HIV infection compounds the lymphopenia associated with cerebral malaria in Malawian children

Figures & data

Figure 1 Median values (plotted on a log scale).

Notes: Platelet counts (A), leukocyte counts (B), percentage (C), and absolute counts (D) of different lymphocyte subsets, percentages of T, NK, and γδ T lymphocytes expressing activation markers CD69 and HLA-DR (E) and memory CD4⁺ and CD8⁺ shown by expression of CD45RO and memory B cells by expression of CD27 (F) in peripheral blood at acute presentation in Malawian children either coinfected with HIV and cerebral malaria (red dots) or with cerebral malaria only (black dots).
Abbreviations: B, B cells; gdT, gamma/delta T cells; M, memory cells; NK, Natural killer cells; T, T cells; WBCs, white blood cells.

Figure 2 Median values (plotted on a log scale).

Notes: Platelet counts (A), leukocyte counts (B), percentage (C), and absolute counts (D) of different lymphocyte subsets, percentages of T, NK, and γδ T lymphocytes expressing activation markers CD69 and HLA-DR (E) and memory CD4⁺ and CD8⁺ shown by expression of CD45RO and memory B cells expressed by CDD27 (F) in peripheral blood at acute presentation in Malawian children either coinfected with HIV and severe malarial anemia (red dots) or with severe malarial malaria only (black dots).
Abbreviations: B, B cells; gdT, gamma/delta T cells; M, memory cells; NK, Natural killer cells; T, T cells; WBCs, white blood cells.

Table 1 Medians (25th and 75th percentiles) of different leukocyte and lymphocyte subsets

