Research Article

The complex relationship of exposure to new Plasmodium infections and incidence of clinical malaria in Papua New Guinea

Swiss Tropical and Public Health Institute, Switzerland
University of Basel, Switzerland
Walter and Eliza Hall Institute of Medical Research, Australia
Papua New Guinea Institute of Medical Research, Papua New Guinea
University of Melbourne, Australia
Burnet Institute, Australia
ISGlobal, Barcelona Centre for International Health Research, Hospital Clínic-University of Barcelona, Spain
Institut Pasteur, France

Sep 1, 2017

https://doi.org/10.7554/eLife.23708

Open access
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Figures
Tables
Additional files

4 figures, 6 tables and 5 additional files

Figures

Figure 1 with 2 supplements

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*P. falciparum* and *P. vivax* _molFOB (A), prevalence by qPCR (B) and LM (C) by week of follow-up.

Blue lines, *P. falciparum*; red lines, *P. vivax*; solid lines, placebo arm; dashed lines, PQ arm. Open circles in (B) mark enrolment qPCR prevalence for each species.

https://doi.org/10.7554/eLife.23708.005

Figure 1—figure supplement 1

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Definition of new infections for calculating _molFOB.

Definition of *P. falciparum* new infections in two exemplary children is shown. The study design and timelines of follow-up are shown in upper panel: enrolment visit (‘E’), followed by radical treatment (black bar ‘T’) and 235 days of follow-up. The presence of *P. falciparum* clones by sampling visit is visualized below. Columns represent sampling visits, rows represent *P. falciparum msp2* alleles, that is distinct *P. falciparum* clones. Grey solid circles, *P. falciparum* negative sample, grey open circle, missing sample due to missed follow-up visit; red circle: sample positive for respective *Pf-msp2* allele. New infections were defined as a positive sample preceded by two samples negative for this allele (black rectangles), excluding missed samples (see Child 2, allele F, days 120–200). The time point of new infections is marked by arrows for the two children.

https://doi.org/10.7554/eLife.23708.006

Figure 1—figure supplement 2

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*P. ovale* and *P. malariae* prevalence by qPCR during follow-up.

Purple lines, *P. ovale*; green lines, *P. malariae*; solid lines, Placebo arm; dashed lines, PQ arm. Open circles mark enrolment qPCR prevalence for each species.

https://doi.org/10.7554/eLife.23708.007

Figure 2

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Distribution of *P.falciparum* _molFOB (A) and *P. vivax* _molFOB by treatment arm (B).

Relative frequencies among the 466 children are shown.

https://doi.org/10.7554/eLife.23708.009

Figure 3

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Heterogeneity in _molFOB (**A, B**) and clinical episode risk (**C, D**) of *P.falciparum* (**A, C**) and *P. vivax* (**B, D**).

Upper panels show the kriging fit of model predictions of _molFOB and clinical episode risk of children in both treatment arms. Lower panels show the standard error relative to the kriging estimate. Dots represent study participants’ houses and are color-coded according to village. Black lines: vehicle-accessible road; dark grey lines: vehicle-inaccessible road; light grey lines: river; red/white cross: health center or aid post; grey square: school or enrolment location. Maps were prepared using ArcGIS 10.2 (Esri, USA).

https://doi.org/10.7554/eLife.23708.010

Figure 4

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The incidence of *P.falciparum* (A) and *P. vivax* (B) clinical episodes relative to _molFOB.

Mean clinical episode incidence is shown as bars (left axis) and proportion of clinical episode incidence divided by _molFOB as connected dots (right axis). Error bars represent 95% CIs. p-values refer to the differences between groups in the proportion of clinical episodes and new infections, assessed by Chi² or Fisher’s exact test.

https://doi.org/10.7554/eLife.23708.014

Tables

Table 1

Characteristics of study participants by village.

https://doi.org/10.7554/eLife.23708.003

Village	N	% female	Mean age (±SD)	Mean weight (±SD)	% LLIN use at enrolment^*	Mean LLIN use during follow-up^† (%, range)	Mean Hb (±SD)
Amahup	119	53	7.6 (±1.5)	19.8 (±3.3)	99	99 (50–100)	11.1 (±1.0)
Albinama	99	43	7.7 (±1.5)	20.0 (±3.3)	95	97 (78–100)	11.7 (±1.8)
Balanga	54	59	7.8 (±1.6)	19.8 (±4.3)	96	99 (83–100)	11.3 (±1.1)
Balif	93	51	7.8 (±1.5)	20.3 (±3.3)	91	99 (69–100)	11.7 (±1.2)
Bolumita	70	50	7.4 (±1.7)	19.3 (±2.9)	77	92 (56–100)	10.7 (±1.0)
Numangu	31	55	7.4 (±1.6)	19.2 (±4.6)	100	100 (92–100)	12.1 (±1.4)
Total	466	51	7.6 (±1.5)	19.8 (±3.5)	93	100 (50–100)	11.4 (±1.4)

* LLIN use in the night preceding enrolment.

