100
• anti-HBs< 100
• No infection
• HCV: yes/no, if yes: treatment and how often
• Lues: yes/ no: if yes: treatment and how often
• Chlamydia/Go: yes/ no: if yes: treatment and how often

1. COVID-19 infection before or after start cART:

• Was a COVID-19 test done and if so type of test if known (PCR or antigen test), date of test and result of laboratory test for COVID-19 (positive or negative)
• COVID-19 Serology results
• Register COVID-19-related hospital admissions
• COVID-19 vaccination: type and date(s)
• Side effects of COVID-19 vaccination:
• Mild: overall malaise, fatigue, muscle ache, pain at injection site, shivers, headache.
• Moderate: mild symptoms to the extend (self)medication/pain relief is required.
• Severe: fever >38.5 degrees Celsius and/or one or more days bedridden

1. cART regimes in the last 2 years

• Reasons for switching available from 'Stichting HIV Monitoring'

1. Prescribed co-medication
2. Full current medication list

Physical examination

• Signs of Kaposi sarcoma
• Lymphadenopathy
• For determination of medication levels: when was the last date and time HIV-medication was taken?
• Other abnormalities: …

Cardiovascular risk profile:

• D:A:D (R) and Framingham scores
• ECG

Laboratory results of date closest to follow-up visit date:

• Full blood count
• Liver enzymes (ALAT, AF, bilirubin)
• Creatinine
• HIV-RNA
• CD4 and CD8 counts
• Glucose
• Lipid profile
• TPHA/VDRL
• HCV serology
• If positive, HCV PCR
• HPV diagnostics in females (PAP, PCR)
• Urine diagnostics