The risk of Plasmodium vivax parasitaemia after P. falciparum malaria: An individual patient data meta-analysis from the WorldWide Antimalarial Resistance Network.

BACKGROUND: There is a high risk of Plasmodium vivax parasitaemia following treatment of falciparum malaria. Our study aimed to quantify this risk and the associated determinants using an individual patient data meta-analysis in order to identify populations in which a policy of universal radical cure, combining artemisinin-based combination therapy (ACT) with a hypnozoitocidal antimalarial drug, would be beneficial. METHODS AND
FINDINGS: A systematic review of Medline, Embase, Web of Science, and the Cochrane Database of Systematic Reviews identified efficacy studies of uncomplicated falciparum malaria treated with ACT that were undertaken in regions coendemic for P. vivax between 1 January 1960 and 5 January 2018. Data from eligible studies were pooled using standardised methodology. The risk of P. vivax parasitaemia at days 42 and 63 and associated risk factors were investigated by multivariable Cox regression analyses. Study quality was assessed using a tool developed by the Joanna Briggs Institute. The study was registered in the International Prospective Register of Systematic Reviews (PROSPERO: CRD42018097400). In total, 42 studies enrolling 15,341 patients were included in the analysis, including 30 randomised controlled trials and 12 cohort studies. Overall, 14,146 (92.2%) patients had P. falciparum monoinfection and 1,195 (7.8%) mixed infection with P. falciparum and P. vivax. The median age was 17.0 years (interquartile range [IQR] = 9.0-29.0 years; range = 0-80 years), with 1,584 (10.3%) patients younger than 5 years. 2,711 (17.7%) patients were treated with artemether-lumefantrine (AL, 13 studies), 651 (4.2%) with artesunate-amodiaquine (AA, 6 studies), 7,340 (47.8%) with artesunate-mefloquine (AM, 25 studies), and 4,639 (30.2%) with dihydroartemisinin-piperaquine (DP, 16 studies). 14,537 patients (94.8%) were enrolled from the Asia-Pacific region, 684 (4.5%) from the Americas, and 120 (0.8%) from Africa. At day 42, the cumulative risk of vivax parasitaemia following treatment of P. falciparum was 31.1% (95% CI 28.9-33.4) after AL, 14.1% (95% CI 10.8-18.3) after AA, 7.4% (95% CI 6.7-8.1) after AM, and 4.5% (95% CI 3.9-5.3) after DP. By day 63, the risks had risen to 39.9% (95% CI 36.6-43.3), 42.4% (95% CI 34.7-51.2), 22.8% (95% CI 21.2-24.4), and 12.8% (95% CI 11.4-14.5), respectively. In multivariable analyses, the highest rate of P. vivax parasitaemia over 42 days of follow-up was in patients residing in areas of short relapse periodicity (adjusted hazard ratio [AHR] = 6.2, 95% CI 2.0-19.5; p = 0.002); patients treated with AL (AHR = 6.2, 95% CI 4.6-8.5; p < 0.001), AA (AHR = 2.3, 95% CI 1.4-3.7; p = 0.001), or AM (AHR = 1.4, 95% CI 1.0-1.9; p = 0.028) compared with DP; and patients who did not clear their initial parasitaemia within 2 days (AHR = 1.8, 95% CI 1.4-2.3; p < 0.001). The analysis was limited by heterogeneity between study populations and lack of data from very low transmission settings. Study quality was high.
CONCLUSIONS: In this meta-analysis, we found a high risk of P. vivax parasitaemia after treatment of P. falciparum malaria that varied significantly between studies. These P. vivax infections are likely attributable to relapses that could be prevented with radical cure including a hypnozoitocidal agent; however, the benefits of such a novel strategy will vary considerably between geographical areas.

Introduction

Malaria continues to exert a huge global health burden, with the latest estimates suggesting there are more than 228 million cases per year, associated with 405,000 deaths [1]. In 2015, the World Health Assembly set a target to reduce malaria prevalence by 90% by 2030 [2]. Although the burden of malaria has fallen in many regions, global estimates of malaria cases have plateaued and even risen in some areas over the past 5 years [1]. There has also been a relative rise in the proportion of malaria due to Plasmodium vivax outside of sub-Saharan Africa [3]. P. vivax is more difficult to eliminate than P. falciparum because it can form dormant liver stages (hypnozoites) that can reactivate weeks to months after the initial infection. Treatment of both the blood and liver stages is referred to as radical cure. If the proposed ambitious malaria elimination targets are to be achieved, innovative strategies are needed to provide safe and effective radical cure to the high proportion of individuals harbouring occult as well as circulating P. vivax parasites [4].

Where P. vivax and P. falciparum are coendemic (Asia, the Horn of Africa, and the Americas), reports have documented a high risk of P. vivax parasitaemia following treatment of P. falciparum infection [5, 6]. A recent meta-analysis of P. falciparum clinical trials revealed that within 63 days, 24% of patients presenting with P. falciparum had a recurrence with P. vivax, and almost 70% of all parasitological recurrences were due to P. vivax [7]. The high risk of P. vivax has been hypothesised to be due to reactivation of the dormant liver stages of parasites, although the mechanisms underlying this are not well understood. In areas where the risk of P. vivax parasitaemia is high, broadening the indication for hypnozoitocidal treatment to include patients presenting with uncomplicated malaria due to either P. vivax or P. falciparum (universal radical cure) has potential to reduce the subsequent risk of vivax parasitaemia and its ongoing transmission [8].

Radical cure with 8-aminoquinoline compounds can reduce the risk of P. vivax relapse significantly; however, both primaquine and tafenoquine can cause haemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency [9]. Thus, the risk versus benefit of a policy of universal radical cure will depend upon quantifying the inherent risk of P. vivax recurrence and associated cofactors in different endemic settings, an approach that is more amenable to an individual patient data meta-analysis than our previous study-level systematic review [7].

The aim of this individual patient data meta-analysis was to define the risk of vivax parasitaemia after falciparum infection in different coendemic environments and to explore the factors underlying these risks in order to identify populations in which a policy of universal radical cure, with blood schizontocidal treatment plus either primaquine or tafenoquine, would be most beneficial.

Methods

Search strategy and selection criteria

A previous systematic review was used to identify suitable studies [7]. In brief, Medline, Embase, Web of Science, and the Cochrane Database of Systematic Reviews were searched for prospective studies published between 1 January 1960 and 5 January 2018 in any language that included supervised treatment of patients with uncomplicated P. falciparum infection (including monoinfection and mixed P. falciparum and P. vivax infection) located in areas coendemic for P. falciparum and P. vivax (S1 Box). Countries were considered coendemic if indigenous P. falciparum and P. vivax cases were reported or suspected in 2016 [10].

