Additional file 1: Supplementary Figure S1. Sequential gating
approach for the measurement of CCR2 expression by CD14+
monocytes. The sequential gating approached used was as follows: First,
the viable (7-AAD negative; region ‘Viable”) cells were identified using a
7-AAD vs SS Log density plot. A “Viable” region was created around the
7-AAD negative cells. Gated on the “Viable”cells, a SSLog vs FS plot was
used to capture intact cells in the “E” region. CD14+ monocytes were
identified (“CD14+” region) using a CD14 vs SS Log density plot that were
gated on viable, intact cells (“E” region). CD14+ monocytes that express
CCR2 were quantified using a CD192 (CCR2) vs SS Log plot. The proportion
of CD14+/CCR2+ cells were captured in the “CD14+ CCR2+” region.
The gating strategy followed to quantify CCR2 expression by CD16+ neutrophils
was similar to what was described for CD14+ monocytes, but instead
of identifying CD14+ monocytes, CD16+ neutrophils were
identified (“CD16+” region) using a CD16 vs SS Log density plot that were
gated on viable, intact cells (“E” region). CD16+ neutrophils that express
CCR2 were quantified using a CD192 (CCR2) vs SS Log plot. The proportion
of CD14+/CCR2+ cells was captured in the “CD16+ CCR2+” region.
Additional file 2: Supplementary Figure S2. Sequential gating
approach for the measurement of CCR2 expression by monocyte
subpopulations. Doublets and debris were removed (Region ‘K’) using a
FS Area vs FS Height density plot. A 7-AAD vs SS Log density plot, gated
on ‘K’ was used to exclude all non-viable cells. Viable cells were captured
in region ‘Viable’. Viable CD14+ monocytes were identified (Region
‘CD14+ Monocytes’) using a CD14 APC vs SS Log density plot. Monocyte
sub-populations were identified using a CD16 FITC vs CD14 PE density
plot gated on viable, CD14+ monocytes. Four monocyte sub-populations
were identified: CD14+/CD16-; CD14++/CD16-; CD14+/CD16+; and
CD14++/CD16+. The percentage CCR2+ monocytes present in each of
the respective monocyte sub-populations were identified using CD195
(CCR2) PE vs SS Log two-parameter plots gated on the respective subpopulations.
The overlay plots within the black bordered square indicates
the strategy used to determine CCR2 expression of the different monocyte
subsets. The negative/positive staining boundaries were determined
based on the negative expression of CCR2 by CD16++/CD14- neutrophils
(indicated in red in the overlay plots). The CCR2+ populations are indicated
in blue.
Additional file 3: Supplementary Figure S3. Maternal intake of
estimated average requirements for macronutrients, vitamins and
minerals for mothers who report on household food security
circumstances. Maternal reports of food insecurity did not associate with
intake levels of macronutrients or minerals. Maternal reports of
experiencing food runout or inability to afford balanced meals associated
with lower intake of vitamin B12 (p=0.01; p=0.04). Many women,
irrespective of food security reports, are at risk of inadequate
macronutrient, vitamin and mineral intakes. Percent intake of EARs for 36
nutrients were calculated for lactating women 14-18, 19-30 or 31-50 years
of age [37]. Calculations for EAR for total protein considered maternal
weight at time of dietary recall. Data are % intake of EAR reported in maternal
dietary recall for macronutrients, *p<0.05 [ANOVA for normal distribution/
equal variance; Kruskal-Wallis/Wilcoxon test for nonparametric
data; or Welch’s test for normal data/unequal variance]). CHO =
carbohydrates.
Additional file 4: Supplementary Figure S4. Cooccurrence of maternal
HIV and food insecurity may increase risk of stunting at birth. Amongst
infants whose mothers report worrying about food runout, risk of
stunting at birth is greater for HEU compared to HUU infants (e; RR=4.90
[0.76, 31.5], ARD=0.56 [0.17, 0.94], p=0.0498). The red line represents the
proportion of infants who had stunting at birth or 12 weeks PP. Mosaic
plots are proportion (%) of HUU or HEU infants who have stunting (<-2
SD length-for-age standardised according to WHO child growth standards
[28]) at birth and 12 weeks old. HUU = HIV-unexposed, uninfected
infant; HEU = HIV-exposed, uninfected infant. RR = Relative risk. ARD =
Absolute risk difference.
Additional file 5: Supplementary Figure S5. Food insecurity may
associate with low attainment of GMCD milestones for HUU and HEU
infants. Infants whose mothers reported household food insecurity did
not attain 1-3 month GMCD milestones (A, C, E) for receptive language,
large movement, relating and response behaviour or play activities, or 3-5
month GMCD milestones (B, D, F) for fine movement or relating and response
behaviour in the same proportion as the international
standardization sample. Maternal reports of food insecurity did not associate
with risk of not attaining all 1-3 month or 3-5 month GMCD milestones
(A-F, [p>0.05], Fisher’s exact 2-Tail). Data are proportion (%) of
infants who attained all age-appropriate GMCD milestones. The horizontal
dotted line represents the GMCD standardised international sample proportion
(85%) of infants who attained all milestones in that age category,
when they were in that age range. The numbers underneath the bars
represent the number of infants attaining all milestones for each milestone.
GMCD = Guide for monitoring child development; HUU = HIVunexposed,
uninfected infant; HEU = HIVexposed, uninfected infant.
Additional file 6: Supplementary Table S1. Flow cytometry reagent
list (including lasers and detectors used). Supplementary Table S2.
Flow cytometry compensation matrix. Supplementary Table S3.
Maternal nutrient intakes from one 24-hour dietary recall for mothers
with and without HIV who attended follow up. Supplementary Table
S4. Maternal nutrient intake from one 24-hour dietary recall for mothers
who report experiencing food insecurity compared to those who do not
experience food insecurity.