† Information on LLIN use in the previous night was collected at each follow-up visit and averaged across follow-up per participant. Mean LLIN use by village was calculated from the averaged individual LLIN use.
Hb: Haemoglobin.

Table 2

Plasmodium infection status at enrolment by village.

https://doi.org/10.7554/eLife.23708.004

Village	P. falciparum					P. vivax					P. malariae			P. ovale
Village	N pos.	Prevalence by qPCR (CI₉₅)	% mixed^*	Mean^† density (IQR)	Mean MOI^‡ (range)	N pos.	Prevalence by qPCR (CI₉₅)	% mixed^*	Mean^† density (IQR)	Mean MOI^‡ (range)	N pos.	Prevalence by qPCR (CI₉₅)	% mixed^*	N pos.	Prevalence by qPCR (CI₉₅)	% mixed^*
Albinama	18	18 (11–27)	72	131 (38–189)	1.4 (1–4)	54	55 (44–65)	24	3 (1–17)	1.8 (1–7)	9	9 (5–17)	67	5	5 (2–12)	100
Amahup	14	12 (7–19)	57	56 (14–105)	1.6 (1–5)	46	39 (30–48)	24	3 (1–29)	2.2 (1–7)	12	10 (6–17)	83	0	0
Balanga	15	28 (17–42)	67	79 (30–848)	1.7 (1–5)	23	43 (30–57)	43	2 (1–28)	2.0 (1–7)	9	17 (8–30)	56	2	3 (0–14)	50
Balif	8	9 (4–17)	63	64 (10–325)	2.0 (1–4)	35	38 (30–48)	14	2 (1–14)	1.9 (1–6)	7	8 (3–15)	57	0	0
Bolumita	50	71 (59–81)	80	331 (62–1988)	2.2 (1–8)	47	67 (55–78)	81	3 (2–27)	2.9 (1–10)	28	40 (29–52)	89	8	11 (5–22)	100
Numangu	8	26 (13–45)	75	192 (30–848)	1.1 (1–2)	18	58 (39–75)	28	3 (1–25)	1.6 (1–5)	4	13 (4–31)	50	0	0
Overall	113	24 (20–28)	73	163 (20–1103)	1.9 (1–8)	223	48 (43–52)	37	3 (1–23)	2.2 (1–10)	69	15 (12–18)	75	15	3 (2–5)	93
p-value^§		<0.001	0.034	0.086	0.047		<0.001	<0.001	0.947	0.020		<0.001	0.086		<0.001	0.133

* % of infections by qPCR that are mixed-species infections.

† Geometric mean of species-specific 18S rRNA copy numbers per µl blood.
‡ MOI, multiplicity of infection: number of Pf-msp2 and Pv-msp1F3 alleles per infection.

§ Differences between villages were tested for using Chi² and Fisher’s exact test (prevalence, proportion mixed) or Kruskal-Wallis test (MOI, log₁₀-transformed parasite density).

Table 3

Multivariable predictors for time to recurrent blood-stage infection with Plasmodium species by qPCR