In the original systematic review, studies were included if they explicitly reported the presence or absence of recurrent parasitaemia. Studies were excluded if patients were followed less than 28 days, if patients were only followed passively, or if the full text manuscript was unavailable. Because primaquine and tafenoquine are contraindicated in pregnant women and not a clinical priority in patients presenting with severe malaria, studies only enrolling these patients were also excluded from the analysis. Identification of studies and extraction of data were undertaken by 2 independent authors, with discrepancies resolved by discussion with a third author.

To improve the generalisability of the results, the inclusion criteria for this individual patient data meta-analysis were restricted further to include only studies in which patients were treated with 1 of 4 widely used ACTs: artemether-lumefantrine (AL), artesunate-amodiaquine (AA), artesunate-mefloquine (AM), or dihydroartemisinin-piperaquine (DP). Furthermore, because it was not always apparent whether recurrent P. vivax was systematically documented during follow-up, studies were only included if at least 1 episode of P. vivax parasitaemia during follow-up was reported in the manuscript. Additional study-level exclusions were if studies only included travellers or soldiers or only patients with hyperparasitaemic infection, because these either reflected malaria acquired from diverse locations or a biased population of patients attending a clinic in a malaria-endemic setting. Investigators of eligible studies were invited to participate in this analysis and share their study’s individual patient data with the WorldWide Antimalarial Resistance Network (WWARN) repository. Shared data were anonymised and standardised using the WWARN Data Management and Statistical Analysis Plans [11].

In addition to the systematic review, the pre-existing WWARN repository was searched for studies of uncomplicated P. falciparum monoinfection or mixed infection in which at least 1 patient was recorded as having P. vivax parasitaemia during study follow-up. This was undertaken to identify unpublished studies and published studies that did not report P. vivax parasitaemia during follow-up in the manuscript. Patient data from eligible studies were collated together with those from the systematic review for analysis.

Data collection and definitions

Demographic and clinical data including age, sex, weight, baseline species and parasite density, presence and species of parasites at follow-up, baseline gametocytaemia, baseline body temperature, and haemoglobin (Hb) or haematocrit at baseline and follow-up were included in the data set. Patient-level data were excluded if baseline parasitaemia was not available or did not include P. falciparum species, patients were treated with adjunctive antimalarials, or there was an enrolment violation according to supplied study data.

Haematocrit was converted to Hb using the formula Hb = (Haematocrit − 5.62)/2.6 [12]. The maximum value was used when multiple Hb measurements were recorded on a single day. The nadir Hb following acute P. falciparum malaria occurs between day 3 and 7, with most studies measuring the first follow-up Hb 7 days after starting antimalarial treatment [13]. The early decline and recovery of Hb was therefore assessed from the Hb measured between days 6 and 8 inclusive. Anaemia was defined as a Hb concentration less than 10 g/dL. A high parasite count was defined as a parasite count greater than 100,000 parasites per μL. Based on geographical location, studies were categorised into long or short P. vivax relapse periodicity using data from the Malaria Atlas Project (MAP) [14]. A median time to relapse of ≤47 days was categorised as short relapse periodicity. Subnational malaria parasite incidence was estimated from MAP models [15].

Data regarding the supervision of drug administration were extracted from publications or study protocols. Drug administration was categorised as ‘supervised’ if the entire treatment regimen was supervised, ‘partially supervised’ if only some treatment doses were supervised, and ‘not specified if data were not available. The year of enrolment was defined in the first instance from individual patient data or, if unavailable, from the median year during which patients were enrolled into the study or as 2 years prior to the year of publication. The study was registered in the International Prospective Register of Systematic Reviews (PROSPERO: CRD42018097400), and the protocol is available at www.wwarn.org/working-together/study-groups/vivax-after-falciparum-study-group (S1 PRISMA Checklist).

Outcomes

The primary outcome was the risk of P. vivax parasitaemia (P. vivax monoinfection or mixed infection) between days 7 and 42. Secondary outcomes were the risk of P. vivax parasitaemia between days 7 and 28 and days 7 and 63.

Statistical analysis

Statistical analyses were undertaken using Stata software, version 15.1 (Stata Corporation, College Station, TX, USA) and R version 3.5.1 (R Foundation for Statistical Computing, Vienna, Austria), according to an a priori statistical analysis plan [16]. The cumulative risk of peripheral parasitaemia was calculated using Kaplan–Meier survival analyses. Patients were right-censored at their first recurrence, day of last follow-up, the day prior to a blood smear result gap >18 days, or the day of the planned outcome assessment [11]. Following peer review, prediction intervals were calculated for the cumulative risk of P. vivax parasitaemia [17].

To estimate the association between ACTs with the rate of P. vivax parasitaemia, Cox’s proportional hazards regression was used with shared frailty for study sites and adjustment for age, sex, baseline parasitaemia, regional relapse periodicity, P. falciparum gametocytes, mixed infection at baseline, and Hb. The proportional hazards assumption was assessed visually using plots of observed versus expected values. Body weight and geographical location were not included because of collinearity with age and relapse periodicity, respectively. To investigate the association between the change in Hb and the rate of P. vivax parasitaemia, the absolute fall in Hb between day 0 and day 7 was included in the multivariable Cox regression model.

The association between day of first parasite clearance (defined as parasite count below the microscopic limit of detection) and risk of P. vivax parasitaemia between days 7 and 63 was assessed by Cox’s proportional hazards regression with shared frailty for study sites and adjustment for age, sex, baseline parasitaemia, regional relapse periodicity, P. falciparum gametocytes, mixed infection at baseline, Hb at baseline, and treatment. Patients treated with single low-dose primaquine were excluded. A subgroup analysis restricted data to patients treated with AL with a minimum of 42 days of follow-up to reduce the impact of slowly eliminated partner drugs with prolonged post-treatment prophylactic effect on outcome assessment. This subgroup was used to explore the association between the background incidence of P. falciparum and P. vivax and the risk of P. vivax parasitaemia. Following peer review, additional subgroup analyses were included for the other ACTs.

Bias related to individual studies was assessed using a tool developed by the Joanna Briggs Institute (S1 Text) [18] and in a sensitivity analysis, with removal of 1 study at a time and calculation of the coefficient of variation. Included and targeted studies that were not included were compared using baseline characteristics.

Ethics

All data included in this analysis were obtained in accordance with ethical approvals from the country of origin. The data are fully anonymised and cannot be traced back to identifiable individuals. This systematic review did not require separate ethical approval according to the guidelines of the Oxford Central University Research Ethics Committee.

Results

There were 153 clinical trials enrolling P. falciparum patients published between 1 January 1960 and 5 January 2018 and identified as part of the previous systematic review [7]. Of these, 51 (33.3%) studies enrolling 15,903 patients between 1991 and 2016 included treatment with the 4 major ACTs and were eligible for inclusion in this analysis. Individual patient data were available for 9,410 (59.2%) patients from 26 studies [19-44] (S1 Table and S2 Table). An additional 32 published and 2 unpublished studies with individual patient data in the WWARN repository were screened for inclusion, of which 16 studies were identified as also being eligible for the analysis [45-58]. In total, 42 studies enrolling 15,341 eligible patients were included in the analysis (Fig 1, S2 Table and S3 Table).

