Skip to main content

Table 3 Summary of the tests done in mothers and infants at different time points

From: The University of Zimbabwe College of Health Sciences (UZ-CHS) BIRTH COHORT study: rationale, design and methods

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 Determineâ„¢ 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

Antibody titres EPI will be measured [74, 75].

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 Fortessaâ„¢ 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.

  1. ALP alkaline phosphatase, AST aspartate aminotransferase, BM breast milk, CD cluster of differentiation, CK-MB creatine kinase-myocardial band, CMV cytomegalovirus, EPI expanded program for immunization, GC gas chromatography, GGT gamma-glutamyl transferase; Glut1 glucose transporter-1, HbA1c glycosylated haemoglobulin, HBcAb hepatitis B core antibody, HBeAb hepatitis B e-antibody, HBeAg hepatitis B e-antigen, HBsAb hepatitis B surface antibody, HBsAg hepatitis B surface antigen, HBV hepatitis B virus, HCV hepatitis C virus, HDL high density lipoprotein, HEI HIV exposed infected, HELLP haemolysis, elevated liver enzymes and low platelet count, HEU HIV exposed but uninfected, HIB Haemophilus influenza type b, HIV human immunodeficiency virus, HLA human leukocyte antigen, HPLC high-pressure liquid-chromatography, HUU HIV unexposed and uninfected, IGRAs interferon gamma release assays, KIR killer cell immunoglobulin-like receptor, LCPUFA long chain poly unsaturated fatty acids, LDH lactate dehydrogenase, LDL low density lipoprotein, MIPs mother-infant pairs, MS mass spectrometer, PUFA poly unsaturated fatty acids, QToF quad time of flight, TB tuberculosis, TQMS triple quadrupole mass spectrometer, TST tuberculin skin test, TTP thrombotic thrombocytopenic purpura, UHPLC ultra-high-pressure liquid-chromatography, UPLC ultra-pressure liquid-chromatography