	Cell type	HIV-infected, non-malaria, group 1 (n=4)	HIV-uninfected, non-malaria, group 2 (n=42)	HIV-infected CM, group 3 (n=9)	HIV-uninfected CM, group 4 (n=29)	Overall P-value	P-value, 1 vs 2	P-value, 3 vs 4	P-value, 1 vs 3	P-value, 2 vs 4
Absolute count (¥1,000)/μL	Platelets	371.5 (338–475)	364 (218–551)	96.5 (69.75–183.37)	83 (36.5–215.8)	<0.0001	0.9069	0.5301	0.0283	<0.0001
	WBCs	8.7 (5.23–13.38)	8.25 (7.03–10.93)	10.20 (7.85–13.75)	11.20 (7.90–15.30)	0.1245	0.8915	0.7853	0.6042	0.0258
	Neutrophils	2.4 (1.65–3.15)	2.1 (1.4–2.6)	6.30 (5.23–12.35)	7.40 (4.6–12.50)	<0.0001	0.7111	0.8923	0.0162	<0.0001
	Lymphocytes	2.75 (2.63–5.05)	5.25 (3.98–6.33)	1.10 (0.85–1.73)	2.01 (1.50–3.2)	<0.0001	0.0388	0.0638	0.0283	<0.0001
	Monocytes	1.85 (0.90–1.98)	0.9 (0.6–1.1)	0.50 (0.33–1.08)	0.5 (0.24–1.03)	0.0452	0.0967	0.7687	0.0283	0.0491
Percentage of TLC	T cells	69.23 (67.67–71.35)	65.03 (56.59–70.35)	51.63 (41.02–66.12)	66.13 (53.88–69.95)	0.1149	0.1334	0.0925	0.0755	0.9022
	CD4^+ T cells	22.72 (19.36–26.0)	35.45 (31.61–40.08)	15.15 (11.34–30.71)	34.09 (28.17–40.17)	0.0006	0.0037	0.0049	0.7105	0.5785
	CD8^+ T cells	44.43 (37.73–47.80)	24.53 (20.06–28.83)	22.47 (19.60–36.12)	22.18 (17.83–27.18)	0.0062	0.0015	0.3539	0.0196	0.2172
	B cells	17.37 (12.76–25.32)	29.60 (23.24–34.98)	29.42 (26.12–43.02)	29.52 (20.94–42.21)	0.1069	0.0276	0.5360	0.8252	0.7388
	NK cells	7.77 (3.28–12.82)	6.35 (3.91–9.16)	5.12 (3.55–7.16)	5.19 (2.54–9.02)	0.4684	0.7404	0.8368	0.0196	0.2305
	γδ T cells	5.25 (4.21–6.50)	3.68 (2.43–5.05)	4.13 (3.03–6.44)	4.66 (3.01–8.67)	0.2456	0.1144	0.7443	0.4140	0.1027
Absolute count/μL	T cells	2,946 (1,069–5,256)	3,317 (2,600–4,171)	620 (523–993)	1,208 (937–1,964)	<0.0001	0.7404	0.0060	0.0336	<0.0001
	CD4^+ T cells	885 (406–1,495)	1,763 (1,387–2,312)	289 (125–460)	717 (498–1,112)	<0.0001	0.0148	0.0008	0.0336	<0.0001
	CD8^+ T cells	1,923 (611–3,532)	1,304 (838–1,548)	352 (188–549)	442 (310–698)	<0.0001	0.4708	0.2572	0.0503	<0.0001
	B cells	724 (373–967)	2,983 (2,398–3,855)	499 (253–707)	993 (789–1,723)	<0.0001	0.0011	0.0014	0.4140	<0.0001
	NK cells	405 (65–792)	311 (196–550)	56 (38–121)	111 (65–163)	<0.0001	0.800	0.1309	0.1483	<0.0001
	γδ T cells	193 (72–479)	198 (143–258)	58 (33–71)	116 (55–234)	0.0021	0.8915	0.0464	0.0503	0.0229
Activation status (%)	CD69^+ T cells	32.25 (23.18–35.14)	21.08 (16.25–28.04)	36.56 (32.37–48.71)	37.09 (28.29–51.85)	<0.0001	0.0899	0.9726	0.1063	<0.0001
Activation status (%), memory status (%)	CD69^+ γδ T cells	34.24 (27.21–38.67)	23.83 (18.47–32.36)	52.41 (45.34–73.88)	60.13 (50.06–68.35)	<0.0001	0.0828	0.4923	0.0196	<0.0001
	CD69^+ NK cells	26.40 (22.29–38.96)	25.77 (19.67–34.52)	72.65 (50.24–79.44)	73.36 (54.62–81.80)	<0.0001	0.9844	0.7573	0.0028	<0.0001
	HLA-DR^+ NK cells	30.75 (24.93–36.52)	25.67 (15.92–31.49)	32.81 (16.93–39.99)	22.50 (12.90–30.5)	0.2239	0.2666	0.1223	1.000	0.3287
	HLA-DR^+ γδ T cells	32.89 (27.62–35.86)	26.85 (16.38–33.65)	35.29 (26.27–44.16)	16.43 (9.46–21.9)	0.0009	0.2344	0.0020	0.8252	0.0044
Memory status (%)	Memory CD4^+ T cells	31.58 (29.02–36.31)	26.84 (22.63–33.70)	51.38 (40.90–77.73)	45.10 (37.33–53.69)	<0.0001	0.2195	0.1592	0.0503	<0.0001
	Memory CD8^+ T cells	37.90 (34.28–39.05)	16.38 (11.96–22.27)	37.47 (16.78–53.46)	16.95 (12.33–27.24)	0.0038	0.0022	0.0590	0.7105	0.5012
	Memory B cells	13.53 (10.79–15.75)	15.39 (9.46–23.87)	33.64 (18.77–44.82)	21.10 (14.01–24.70)	0.0132	0.5987	0.0688	0.0112	0.1130
Parasites/mL blood, n		N/A	N/A	127,969 (1,986–295,385)	66,286 (1,764–517,050)	N/A	N/A	0.4936	N/A	N/A

Note: All groups presented either as percentages or absolute counts following a global statistical analysis of data from all four groups plus separate group analyses. Abbreviations: CM, cerebral malaria; NA, not applicable; TLC, total leukocytes; WBCs, white blood cells.

