Assessments | Method/threshold value(s) | Equipment/ kits/comment |
---|---|---|
HIV-1/2 diagnosis: All HIV uninfected women at enrolment and every 6 months. All HIV exposed infants from 18 months of age | Qualitative rapid immunochromatographic, whole blood serial testing algorithm according to the national guidelines for HIV testing and counselling [66]. | - Initial screening; SD Bioline HIV-1/2 3.0 (Standard Diagnostics Inc., Kyonggi-do, South Korea. -Confirmation; Alere Determine™ HIV-1/2 (Abbott -Diagnostics, Lake Bluff, USA). -Tie breaker; Western blotting, The Orasure kit, Epitope Inc., USA |
Early infant HIV diagnosis: All HIV exposed infants from delivery, at every visit until weaning or diagnosis of HIV infection up to 18 months of age [67]. | HIV-1 RNA Qualitative Test on dried blood spots (DBS), according to the manufacturer’s instructions. | COBAS1 AmpliPrep/ COBAS1 TaqMan1 HIV-1 RNA Qualitative Test (Roche Molecular System Inc., Branchburg, NJ, USA. |
HIV-1 RNA quantification: In plasma, of all HIV infected women at enrolment and exit In BM colostrum, and every 6 months All HIV infected infants’ earliest positive DBS sample and at exit | According to the manufacturer’s recommendations. The lower detection limit of the assay; 20 copies/mL | Automated TaqMan Roche Amplicor 1.5 Monitor Test (Cobas AmpliPrep/Cobas TaqMan, Roche Diagnostics, Branchburg NJ, USA. |
Immune status assessment: All HIV infected women at enrolment and exit All HIV infected infants earliest positive whole blood sample and at exit | CD4 + T-lymphocyte (absolute) count EDTA whole blood sample CD4+ T-lymphocyte (percentage). | Partec Cyflow counter (Munster, Germany) be performed within 6 h of whole blood collection. |
Maternal ART levels: Efavirenz, Lamivudine, Tenofovir will be tested enrolment, and every 12 months in all mothers on ART | Different compartments, plasma, breast milk, urine and amniotic-fluid as previously described [68]. | Agilent Technologies, Palo Alto High performance liquid chromatography, Agilent 6430 Triple Quadrupole LC-MS/MS system, CA, USA. |
Syphilis serology: All mothers at enrolment and exit All exposed infants from delivery and at every visit HBV/HCV serology: All mothers at enrolment and exit All exposed infants from delivery and at every visit (DBS) HBV DNA Assays: Host genetics of HBV infection susceptibility CMV serology: All mothers at enrolment and exit CMV DNA test: All mothers at enrolment and exit All exposed infants from delivery and at every visit (DBS) Host genetics of CMV infection/ transmission | Detection of anti-Treponema pallidum antibodies and a non-Treponemal slide agglutination test for the semi-quantitative detection of plasma reagins in serum. HBsAg detection, Profiling; HBsAg, HBsAb, HBeAg, HBeAb & HBcAb Plasma, breast milk HBV DNA for occult hepatitis determination HBV plasma DNA levels HLA, KIR typing (whole blood) CMV IgG, IgM levels, avidity assays. CMV DNA in plasma, amniotic fluid breast milk and urine CMV DNA in DBS HLA, KIR typing | Qualitative rapid immunochromatographic and, SD Bioline 3.0, SD Biosensor, Korea Fortress diagnostics Ltd., UK Screening; SD Bioline, Korea Confirmation; Quality kit, USA Tie breaker: Acon, USA HBV profile All positives: 5 profile (CTK, USA) HBV TaqMan PCR Kit, Norgen Biotek, Norway COBAS1 AmpliPrep/ COBAS1 TaqMan1 HBV DNA Quantitative Test (Roche Molecular System Inc., Branchburg, NJ, USA As previously described (112). PerkinElmer kits, Euroimmun, South Africa RealStar CMV PCR kit v1.0 (Altona Diagnostics, Hamburg, Germany), following isolation of viral DNA using the QIAamp MinElute Virus Spin Kit (Qiagen, Hilden, Germany). As previously described [69]. |
Clinical biochemistry: All mothers serum at enrolment and exit All infants serum within, 6 and 24 months of age | Renal; creatinine, urea, uric acid, Urinary protein and albumin eGFR [70]. Liver enzymes Including AST, GGT, bilirubin, LDH, ALP, Cardiac markers; troponin CKMB. Lipids; total and LDL/HDL cholesterol. Bone profile; calcium, magnesium, phosphate. | Beckman Coulter AU680 chemistry analyser (Krefeld, Germany) Urinalysis strips, Cypress Diagnostics. HELLP syndrome for acute kidney injury assessment LDH: AST ratio > 22 distinguishing TTP from HELLP syndrome. Keiser SD [71]. All results to determine antenatal reference ranges associated favourable outcome of pregnancy |
Full blood counts: All mothers at enrolment and exit All infants within 10 days of life, 6 and 24 months of age | Maternal haemoglobin concentration < 11.0 g/dl and, and varying with age for infants, < 13.5 g/dl) within the first month of life and < 10.7 g/dl from 9 weeks of age [72]. | Mindray© Haematology 3-part differential, 16 parameters BC3600 Analyser (Shenzhen, China) Determine antenatal reference ranges associated favourable outcome of pregnancy |