https://doi.org/10.7554/eLife.23708.008

Variable	P. vivax			P. falciparum			P. malariae			P. ovale
Variable	AHR^*	CI₉₅	p-value	AHR^*	CI₉₅	p-value	AHR^*	CI₉₅	p-value	AHR^*	CI₉₅	p-value
PQ treatment	0.18	0.13–0.25	<0.001	0.73	0.52–1.02	0.064	0.51	0.22–1.19	0.121	0.31	0.12–0.75	0.010
Age	0.95	0.87–1.04	0.247	1.05	0.94–1.17	0.361	0.98	0.75–1.29	0.905	0.96	0.74–1.26	0.793
LLIN use at enrolment	0.62	0.39–0.98	0.043	0.84	0.49–1.44	0.531	1.33	0.33–6.09	0.715	0.95	0.26–3.43	0.936
Hb at enrolment (g/dl)	0.88	0.80–0.98	0.019	0.90	0.80–1.02	0.099	0.83	0.61–1.12	0.224	0.92	0.66–1.28	0.634
Village
Albinama (ref)	1			1			1			1
Amahup	0.45	0.29–0.71	0.001	0.58	0.31–1.11	0.101	0.34	0.07–1.79	0.205	2.83	0.29–27.48	0.370
Balanga	2.15	1.40–3.31	<0.001	1.81	0.99–3.30	0.054	0.92	0.24–3.60	0.910	7.74	0.85–70.45	0.070
Balif	1.00	0.66–1.54	0.983	0.60	0.30–1.19	0.145	0.24	0.03–2.07	0.193	4.60	0.51–41.41	0.173
Bolumita	3.34	2.09–5.33	<0.001	4.73	2.69–8.30	<0.001	1.21	0.34–4.31	0.770	19.43	2.19–172.37	0.008
Numangu	0.83	0.44–1.59	0.583	2.29	1.17–4.50	0.015	0.82	0.15–4.53	0.823	3.17	0.19–52.41	0.420
Infection status at enrolment (by qPCR)
Uninfected (ref)	1			1			1			1
P. vivax	1.27	0.91–1.78	0.165	1.37	0.86–2.20	0.186	0.92	0.20–4.18	0.913	2.17	0.68–6.97	0.192
P. falciparum	1.36	0.84–2.19	0.205	1.56	0.86–2.82	0.145	3.54	0.85–14.72	0.083	1.25	0.26–5.90	0.779
P. malariae	0.83	0.38–1.85	0.655	0.99	0.38–2.56	0.977	6.35	1.31–30.81	0.022	1.58	0.17–14.30	0.676
Mixed P.f. or P.v.^†	1.74	1.14–2.65	0.010	2.08	1.25–3.48	0.005	3.37	0.88–12.90	0.076	2.03	0.55–7.53	0.287

* AHRs were modeled using Cox proportional hazard regression.

† Mixed infection including P. falciparum or P. vivax infection in conjunction with one or more other Plasmodium spp.
PQ: Primaquine; LLIN: long-lasting insecticide-treated net; Hb: haemoglobin.

Table 4

Multivariable predictors of Pv- and Pf-_molFOB per follow-up interval.

Model predictions from this model were used for mapping _molFOB in Figure 3A.

https://doi.org/10.7554/eLife.23708.011

Variable	P. vivax						P. falciparum
	PQ arm			Placebo arm			Combined arms
	IRR^*	CI₉₅	p-value	IRR^*	CI₉₅	p-value	IRR^*	CI₉₅	p-value
PQ treatment	n.a.^†	n.a.	n.a.	n.a.	n.a.	n.a.	0.89	0.65–1.22	0.474
New P. falc. infections in interval^‡	1.32	0.92–1.89	0.134	1.10	0.85–1.42	0.466	n.a.	n.a.	n.a.
New P. vivax infections in interval^‡	n.a.	n.a.	n.a.	n.a.	n.a.	n.a.	1.15	0.97–1.36	0.100
Age	0.86	0.74–1.01	0.059	0.95	0.87–1.04	0.305	1.03	0.92–1.14	0.640
LLIN use at enrolment	0.96	0.51–1.79	0.897	0.62	0.43–0.91	0.013	1.07	0.7–1.62	0.755
Hb at enrolment (g/dl)	0.85	0.72–1.01	0.063	0.91	0.85–0.99	0.025	0.85	0.75–0.97	0.013
Village
Albinama (ref)	1			1			1
Amahup	0.02	0–0.11	<0.001	0.56	0.34–0.91	0.020	0.52	0.25–1.07	0.074
Balif	0.85	0.4–1.8	0.664	1.74	1.16–2.61	0.007	1.81	0.98–3.35	0.059
Balanga	0.28	0.1–0.82	0.020	1.13	0.73–1.73	0.590	0.75	0.37–1.52	0.423
Bolumita	1.52	0.73–3.17	0.268	2.67	1.83–3.9	<0.001	6.05	3.32–11.05	<0.001
Numangu	0.5	0.15–1.68	0.264	0.76	0.4–1.43	0.394	2.8	1.39–5.64	0.004
Study Day
Day 0–35 (ref)	1			1			1
Day 36–80	1.37	0.54–3.48	0.509	1.99	1.39–2.84	<0.001	2.42	1.44–4.07	0.001
Day 81–175	1.34	0.57–3.12	0.503	0.89	0.61–1.3	0.538	1.13	0.7–1.84	0.616
Day > 175	0.65	0.25–1.69	0.374	0.56	0.38–0.83	0.004	0.87	0.48–1.56	0.643

*IRRs were modeled per sampling interval using negative binomial generalized estimating equations allowing for repeated visits with log-link and an exchangeable correlation structure.