Table 2 Medians (25% and 75% percentiles) of different leukocyte and lymphocyte subsets

	Cell type	HIV-infected, non-malaria, group 1 (n=4)	HIV-uninfected, non-malaria, group 2 (n=42)	HIV-infected, SMA, group 3 (n=5)	HIV-uninfected, SMA, group 4 (n=30)	Overall P-value	P-value, 1 vs 2	P-value, 3 vs 4	P-value, 1 vs 3	P-value, 2 vs 4
Absolute count (¥1,000)/μL	Platelets	372 (338–475)	364 (218–551)	112 (70–237)	98 (64.5–176)	<0.0001	0.9069	0.6502	0.0159	<0.0001
	WBCs	8.7 (5.23–13.38)	8.25 (7.03–10.93)	7.7 (5.5–13.9)	13.10 (8.63–19.25)	0.0027	0.8915	0.1033	0.9048	0.0003
	Neutrophils	2.4 (1.65–3.15)	2.1 (1.4–2.6)	2.5 (2.4–3.15)	4.48 (2.48–7.35)	0.0001	0.7111	0.1203	0.7302	<0.0001
	Lymphocytes	2.75 (2.63–5.05)	5.25 (3.98–6.33)	2.5 (2.0–4.75)	5.6 (3.4–11.13)	0.0271	0.0388	0.0488	0.4127	0.3831
	Monocytes	1.85 (0.90–1.98)	0.9 (0.6–1.1)	0.90 (0.70–1.55)	1.65 (1.0–3.33)	0.0001	0.0967	0.0780	0.2663	<0.0001
Percentage of TLC	T cells	69.23 (67.67–71.35)	65.03 (56.59–70.35)	58.29 (51.84–62.04)	62.99 (51.94–66.39)	0.0747	0.1334	0.2203	0.0159	0.3699
	CD4^+ T cells	22.72 (19.36–26.0)	35.45 (31.61–40.08)	14.0 (13.12–21.82)	29.32 (23.26–33.24)	<0.0001	0.0037	0.0011	0.1905	0.0007
	CD8^+ T cells	44.43 (37.73–47.80)	24.53 (20.06–28.83)	39.14 (33.98–42.60)	26.81 (19.21–30.05)	0.0002	0.0015	0.0028	0.2857	0.6684
	B cells	17.37 (12.76–25.32)	29.60 (23.24–34.98)	24.20 (15.57–34.26)	31.05 (27.94–37.34)	0.0602	0.0276	0.3704	0.2857	0.2855
	NK cells	7.77 (3.28–12.82)	6.35 (3.91–9.16)	3.74 (2.63–5.51)	6.67 (3.48–9.72)	0.3350	0.7404	0.1039	0.2857	0.8775
	γδ T cells	5.25 (4.21–6.50)	3.68 (2.43–5.05)	6.60 (4.43–9.56)	5.85 (3.27–11.56)	0.0203	0.1144	0.7954	0.5556	0.0109
Absolute count/μL	T cells	2,946 (1,069–5,256)	3,317 (2,600–4,171)	2,874 (1,287–4,004)	3,446 (2,018–7,466)	0.5823	0.7404	0.2680	0.9048	0.5113
	CD4^+ T cells	885 (406–1,495)	1,763 (1,387–2,312)	740 (495–1,445)	1,651 (954–2,836)	0.0099	0.0148	0.0320	1.000	0.6193
	CD8^+ T cells	1,923 (611–3,532)	1,304 (838–1,548)	1,892 (943–2,264)	1,581 (750–2,659)	0.5938	0.4708	0.9062	0.7302	0.3040
	B cells	193 (72–479)	198 (143–258)	732 (455–2,404)	2,757 (1,653–4,871)	0.0008	0.0011	0.0196	0.5556	0.8505
	NK cells	724 (373–967)	2,983 (2,398–3,855)	139 (102–245)	358 (182–601)	0.1513	0.800	0.0359	1.000	0.5796
	γδ T cells	405 (65–792)	311 (196–550)	210 (157–544)	432 (121–799)	0.1027	0.8915	0.5557	0.5556	0.0195
Activation status (%)	CD69^+ T cells	32.25 (23.18–35.14)	21.08 (16.25–28.04)	54.71 (45.84–68.20)	28.46 (23.04–36.34)	0.0005	0.0899	0.0032	0.0159	0.0092
	CD69^+ γδ T cells	34.24 (27.21–38.67)	23.83 (18.47–32.36)	74.55 (57.8–82.68)	51.94 (37.94–60.60)	<0.0001	0.0828	0.0152	0.0159	<0.0001
	CD69^+ NK cells	26.4 (22.29–38.96)	25.77 (19.67–34.52)	61.98 (56.61–77.40)	49.43 (37.14–57.07)	<0.0001	0.9844	0.0222	0.0159	<0.0001
	HLA-DR^+ NK cells	30.75 (24.93–36.52)	25.67 (15.92–31.49)	28.08 (22.39–42.37)	23.79 (14.4–35.2)	0.4980	0.2666	0.3339	1.000	0.8775
	HLA-DR^+ γδ T cells	32.89 (27.62–35.86)	26.85 (16.38–33.65)	48.01 (37.4–72.01)	36.98 (23.61–56.34)	0.0022	0.2344	0.2481	0.111	0.0032
Memory status (%)	Memory CD4^+ T cells	31.58 (29.02–36.31)	26.84 (22.63–33.70)	40.0 (18.65–58.49)	27.57 (21.09–35.06)	0.5681	0.2195	0.3339	0.7302	0.9227
	Memory CD8^+ T cells	37.90 (34.28–39.05)	16.38 (11.96–22.27)	45.88 (39.38–52.21)	14.42 (9.62–23.16)	0.0038	0.0022	0.0004	0.111	0.3345
	Memory B cells	13.53 (10.79–15.75)	15.39 (9.46–23.87)	22.19 (16.75–43.87)	14.90 (10.12–20.90)	0.0132	0.5987	0.0403	0.0635	0.7190
Parasites/mL blood, n		N/A	N/A	69,406 (7,497–258,632)	13,448 (149–295,598)	N/A	N/A	0.0.0625	N/A	N/A