Fig 1

Study flowchart.

AA, artesunate-amodiaquine; ACT, artemisinin-based combination therapy; AL, artemether-lumefantrine; AM, artesunate-mefloquine; DP, dihydroartemisinin-piperaquine; WWARN, WorldWide Antimalarial Resistance Network.

Of the studies included, 2,711 (17.7%) patients were treated with AL (13 studies), 651 (4.2%) with AA (6 studies), 7,340 (47.8%) with AM (25 studies), and 4,639 (30.2%) with DP (16 studies) (Fig 1 and S2 Table). In 2 studies, 448 patients were coadministered a single dose of primaquine. Patients were followed for 28 days in 3 studies (n = 670 patients), 42 days in 25 studies (n = 6,510 patients), 56 days in 1 study (n = 2,072 patients), 63 days in 11 studies (n = 5,653 patients), and a mixture of 28 and 63 days in 2 studies (436 patients).

The patients’ baseline characteristics are presented in Table 1. Overall, 14,146 (92.2%) patients had P. falciparum monoinfection and 1,195 (7.8%) mixed infection with P. falciparum and P. vivax confirmed by blood film microscopy. The median age was 17.0 years (interquartile range [IQR] = 9.0–29.0 years; range = 0–80 years), with 1,584 (10.3%) patients younger than 5 years. Most patients (14,258; 92.9%) were recruited in areas with short relapse periodicity, and 82.5% (12,652) were enrolled in the Greater Mekong Subregion (S1 Fig). All 684 (4.5%) patients enrolled in the Americas were treated with AM or DP, whereas in Africa, all patients (120; 0.8%) were treated with AL. The 14,537 (94.8%) patients enrolled in Asia were treated with 1 of the 4 main ACTs (Table 1). The median parasite density at baseline was 7,544/μL (IQR = 2,000–28,021; range = 7–479,280). Baseline Hb data were available for 12,184 (79.4%) patients, of whom 27.6% (3,358) were anaemic (Hb <10 g/dL). No issues impacting data integrity were identified during collation of individual patient data. Studies that were targeted but for which data were not available were more likely to have been conducted in Africa; these studies had a similar median age and sex distribution to those included (S4 Table and S5 Table). Study quality was generally high (S1 Text).

Table 1

Demographics and baseline characteristics.

	AL, n (%) (n = 2,711)	AA, n (%) (n = 651)	AM, n (%) (n = 7,340)	DP, n (%) (n = 4,639)	Overall, n (%) (n = 15,341)
Sex*
Female	958 (35.4%)	252 (38.8%)	3,015 (41.1%)	1,391 (30.2%)	5,616 (36.7%)
Male	1,752 (64.6%)	398 (61.2%)	4,323 (58.9%)	3,214 (69.8%)	9,687 (63.3%)
Age, years†
Median (IQR)	18.0 (9.0–28.0)	16.0 (8.0–28.0)	14.0 (8.0–25.0)	22.0 (12.0–32.0)	17.0 (9.0–29.0)
≥15	1,596 (58.9%)	352 (54.2%)	3,535 (48.2%)	3,275 (70.8%)	8,758 (57.1%)
5 to <15	753 (27.8%)	252 (38.8%)	2,994 (40.8%)	984 (21.3%)	4,983 (32.5%)
<5	361 (13.3%)	45 (6.9%)	810 (11.0%)	368 (8.0%)	1,584 (10.3%)
Weight, kg‡
Median (IQR)	44.0 (21.0–52.0)	38.0 (20.5–49.4)	35.9 (18.3–49.0)	42.5 (19.0–53.0)	39.0 (19.0–50.0)
5 to <15	350 (13.3%)	67 (10.3%)	1,008 (14.6%)	443 (16.6%)	1,868 (14.5%)
15 to <25	418 (15.8%)	134 (20.7%)	1,575 (22.8%)	444 (16.7%)	2,571 (20.0%)
25 to <35	237 (9.0%)	80 (12.3%)	783 (11.3%)	210 (7.9%)	1,310 (10.2%)
35 to <45	361 (13.7%)	126 (19.4%)	1,004 (14.5%)	309 (11.6%)	1,800 (14.0%)
45 to <55	833 (31.6%)	155 (23.9%)	1,693 (24.5%)	677 (25.4%)	3,358 (26.1%)
55 to <80	438 (16.6%)	86 (13.3%)	837 (12.1%)	569 (21.4%)	1,930 (15.0%)
≥80	2 (0.1%)	0 (0.0%)	9 (0.1%)	12 (0.5%)	23 (0.2%)
Relapse periodicity
Long	120 (4.4%)	202 (31.0%)	499 (6.8%)	262 (5.6%)	1,083 (7.1%)
Short	2,591 (95.6%)	449 (69.0%)	6,841 (93.2%)	4,377 (94.4%)	14,258 (92.9%)
Geographical region
Asia-Pacific	2,591 (95.6%)	651 (100.0%)	6,918 (94.3%)	4,377 (94.4%)	14,537 (94.8%)
The Americas	0 (0.0%)	0 (0.0%)	422 (5.7%)	262 (5.6%)	684 (4.5%)
Africa	120 (4.4%)	0 (0.0%)	0 (0.0%)	0 (0.0%)	120 (0.8%)
Treatment supervision
Not specified	67 (2.5%)	202 (31.0%)	397 (5.4%)	0 (0.0%)	666 (4.3%)
Supervised	1,843 (68.0%)	294 (45.2%)	5,987 (81.6%)	2,182 (47.0%)	10,306 (67.2%)
Partial supervised	801 (29.5%)	155 (23.8%)	956 (13.0%)	2,457 (53.0%)	4,369 (28.5%)
P. falciparum gametocytes present§	238/1,959 (12.1%)	93/428 (21.7%)	697/5,765 (12.1%)	495/3,568 (13.9%)	1,523/11,720 (13.0%)
Mixed infection	183/2,711 (6.8%)	42/651 (6.5%)	712/7,340 (9.7%)	258/4,639 (5.6%)	1,195/15,341 (7.8%)
Baseline parasitaemia¶
Median (IQR)	9,120 (1,900–39,600)	8,680 (2,364–24,507)	7,213 (1,976–25,622)	7,574 (2,048–25,120)	7,544 (2,000–28,022)
≤100,000 parasites per μL	2,507 (92.5%)	638 (98.0%)	6,610 (94.2%)	4,290 (94.5%)	14,045 (94.2%)
>100,000 parasites per μL	204 (7.5%)	13 (2.0%)	404 (5.8%)	250 (5.5%)	871 (5.8%)
Hb, g/dL; mean (SD)||	10.4 (2.7)	10.7 (2.4)	10.8 (2.5)	10.9 (2.7)	10.8 (2.6)
Anaemic, Hb < 10 g/dL	721/2,586 (27.9%)	212/530 (40.0%)	1,655/6,481 (25.5%)	770/2,587 (29.8%)	3,358/12,184 (27.6%)
Fever, temperature >37.5°C**	1,331/2,547 (52.3%)	332/440 (75.5%)	3,713/6,565 (56.6%)	1,099/2,221 (49.5%)	6,475/11,773 (55.0%)

*Data not available for 38 patients.