Maternal blood random glucose and HbA1c: Once in pregnancy at enrolment | Cut off values for random glucose of 110 mg/dl (6.1 mmol/L) within 2 h of a meal or 101 mg/dl (5.6 mmol/L) more than 2 h postprandial. A cut off for HbA1c of 6.5% will identify undiagnosed diabetes mellitus whilst an HbA1c ≥5.6% will mark individuals with an increased risk for future diabetes [73]. | Mindray BS200 Chemistry Analyser (Mindray, Shenzen, China) Determine antenatal reference ranges associated favourable outcome of pregnancy |
Intestinal pathogens: All mothers at enrolment and exit All Infants at exit | Maternal and infant stool samples will be screened for, H pylori, intestinal protozoa and helminths trophozoites and ova. | Direct wet mount microscopy and the formal-ether concentration technique [124]. |
Humoural immune responses to EPI vaccines Infants At 6 weeks, 12 and 24 months of age | The Bio-Plex® 200 system Luminex analyser, Bio-Rad, CA, USA | |
Plasma inflammation and cell stress biomarkers: In a subgroup of MIPs with different durations of exposures to ART from pregnancy to 12 months of age | At least 25 inflammatory chemokines and cytokines measurements. | The Bio-Plex® 200 system Luminex analyser, Bio-Rad, CA USA |
Plasma metabolomics: >  400 analytes, in a subgroup of MIPs with different durations of exposures to ART from pregnancy to 12 months of age to assess altered amino acid and lipid metabolism | 3 platforms -Biogenic amines (Amino acids, catecholamines & polyamines), Signalling lipids (Sphingosine-1-phosphate etc., Positive lipids (Lysophospholipids, phospholipids, cholesterol esters, di/triglycerides & sphingomyelins) as described [76]. | UPLC; TQMS; QToF –HPLC; UHPLC –; MS/MS – Triple Quadrupole Mass Spectrometer. |
Infants atopic sensitization Infant blood samples from 6, 24 and 96 weeks. | Total and specific serum IgE against food/aero-allergens will be assessed in selected Infants with specific serum IgE levels > 0.3 IU/mL will be considered sensitised [77, 78]. | Immuno-assay analyser (Thermofisher, Freiburg, Germany). |
Breast milk characterisation Maternal milk from delivery up to weaning time | Micro−/macronutrients, vitamins, oligosaccharides, immunological biomarkers, essential fatty PUFAs such aslinoleic acid and α-linolenic acid: precursors of LCPUFA e.g. arachidonic acid and docosohexaanoic acid, from birth until weaning [79]. | Gas chromatography, GC-CP3800, Varian, USA) equipped with a flame ionization detector and a fused silica capillary column 50 m × 0.25 mm (I.D. CP-SIL 88 for FAME), Pennsylvania (PA) 16,823–0048, USA). |
Lab based Sub studies assessments | ||
Plasma inflammatory biomarkers In a subgroup of MIPs from pregnancy, breastfeeding period and after weaning | −7 panels, including cytokines, chemokines, vascular injury, angiogenesis, Th17. | V-PLEX Human Biomarker 54-Plex Kit, ECL Technology, Maryland, USA. |
Multiplexed 16S sequencing In a subgroup of MIPs from pregnancy, breastfeeding period and after weaning | 16S rRNA gene segment spanning V5 and V6 regions in stool, plasma and breast milk as previously described [80]. | Illumina MiSeq/Ion Torrent PGM sequencer. |
Breast milk (whole) immune-metabolomics In MIPs with adverse outcomes, from colostrum, and at least 3 later points. | Non-targeted injection analysis. | Mass spectrometry (TQF-MS), Agilent 6550, Agilent Technologies Inc., Santa Clara, CA. |
Maternal mycotoxins biomarkers In pregnancy, at delivery and exit | Level of mycotoxin biomarkers including aflatoxins B1 and 2 in urine, amniotic-fluid and milk | Measurement will be performed on Agilent 6430 Triple Quadrupole LC-MS/MS system, Palo Alto, CA, USA |
Natural killer (NK) cells characterisations In a subgroup of HEU (with short, medium and long term ART exposures) versus HUU infants two time points between 12 and 24 months of age. | NK cells surface marker staining: CD2, CD3, CD16, CD56, CD69, KIR and mitochondria functional assays following PBMC isolation, including nutrient transporter expression on NK cells: Glut1, CD98, and CD71. Mitochondrial function of NK cells will be done using mitotracker green and mitotracker deep red assays. | The BD LSR Fortessa™ cell analyser, multicolour (16 colour) flow cytometer assays, BD Sciences |
Maternal latent TB testing: In a subgroup of HIV infected and uninfected in pregnant women and 6 weeks postpartum | CD4/CD8 IGRAs against TB antigens (ESAT-6/CFP-10/TB-7.7 against TST/ Mantoux at 0.1 ml dose. | Qiagen QuantiFERON gold plus kit, USA, Tuberculin purified protein derivative (PPD) Mantoux, TubersolR, Sanofi Pasteur, in tween solution, 5 US units per test |
Salivary cortisol levels In a subgroup of MIP at 6 months follow up visit | Test done according to the manufacturer’s recommendations. Sensitivity is 0.007μg/dl. | Salimetrics, LLC competitive enzyme immunoassay kit, Carlsbad, CA USA. |
Helicobacter pylori testing In a subgroup of MIP from pregnancy to exit | Quantitative ELISA; H. pylori IgG IgA, IgM antibodies and detection of H pylori antigens from birth to 24 months according to the manufacturer’s recommendations H pylori Ag Sensitivity:0.165 ng/ml. | Sandwich enzyme Immunoassay, Colorimetric test ELISA, Monobind Lake Forest, CA USA. |