Note: All groups presented either as percentages or absolute counts following a global statistical analysis of data from all four groups plus separate group analyses.

Abbreviation: SMA, severe malarial anemia; NA, not applicable; TLC, total leukocytes; WBCs, white blood cells.

Table S1 Monoclonal antibodies used and corresponding cell populations

FITC	PE	PerCP	APC	Identity of subsets
moGI	moGI	moGI	moGI	Isotype control
N/A	CD27	N/A	CD19	Memory/naïve B cells
HLA-DR	CD56	CD3	CD69	T cells, Natural killer cells, Natural Killer T cells cell-activation status
TCRγ/δ	HLA-DR	N/A	CD69	γδ^+ T cells, activation status
N/A	CD4	CD3	CD8	CD4^+ and CD8^+ T cells
CD45RA	CD45RO	CD4	CD8	Memory/naïve CD4^+ and CD8^+ T cells
N/A	N/A	N/A	N/A	Negative control

Abbreviations: APC, allophycocyanin; FITC, fluorescein isothiocyanate; NA, not applicable; PE, phycoerythrin; PerCP, peridinin chlorophyll protein; T, T cells; NK = Natural Killer Cells; NKT = Natural Killer T cells.

Table S2 Monoclonal antibodies and names of suppliers

Monoclonal antibody	Product number	Supplier
Anti-CD3-PerCP	560835	BD
Anti-CD4-FITC	555346	BD
Anti-CD8-APC	561952	BD
Anti-CD19-APC	641395	BD
Anti-CD25-APC	560987	BD
Anti-CD27-PE	340425	BD
Anti-CD28-FITC	555728	BD
Anti-CD38-APC	560239	BD
Anti-CD45-PerCP	555483	BD
Anti-CD45RA-FITC	555488	BD
Anti-CD45RO-PE	555493	BD
Anti-CD56-PE	555516	BD
Anti-CD57-FITC	555619	BD
Anti-CD69-APC	555533	BD
Anti-HLA-DR-FITC	555560	BD
Anti-HLA-DR-PE	555812	BD
Anti-TCR-γ/δ-FITC	347903	BD
Anti-TCR-Vδ2-PE	555739	BD
Anti-moGI-FITC	555909	BD
Anti-moGI-PE	555749	BD
Anti-moGI-PerCP	555751	BD
Anti-moGI-APC	550795	BD

Abbreviations: APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PerCP, peridinin chlorophyll protein.