†Data not available for 16 patients.

‡Data not available for 2,481 patients.

§Data not available for 3,623 patients.

¶Parasite count data not available for 427 patients.

||Data not available for 3,159 patients.

**Data not available for 3,570 patients.

Abbreviations: AA, artesunate-amodiaquine; AL, artemether-lumefantrine; AM, artesunate-mefloquine; DP, dihydroartemisinin-piperaquine; Hb, haemoglobin; IQR, interquartile range; n, number; SD, standard deviation.

The overall risk of P. vivax parasitaemia

Recurrent parasitaemia between day 7 and 42 was recorded in 2,020 (13.2%) patients, of whom 1,047 (51.8%) had P. vivax monoinfection, 99 (4.9%) mixed P. vivax and P. falciparum infection, and 874 (43.3%) P. falciparum monoinfection. The overall cumulative risks of recurrent parasitaemia of any species were 8.1% (95% CI 7.7–8.6) by day 28, 16.8% (16.2–17.5) by day 42, and 30.5% (29.4–31.6) by day 63. The corresponding cumulative risks of P. vivax parasitaemia (monoinfection or mixed infection) were 3.7% (3.4–4.1), 10.2% (9.6–10.7), and 22.2% (21.1–23.3), respectively, and for P. falciparum parasitaemia (monoinfection) were 4.5% (4.2–4.9), 7.3% (6.9–7.8), and 10.4% (9.8–11.2), respectively.

At day 42, the cumulative risk of P. vivax parasitaemia was 31.1% (95% CI 28.9–33.4; prediction interval) after AL, 14.1% (10.8–18.3) after AA, 7.4% (6.7–8.1) after AM, and 4.5% (3.9–5.3) after DP. By day 63, the risk of P. vivax parasitaemia had risen to 39.9% (36.6–43.3) after AL, 42.4% (34.7–51.2) after AA, 22.8% (21.2–24.4) after AM, and 12.8% (11.4–14.5) after DP (Fig 2). There was substantial heterogeneity in risk between studies (Fig 3, Fig 4, Fig 5, Fig 6 and S6 Table). Of the 39 studies in which patients were followed for at least 42 days, the risk of P. vivax parasitaemia was ≥20% in 75.0% (9/12) of studies following AL, 20.0% (1/5) following AA, 21.7% (5/23) following AM, and 25.0% (4/16) following DP.

Fig 2

Cumulative risk (Kaplan–Meier analysis) of P. vivax parasitaemia following ACTs. *ACTs (AA, AL, AM, DP).

†P. vivax recurrence includes recurrences with P. vivax monoinfection or mixed-species infection. AA, artesunate-amodiaquine; ACT, artemisinin-based combination therapy; AL, artemether-lumefantrine; AM, artesunate-mefloquine; DP, dihydroartemisinin-piperaquine.

Fig 3

Cumulative risk (Kaplan–Meier analysis) of P. vivax parasitaemia after P. falciparum infection by study for AL. AL, artemether-lumefantrine.

Fig 4

Cumulative risk (Kaplan–Meier analysis) of P. vivax parasitaemia after P. falciparum infection by study for AA. AA, artesunate-amodiaquine.

Fig 5

Cumulative risk (Kaplan–Meier analysis) of P. vivax parasitaemia after P. falciparum infection by study for AM. AM, artesunate-mefloquine.

Fig 6

Cumulative risk (Kaplan–Meier analysis) of P. vivax parasitaemia after P. falciparum infection by study for DP. DP, dihydroartemisinin-piperaquine.

Risk factors for P. vivax parasitaemia

The rate of P. vivax parasitaemia was greatest in children. Compared with adults, children under 5 years had a hazard ratio (HR) = 4.40, 95% CI 3.66–5.29; p < 0.001, and children 5 to <15 years had an HR = 2.19, 1.90–2.53; p < 0.001. Other significant univariable baseline risk factors for P. vivax parasitaemia were high parasite count (>100,000 parasites per μL) (HR = 1.54, 1.24–1.90; p < 0.001), mixed P. falciparum and P. vivax infection (HR = 2.54, 2.14–3.01; p < 0.001), and P. falciparum gametocytaemia (HR = 1.57, 1.24–1.99; p < 0.001). Increasing baseline Hb was a protective factor (HR = 0.88 per 1 g/dL increase, 0.86–0.90; p < 0.001). Patients enrolled into studies conducted in regions of short relapse periodicity had a significantly greater rate of P. vivax parasitaemia than those enrolled into studies conducted in regions of long relapse periodicity (HR = 8.61, 2.34–31.65; p = 0.001) (Table 2).

Table 2

Risk factors for P. vivax parasitaemia after falciparum infection between days 7 and 42 in patients with different ACTs.