† n.a., not applicable.
‡_molFOB in the follow-up interval (time-varying covariate).

PQ: Primaquine; LLIN: long-lasting insecticide-treated net; Hb: haemoglobin.

Table 5

Multivariable predictors for time to P. vivax and P. falciparum clinical episodes.

Model predictions from this model were used for mapping the relative risk of clinical malaria episodes in Figure 3C and D.

https://doi.org/10.7554/eLife.23708.013

Variable	P. vivax			P. falciparum
Variable	AHR^*	CI₉₅	p-value	AHR^*	CI₉₅	p-value
PQ treatment	0.76	0.34–1.68	0.497	1.79	1.05–3.03	0.031
P. vivax _molFOB^‡	1.07	1.04–1.09	<0.001	n.a.	n.a.	n.a.
P. falciparum _molFOB^‡	n.a.	n.a.	n.a.	1.15	1.11–1.21	<0.001
Age	0.62	0.46–0.84	0.002	0.98	0.85–1.13	0. 799
LLIN use at enrolment	0.84	0.24–2.88	0.778	0.44	0.22–0.87	0.018
Hb at enrolment (g/dl)	0.95	0.74–0.67	0.668	0.85	0.71–1.01	0.070
Village
Albinama (ref)	1			1
Amahup	0.89	0.23–3.46	0.871	0.65	0.20–2.08	0.465
Balif	1.48	0.45–4.86	0.518	1.26	0.50–3.14	0.626
Balanga	0.85	0.21–3.53	0.827	1.39	0.59–3.30	0.455
Bolumita	0.99	0.24–4.03	0.987	1.32	0.58–3.03	0.508
Numangu	1.00	0.23–4.31	0.997	4.29	2.06–8.97	<0.001
Infection status at enrolment (by qPCR)
Uninfected (ref)	1			1
P. vivax	0.77	0.29–2.07	0.608	1.64	0.91–2.95	0.101
P. falciparum	1.74	0.59–5.11	0.316	0.97	0.34–2.77	0.954
Mixed P.f. or P.v.	1.59	0.56–4.50	0.381	1.24	0.57–2.68	0.582

* AHRs were modeled using multiple failure Cox proportional hazard regression.

† n.a., not applicable
‡ Average _molFOB until the time of failure (time-varying covariate).

PQ: Primaquine; LLIN: long-lasting insecticide-treated net; Hb: haemoglobin.

Table 6

Multivariable predictors for odds of P. falciparum clinical episodes

https://doi.org/10.7554/eLife.23708.015

	P. falciparum episode
Variable	OR^*	CI₉₅	p-value
PQ treatment	1.42	0.80–2.52	0.226
P. vivax qPCR positive^†	0.35	0.15–0.78	0.011
P. falciparum _molFOB^‡	1.21	1.10–1.34	<0.001
Age	0.93	0.80–1.09	0.370
LLIN at enrolment	0.37	0.16–0.83	0.016
Hb (g/dl) at enrolment	0.88	0.70–1.11	0.292
Village
Albinama (ref)	1
Amahup	0.41	0.12–1.39	0.154
Balif	0.9	0.26–3.08	0.870
Balanga	1.19	0.42–3.39	0.747
Bolumita	1.48	0.42–5.17	0.540
Numangu	4.17	1.64–10.58	0.003
Study Day
Day 0–80	1
Day 81–175	0.99	0.51–1.91	0.972
Day > 175	0.83	0.39–1.75	0.629

* ORs were modeled using a binomial generalized estimating equation with logit link function using an exchangeable correlation structure.

† Determined as P. vivax positive at the same or previous sampling visit.
‡_molFOB in the follow-up interval (time-varying covariate).

PQ: Primaquine; LLIN: long-lasting insecticide-treated net; Hb: haemoglobin.