	Total Patients	Patients with P. vivax Parasitaemia	Risk of P. vivax Parasitaemia at Day 42	Univariable Analyses		Multivariable Analyses*
				Crude HR (95% CI)	p-Value	Adjusted HR (95% CI)	p-Value
Sex
Male	9,687	714	10.32 (9.62–11.06)	1.10 (0.97–1.24)	0.125	1.26 (1.08–1.46)	0.003
Female	5,616	426	9.94 (9.08–10.88)	Reference		Reference
Age, per year increase	15,325	1,140		0.95 (0.95–0.96)	<0.001	–
Age category, years
<5	1,584	313	24.95 (22.63–27.45)	4.40 (3.66–5.29)	<0.001	3.04 (2.39–3.87)	<0.001
5 to <15	4,983	462	11.90 (10.92–12.96)	2.19 (1.90–2.53)	<0.001	1.81 (1.52–2.15)	<0.001
≥15	8,758	365	6.01 (5.44–6.64)	Reference		Reference
Weight per kg increase	12,860	1,116		0.97 (0.96–0.97)	<0.001	–
Relapse periodicity
Short	14,258	1,125	10.85 (10.27–11.47)	8.61 (2.34–31.65)	0.001	6.20 (1.98–19.47)	0.002
Long	1,083	15	1.73 (1.05–2.87)	Reference		Reference
Geographical region
Africa	120	7	6.65 (3.23–13.46)	0.27 (0.03–2.65)	0.261	–
The Americas	684	7	1.11 (0.53–2.31)	0.08 (0.01–0.74)	0.026	–
Asia-Pacific	14,537	1,126	10.74 (10.16–11.35)	Reference		–
P. falciparum gametocytes
Yes	1,523	222	17.84 (15.81–20.09)	1.57 (1.24–1.99)	<0.001	1.40 (1.10–1.79)	0.007
No	10,197	575	8.12 (7.50–8.79)	Reference		Reference
Mixed infection
Yes	1,195	184	21.82 (19.15–24.80)	2.54 (2.14–3.01)	<0.001	2.20 (1.79–2.70)	<0.001
No	14,146	956	9.21 (8.67–9.79)	Reference		Reference
Parasitaemia, parasites per μL
Every 10-times increase	14,916	1,134		1.31 (1.20–1.41)	<0.001	–
>100,000 parasites/μL	871	112	17.68 (14.90–20.91)	1.54 (1.24–1.90)	<0.001	1.59 (1.22–2.08)	0.001
≤100,000 parasites/μL	14,045	1,022	9.93 (9.36–10.52)	Reference		Reference
Hb every 1 g/dL increase	12,184	1,090		0.88 (0.86–0.90)	<0.001	0.94 (0.90–0.97)	<0.001
Anaemic, Hb < 10 g/dL
Yes	3,358	482	18.55 (17.09–20.10)	1.70 (1.49–1.94)	<0.001	–
No	8,826	608	8.94 (8.29–9.65)	Reference		–
Drug
AL	2,711	522	31.08 (28.89–33.40)	5.18 (4.10–6.56)	<0.001	6.23 (4.55–8.53)	<0.001
AA	651	47	14.07 (10.77–18.27)	1.75 (1.19–2.55)	0.004	2.26 (1.40–3.65)	0.001
AM	7,340	423	7.36 (6.71–8.06)	1.37 (1.09–1.72)	0.006	1.40 (1.04–1.89)	0.028
DP	4,639	148	4.50 (3.85–5.27)	Reference		Reference

*Geographical region and weight excluded from multivariable analysis due to collinearity with relapse periodicity and age.

Abbreviations: AA, artesunate-amodiaquine; ACT, artemisinin-based combination therapy; AL, artemether-lumefantrine; AM, artesunate-mefloquine; CI, confidence interval; DP, dihydroartemisinin-piperaquine; Hb, haemoglobin; HR, hazard ratio.

In multivariable analysis, younger afge (adjusted HR [AHR] = 3.04, 95% CI 2.39–3.87, p < 0.001 and AHR = 1.81, 95% CI 1.52–2.15, p < 0.001 comparing age <5 years and age 5 to <15 years to adults, respectively), short relapse periodicity (AHR = 6.20, 95% CI 1.98–19.47; p = 0.002), P. falciparum gametocytaemia (AHR = 1.40, 1.10–1.79; p = 0.007), mixed infection (AHR = 2.20, 1.79–2.70; p < 0.001), high parasite count (AHR = 1.59, 1.22–2.08; p = 0.001), male sex (AHR = 1.26, 95% CI 1.08–1.46; p = 0.003), and low baseline Hb (AHR = 0.94 per 1 g/dL increase, 0.90–0.97; p < 0.001) were independent risk factors for P. vivax parasitaemia (Table 2). Compared with patients treated with DP, the rate of P. vivax parasitaemia was greater after AL (AHR = 6.23, 4.55–8.53; p < 0.001), AA (AHR = 2.26, 1.40–3.65; p = 0.001), and AM (AHR = 1.40, 1.04–1.89; p = 0.028) (Fig 7). Removal of 1 study at a time did not reveal bias related to any individual study (S7 Table).

Fig 7

Risk of P. vivax parasitaemia following falciparum infection between day 7 and day 42 according to treatment.

Dotted lines demonstrate 95% CIs. Figure adjusted for age, sex, baseline parasitaemia, regional relapse periodicity, P. falciparum gametocytes, mixed infection, and baseline Hb, assuming no study-site effect. AA, artesunate-amodiaquine; AL, artemether-lumefantrine; AM, artesunate-mefloquine; DP, dihydroartemisinin-piperaquine; Hb, haemoglobin.

The mean Hb concentration at baseline was 11.8 g/dL (standard deviation [SD] 2.35) compared with 10.9 g/dL (SD 2.03) on day 7 in 6,328 patients with available data. The overall mean fall in Hb between baseline and day 7 was 0.87 g/dL (SD 1.71, range −8.8 to 11.6). The rate of P. vivax parasitaemia did not correlate with the absolute fall in Hb in univariable or multivariable analysis.

Association between risk of P. vivax parasitaemia and parasite clearance of initial P. falciparum infection

Overall, 46.3% (4,260/9,208) of patients were aparasitaemic by day 1 and 89.9% (8,277/9,208) by day 2; S8 Table. The risk of P. vivax parasitaemia between days 7 and 63 was 16.3% (95% CI: 14.7–18.1) in the patients clearing their parasitaemia on day 1, 22.6% (95% CI: 20.7–24.5) in patients clearing on day 2, and 29.2% (95% CI: 25.1–33.7) in patients clearing after this. The rate of P. vivax parasitaemia by day 63 was investigated in a multivariable model controlling for confounding factors. Compared with patients clearing their peripheral P. falciparum parasites by day 1, the rate of P. vivax was significantly greater in those clearing their initial parasitaemia on day 2 (AHR = 1.48, 95% CI 1.27–1.73; p < 0.001) and even greater in those clearing their parasitaemia thereafter (AHR = 1.82, 1.44–2.30; p < 0.001) (Fig 8 and S9 Table). Removal of 1 study at a time did not reveal bias related to any individual study (S10 Table).

Fig 8

Risk of P. vivax parasitaemia following falciparum infection between days 7 and 63 according to treatment and day of parasite clearance: (A) AL, (B) AA, (C) AM, and (D) DP.

Figure adjusted for age, sex, baseline parasitaemia, regional relapse periodicity, P. falciparum gametocytes, mixed infection at baseline, and Hb at baseline, assuming no study-site effect. AA, artesunate-amodiaquine; AL, artemether-lumefantrine; AM, artesunate-mefloquine; DP, dihydroartemisinin-piperaquine; Hb, haemoglobin.

A sensitivity analysis including patients treated with single low-dose primaquine did not change these results substantially (compared with patients who cleared parasitaemia by day 1, AHR = 1.48 [95% CI 1.27–1.73] if cleared on day 2 and AHR = 1.82 [95% CI 1.44–2.29] if cleared thereafter).

Site factors associated with risk of P. vivax parasitaemia following treatment

The study-specific risks of P. vivax during follow-up are presented in Fig 3, Fig 4, Fig 5 and Fig 6. The risk of P. vivax parasitaemia at day 42 was greatest following treatment with AL. Twelve studies across 18 study sites and 7 countries enrolled and treated 2,562 patients with AL who were followed for 42 days or longer (S2 Fig). Nine (75%) studies enrolled children younger than 5 years. The study-specific risk of P. vivax at day 42 varied from 0% to 63.8%. The risk was ≥20% in study sites from Thailand (4 study sites), Papua New Guinea (PNG) (4 study sites), Myanmar (3 study sites), and Indonesia (1 study site); 12%–26% in 3 study sites from Bangladesh; 0%–7% in 2 study sites from Ethiopia; and 2.0% in 1 study from Laos (Fig 3). The corresponding data for the other treatment arms are presented in Fig 4, Fig 5, Fig 6 and S11 Table.

Using subnational data, the estimated incidence of P. falciparum at each site varied from 0.5 to 154 cases per 1,000 person-years, and the background incidence of P. vivax varied from 1.6 to 151 cases per 1,000 person-years. The risk of P. vivax following AL was correlated with the site-specific background incidence of P. vivax (rs = 0.676, p = 0.0029) and incidence of P. falciparum (rs = 0.607, p = 0.0098) (Fig 9). However, after controlling for confounding factors in a Cox regression model, neither of the background incidences were associated significantly with the risk of P. vivax (AHR = 0.99, 0.97–1.02, p = 0.634 and AHR = 1.00, 0.99–1.02, p = 0.629, respectively, for every 1 case increase per 1,000 person-years) (S12 Table). The results were similar for the other ACTs (S13 Table, S14 Table, S15 Table, S3 Fig, S4 Fig, S5 Fig). The year of enrolment was not independently associated with the risk of P. vivax (AHR = 0.93 per year later, 0.84–1.03; p = 0.173).

Fig 9

Risk of P. vivax parasitaemia at day 42 following treatment with AL according to background subnational incidence of P. vivax (A) and P. falciparum (B).

rs = 0.676, p = 0.0029 (A) and rs = 0.607, p = 0.0098 (B). AL, artemether-lumefantrine.

Discussion

Our individual patient data meta-analysis, including more than 15,000 patients, confirms a high risk of P. vivax parasitaemia following treatment of P. falciparum with ACTs; however, there was marked heterogeneity between study populations and sites. The risk of P. vivax was highest in studies undertaken in regions with short relapse periodicity and in patients who were young or presented with mixed-species infections or a high baseline parasitaemia.

Overall, 10% of patients treated with an ACT had P. vivax recurrence by day 42, and this increased to 22% by day 63. The risk of P. vivax was significantly lower in patients treated with an ACT containing a slowly eliminated partner drug such as piperaquine, mefloquine, and amodiaquine [5, 7]. Lumefantrine is eliminated faster, with a half-life of 4 days, and after 16 days (4 half-lives) provides minimal post-treatment prophylaxis against P. vivax relapses [59]. This is reflected by the high risk of P. vivax parasitaemia following AL, which reached 31% by day 42.

The heterogeneity in the risk of P. vivax was highlighted in our recent systematic review of clinical trials undertaken in coendemic areas [7] and has been investigated further in a review of trials from the eastern border of Myanmar [60]. On the Thailand–Myanmar border, the risk of P. vivax parasitaemia within 42 days of treatment of falciparum malaria exceeded 20% between 2003 and 2010 and reached 85% in 1 study. However, after 2010, the risk fell to less than 5%. The authors hypothesise that the decline in the risk of P. vivax was attributable to substantial improvements in malaria control and thus a decline in the proportion of patients presenting with P. falciparum who were coinfected with P. vivax. A key determinant of P. vivax relapse is the number of hypnozoite parasites present in the liver, which varies with local P. vivax transmission intensity, immunity, and sporozoite inoculum [61]. Although the risk of P. vivax was correlated positively with the predicted background incidence of both P. vivax and P. falciparum, this was not apparent after adjusting for confounding factors. We observed no temporal trends indicative of a lower risk of P. vivax parasitaemia in more recent studies. This may relate to the relatively few studies available to compare the use of an individual ACT at a single site, lack of temporally distinct data from multiple studies in the same regions, or heterogeneity between study populations from different locations. Incidence estimates are also inexact, relying on subnational regional data that may not equate to the study site and do not account for seasonal variation of transmission [15]. There is also a risk of inclusion bias, with prospective clinical efficacy studies generally undertaken in regions with a relatively high malaria burden to ensure adequate recruitment. The ability to compare the effect of malaria prevalence between clinical efficacy studies is therefore likely to be limited compared with data from longitudinal cohorts [62].

Our individual patient data meta-analysis confirms an increased risk of recurrence in regions where the duration between P. vivax relapses is short [5, 7] and highlights the additional risks of young age, high baseline parasitaemia, low baseline Hb, and presenting with either mixed-species infection or P. falciparum gametocytes. More than 15% of patients presenting with mixed infection of P. falciparum and P. vivax had P. vivax parasitaemia within 42 days, supporting the World Health Organization (WHO) recommendation for the use of primaquine radical cure in these patients. However, the high risk of P. vivax was also apparent in those presenting with P. falciparum monoinfection. We could not determine whether recurrences were due to relapse or reinfection in patients continuing to reside in a malaria-endemic area. The subnational incidence estimates of P. vivax infection in the study sites included in our study ranged from 0.2 to 151 per 1,000 person-years [15]. If one conservatively assumes 2 weeks post-treatment prophylaxis following antimalarial treatment and the highest risk of reinfection of the included study sites, then the maximum risk of a new infection with P. vivax within 63 days of P. falciparum would be 2.0%. The risks of P. vivax parasitaemia in our analysis were thus at least 6- to 21-fold–greater than could be explained by reinfection alone.

It is possible that patients with mixed infections of P. falciparum with low-level P. vivax parasitaemia were misdiagnosed as P. falciparum monoinfections. However, all of the ACTs included in the analysis have high efficacy against P. vivax [63], and even if 10% of patients had had submicroscopic P. vivax parasitaemia at presentation, the risk of recrudescent P. vivax by day 63 would be no greater than 0.5%. Conversely, we may have underestimated the risk of vivax recurrence because submicroscopic relapses were not quantified and the duration of follow-up was restricted to 63 days, thus preventing detection of later relapses. In a longitudinal cohort in which asymptomatic submicroscopic infections were quantified over a 12-month period, there was a 2.4-fold–higher risk of asymptomatic vivax recurrence following asymptomatic falciparum infections [64].

Our findings are more consistent with the P. vivax parasitaemia during follow-up arising from activation of P. vivax hypnozoites present at the time of the initial presentation with P. falciparum [5, 65, 66]. Importantly, the initial therapeutic response, as measured by parasite clearance, was correlated with the subsequent risk of P. vivax. Patients taking longer to clear their P. falciparum parasitaemia were at almost 2-fold–greater risk of P. vivax than those who cleared their parasitaemia rapidly. Whilst this could reflect vulnerability of a host with low immunity to both slower parasite clearance and recurrent parasitaemia, the increased risk of P. vivax was independent of the patients’ age, a surrogate marker of host immunity. We believe this supports the hypothesis that vivax parasitaemia following falciparum malaria relates to a host–parasite interaction that activates hypnozoites acquired from a prior infection. Haemolysis has been postulated to trigger P. vivax reactivation [66, 67], although we found no correlation between the early fall in Hb concentration and the risk of subsequent vivax parasitaemia.