Additional files

Supplementary file 1 Univariate factors. Table 1. Univariate/PQ-adjusted predictors for time to recurrent blood-stage infection with Plasmodium species by qPCR. Table 2. Univariate (P. vivax) and univariate/PQ-treatment-adjusted (P. falciparum) predictors for Pv- and Pf-_molFOB by follow-up interval. Table 3. Univariate/PQ-treatment-adjusted predictors for time to P. vivax and P. falciparum episodes. Table 4. Univariate/PQ-treatment-adjusted predictors for odds of P. falciparum clinical episodes.: https://doi.org/10.7554/eLife.23708.012
Download elife-23708-supp1-v2.docx
Supplementary file 2 Multivariable predictors for time to recurrent blood-stage infection with Plasmodium species by LM.: https://doi.org/10.7554/eLife.23708.016
Download elife-23708-supp2-v2.docx
Supplementary file 3 Multivariable predictors for P. falciparum and P. vivax density by qPCR during follow-up.: https://doi.org/10.7554/eLife.23708.017
Download elife-23708-supp3-v2.docx
Supplementary file 4 Multivariable predictors of Pv-_molFOB (combining primaquine and placebo arms) per follow-up interval. This model is similar to that presented in Table 4 in the main text but combines the treatment arms for P. vivax. Model predictions from this model were used for mapping molFOI in Figure 3B.: https://doi.org/10.7554/eLife.23708.018
Download elife-23708-supp4-v2.docx
Transparent reporting form: https://doi.org/10.7554/eLife.23708.019
Download elife-23708-transrepform-v2.docx

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Natalie E Hofmann
Stephan Karl
Rahel Wampfler
Benson Kiniboro
Albina Teliki
Jonah Iga
Andreea Waltmann
Inoni Betuela
Ingrid Felger
Leanne J Robinson
Ivo Mueller

(2017)

The complex relationship of exposure to new Plasmodium infections and incidence of clinical malaria in Papua New Guinea

eLife 6:e23708.

https://doi.org/10.7554/eLife.23708

Figures

P. falciparum and P. vivax _molFOB (A), prevalence by qPCR (B) and LM (C) by week of follow-up.

Definition of new infections for calculating _molFOB.

P. ovale and P. malariae prevalence by qPCR during follow-up.

Distribution of P.falciparum _molFOB (A) and P. vivax _molFOB by treatment arm (B).

Heterogeneity in _molFOB (A, B) and clinical episode risk (C, D) of P.falciparum (A, C) and P. vivax (B, D).

The incidence of P.falciparum (A) and P. vivax (B) clinical episodes relative to _molFOB.

Tables

Characteristics of study participants by village.

Plasmodium infection status at enrolment by village.

Multivariable predictors for time to recurrent blood-stage infection with Plasmodium species by qPCR

Multivariable predictors of Pv- and Pf-_molFOB per follow-up interval.

Multivariable predictors for time to P. vivax and P. falciparum clinical episodes.

Multivariable predictors for odds of P. falciparum clinical episodes

Additional files

Supplementary file 1

Supplementary file 2

Supplementary file 3

Supplementary file 4

Transparent reporting form

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P. falciparum and P. vivax molFOB (A), prevalence by qPCR (B) and LM (C) by week of follow-up.

Definition of new infections for calculating molFOB.

P. ovale and P. malariae prevalence by qPCR during follow-up.

Distribution of P.falciparum molFOB (A) and P. vivax molFOB by treatment arm (B).

Heterogeneity in molFOB (A, B) and clinical episode risk (C, D) of P.falciparum (A, C) and P. vivax (B, D).

The incidence of P.falciparum (A) and P. vivax (B) clinical episodes relative to molFOB.

Characteristics of study participants by village.

Plasmodium infection status at enrolment by village.

Multivariable predictors for time to recurrent blood-stage infection with Plasmodium species by qPCR

Multivariable predictors of Pv- and Pf-molFOB per follow-up interval.

Multivariable predictors for time to P. vivax and P. falciparum clinical episodes.

Multivariable predictors for odds of P. falciparum clinical episodes

Supplementary file 1

Supplementary file 2

Supplementary file 3

Supplementary file 4

Transparent reporting form

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

P. falciparum and P. vivax _molFOB (A), prevalence by qPCR (B) and LM (C) by week of follow-up.

Definition of new infections for calculating _molFOB.

Distribution of P.falciparum _molFOB (A) and P. vivax _molFOB by treatment arm (B).

Heterogeneity in _molFOB (A, B) and clinical episode risk (C, D) of P.falciparum (A, C) and P. vivax (B, D).

The incidence of P.falciparum (A) and P. vivax (B) clinical episodes relative to _molFOB.

Multivariable predictors of Pv- and Pf-_molFOB per follow-up interval.