Our study has a number of limitations. The systematic review included all P. falciparum clinical trials between 1960 and 2018, although the analysis was restricted to patients who were treated with an ACT with studies conducted between 1991 and 2018. The analysis included 42 studies, enrolling 15,341 patients in 12 malaria-endemic countries, and the quality of the included studies suggested a low risk of systematic bias (S1 Text); however, there were significant differences in these data and those included in our previous systematic review [7]. Fourteen studies uploaded to the WWARN repository with P. vivax recurrences were not included as part of the 153 studies in the previous review. Ten of these were not included in the initial systematic review because the corresponding manuscripts reported neither the presence nor absence of vivax recurrences. An additional limitation of our current analysis was the inability to include data from 25 targeted studies. Although the patient demographics from these studies were similar to the included studies, they included a greater number of African studies, which might have allowed better generalisability of results to this region. Furthermore, the studies that were included were not equally representative of all vivax-endemic locations, with the majority undertaken in southeast Asia and only a single study included from India. Studies in the WWARN repository were only included into the analysis if they reported a P. vivax recurrence in at least 1 treatment arm. This was justified because in many P. falciparum studies, P. vivax parasitaemia may not be deemed relevant to the efficacy analysis, and data may not be recorded or shared with WWARN. Hence, it is possible that exclusion of these may have systematically biased against studies without any P. vivax recurrence and thus overestimated the risk of P. vivax. Reassuringly, only 3 out of 33 clinical trial arms in an independent systematic review reported no P. vivax recurrences [60].

The rapid elimination of P. vivax will require a greater emphasis on addressing the hidden reservoirs of the parasite, including individuals with asymptomatic carriage of blood- or liver-stage parasites. Our analysis highlights that in coendemic areas, patients presenting with P. falciparum may be at significant risk of carrying dormant hypnozoites. Hence, whilst current WHO guidelines only recommend treatment with a prolonged course of primaquine for patients with P. vivax or a P. vivax–mixed-species infection [68], consideration should be given to offering radical cure, with either primaquine or tafenoquine, with appropriate G6PD testing, to patients presenting with P. falciparum monoinfection in coendemic regions. In view of the heterogeneity in the risk of P. vivax and drug-induced haemolysis between locations, such a strategy should be recommended with caution. The risk factors for P. vivax identified in this analysis may help define populations for whom the benefit of a universal policy of radical cure has greatest benefit but should be guided by complementary prospective clinical efficacy studies, and in coendemic areas, these should include quantifying the subsequent risk of all species of Plasmodia following antimalarial treatment.

PRISMA-IPD.

PRISMA-IPD, Preferred Reporting Items for Systematic Review and Meta-Analyses of individual participant data

(PDF)

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Search strategy.

(PDF)

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Assessment of risk of bias relating to individual studies.

(PDF)

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Map of study-site locations.

Map created using ggplot2 in R.

(TIF)

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Map of cumulative risk of P. vivax parasitaemia at day 42 following AL.

Map created using ggplot2 in R. AL, artemether-lumefantrine

(TIF)

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Risk of AA, artesunate-amodiaquine

(PDF)

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Risk of AM, artesunate-mefloquine

(PDF)

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Risk of DP, dihydroartemisinin-piperaquine

(PDF)

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Reasons for studies not being included in analysis.

(PDF)

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Studies included in analysis.

(PDF)

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Study sites included in analysis.

(PDF)

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Studies targeted for the analysis but not available.

(PDF)

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Comparison of baseline characteristics from included and targeted studies.

(PDF)

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Prediction intervals for cumulative risk of P. vivax parasitaemia following ACTs.

ACT, artemisinin-based combination therapy

(PDF)

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Sensitivity analysis for associations between patient characteristics and rate of P. vivax parasitaemia between days 7 to 42 for the general model.

(PDF)

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Parasite clearance according to treatment.

(PDF)

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Relationship between day of parasite clearance, patient characteristics, and rate of P. vivax parasitaemia between days 7 and 63.

(PDF)

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Sensitivity analysis for associations between patient characteristics and rate of P. vivax parasitaemia between days 7 to 63 for the model including day of parasite clearance.

(PDF)

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Studies with follow-up for 42 days or longer included in analysis of site factors.

(PDF)

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Relationship between patient characteristics and study-site malaria prevalence and rate of P. vivax parasitaemia between days 7 and 42 in patients treated with AL.

AL, artemether-lumefantrine

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Relationship between patient characteristics and study-site malaria prevalence and rate of P. vivax parasitaemia between days 7 and 42 in patients treated with AA. AA, artesunate-amodiaquine.

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Relationship between patient characteristics and study-site malaria prevalence and rate of P. vivax parasitaemia between days 7 and 42 in patients treated with AM. AM, artesunate-mefloquine.

(PDF)

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Relationship between patient characteristics and study-site malaria prevalence and rate of P. vivax parasitaemia between days 7 and 42 in patients treated with DP. DP, dihydroartemisinin-piperaquine.

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1 Jun 2020

Dear Dr Price,

Thank you for submitting your manuscript entitled "The risk of Plasmodium vivax parasitaemia after Plasmodium falciparum malaria: a WWARN individual patient data meta-analysis" for consideration by PLOS Medicine.

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25 Jun 2020

Dear Dr. Price,

Thank you very much for submitting your manuscript "The risk of Plasmodium vivax parasitaemia after Plasmodium falciparum malaria: a WWARN individual patient data meta-analysis" (PMEDICINE-D-20-02368R1) for consideration at PLOS Medicine.

Your paper was evaluated by a senior editor and discussed among all the editors here. It was also discussed with an academic editor with relevant expertise, and sent to independent reviewers, including a statistical reviewer. The reviews are appended at the bottom of this email and any accompanying reviewer attachments can be seen via the link below:

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In light of these reviews, I am afraid that we will not be able to accept the manuscript for publication in the journal in its current form, but we would like to consider a revised version that addresses the reviewers' and editors' comments. Obviously we cannot make any decision about publication until we have seen the revised manuscript and your response, and we plan to seek re-review by one or more of the reviewers.

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Comments from the reviewers:

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Reviewer #1: See attachment

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Reviewer #2: See attachment

Michael Dewey

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Reviewer #3: General comments / Major issues

This is a very well written paper on an important topic in malaria epidemiology that may have implications for Plasmodium vivax (Pv) elimination strategies. The study aims to define the risk factors of Pv parasitemia following a Pf infection in various settings using an individual-level meta-analysis. While this is a well done and interesting meta-analysis, there are number of comments the authors should address.

Major compulsory comments to be addressed:

* My main issue with the paper, as currently written, is a lack of explanation as to the rationale for the study. It is clear the authors hypothesis that a Pf infection, and subsequent treatment, increases the risk of a relapse from a concurrent hypnozoite Pv infection, but the authors offer little rationale as to why they believe this hypothesis to be true. Is it simply because in areas with both Pf and Pv transmission, individuals with a Pf infection would also be at greater risk for harboring a Pv infection because of myriad factors in low and heterogeneous transmission that put specific people at greater risk? Would it have to do with immunity? Declines in hemoglobin following Pf treatment are explored, but are not stipulated a priori as a potential reason for Pv reactivation. Such rationales for the primary hypothesis under study should be clearly stated in the introduction, tested in the methods and results, and then reassessed and interpreted in the discussion.

* One of the major weakness of this study is disentangling what the level of Pv relapse means among individuals with a Pf infection that are treated with various ACTs (e.g. cumulative risk of Pv parasitemia were 3.7%, 10.2% and 22.2%, at days 28, 42 and 63 respectively - lines 322-324). But how does this compare to the underlying rate of Pv parasitemia/relapse at these time points among individuals without a Pf infection who never received treatment? I.e. - What is the additional risk of Pv relapse due to a Pf-treated infection compared to those without a Pf infection? I understand the authors do not have such data on individuals without a Pf infection, but that comparison is what is needed to actually test if the rate of relapse in Pv is higher than what would be expected among those without a Pf clearance. This limitation needs to be clearly stated and discussed. Can the authors offer any insight from the literature into how they think Pv parasitemia/activation levels would differ between those with and without a Pf infection and treatment?

* Could one factor that might explain the potential increased risk of Pv relapse among individuals treated for a Pf infection be the underlying level of Pf vs Pf transmission risk in the study sites - perhaps measured as the ration of Pf:Pv infections at baseline? If areas with much more Pv transmission relative to Pf transmission had higher risk of Pv relapse as compared to areas with higher Pf to Pv transmission at specified follow-ups, to me this would indicate that those with a Pf infection are just at higher risk of any Plasmodium infection. Can the authors include a Pf:Pv ratio indicator, perhaps categorized, as a covariate in the Cox PH models assessing the risk factors of Pv parasitemia? Assuming this could be derived by MAP estimates of PfPR and PvPR for each study site?

* Can the authors more clearly state in the discussion the major limitation of the study that they cannot distinguish between a new Pv infection and a reactivation of a previous infection? Are the levels of parasitemia after 28, 42 and 63 days in line with transmission risk for a new Pv infection over this period in the study sites? I doubt they are and this argument should be made that they are likely reactivations because they are much higher than transmission would likely allow for them to be new Pv infections over these relatively short time periods?

* Did the Cox models also account for malaria transmission season? They should if they have not.

* I would think the underlying level of risk of transmission of Pf and Pv would be a significant risk factor (or potential confounder) when assessing the risk of a Pf infection treated with various ACTs. The authors appear to try and account for this with a covariate in the models for malaria parasite prevalence from MAP (lines 230-231). Why was this then categorized as low / not low at a cut-point of PfPR = 1.5%? As defined, this was not significant in the Cox models? Did they try other cut-points or categories? Why not just keep PfPR continuous - to me that would be the best option for accounting for this very important factor in the analyses.

* The authors state a date range of 1960 - 2018 was used for the systematic review of Pf treatment studies, taken for a previous study. This is a bit disingenuous as ACTS did not become available until well after 1960 (I would imagine in the 1990s at the earliest)? The authors should revise the date ranges for the systematic review accordingly, as well as provide the date ranges of the earliest and latest included studies in their description of the systematic review results.

* It would be helpful if the authors could state how the covariates in the Cox PH models were defined (e.g. those in lines 257 - 259). For example, was mixed infection categorized as yes/no (0/1). Was hemoglobin a continuous variable?

* For the analysis assessing the site-specific risk factors associated with risk of Pv following treatment (section starting on line 378), why was this limited to those treated with AL? Why not include all ACTs and just account for ACT type with a fixed effect covariate in the model?

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Any attachments provided with reviews can be seen via the following link:

[LINK]

Submitted filename: Reviewer_Plos Med.docx

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Submitted filename: hossain.pdf

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23 Jul 2020

Submitted filename: WWARN Pv after Pf_Response.docx

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24 Aug 2020

Dear Dr. Price,

Thank you very much for re-submitting your manuscript "The risk of Plasmodium vivax parasitaemia after Plasmodium falciparum malaria: a WWARN individual patient data meta-analysis" (PMEDICINE-D-20-02368R2) for review by PLOS Medicine.

I have discussed the paper with my colleagues and the academic editor and it was also seen again by three reviewers. I am pleased to say that provided the remaining editorial and production issues are dealt with we are planning to accept the paper for publication in the journal.

The remaining issues that need to be addressed are listed at the end of this email. Any accompanying reviewer attachments can be seen via the link below. Please take these into account before resubmitting your manuscript:

[LINK]

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In revising the manuscript for further consideration here, please ensure you address the specific points made by each reviewer and the editors. In your rebuttal letter you should indicate your response to the reviewers' and editors' comments and the changes you have made in the manuscript. Please submit a clean version of the paper as the main article file. A version with changes marked must also be uploaded as a marked up manuscript file.

Please also check the guidelines for revised papers at http://journals.plos.org/plosmedicine/s/revising-your-manuscript for any that apply to your paper. If you haven't already, we ask that you provide a short, non-technical Author Summary of your research to make findings accessible to a wide audience that includes both scientists and non-scientists. The Author Summary should immediately follow the Abstract in your revised manuscript. This text is subject to editorial change and should be distinct from the scientific abstract.

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We ask every co-author listed on the manuscript to fill in a contributing author statement. If any of the co-authors have not filled in the statement, we will remind them to do so when the paper is revised. If all statements are not completed in a timely fashion this could hold up the re-review process. Should there be a problem getting one of your co-authors to fill in a statement we will be in contact. YOU MUST NOT ADD OR REMOVE AUTHORS UNLESS YOU HAVE ALERTED THE EDITOR HANDLING THE MANUSCRIPT TO THE CHANGE AND THEY SPECIFICALLY HAVE AGREED TO IT.

Please ensure that the paper adheres to the PLOS Data Availability Policy (see http://journals.plos.org/plosmedicine/s/data-availability), which requires that all data underlying the study's findings be provided in a repository or as Supporting Information. For data residing with a third party, authors are required to provide instructions with contact information for obtaining the data. PLOS journals do not allow statements supported by "data not shown" or "unpublished results." For such statements, authors must provide supporting data or cite public sources that include it.

If you have any questions in the meantime, please contact me or the journal staff on plosmedicine@plos.org.

We look forward to receiving the revised manuscript by Aug 31 2020 11:59PM.

Sincerely,

Artur Arikainen

Associate Editor

PLOS Medicine

plosmedicine.org

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Requests from Editors:

1. Title: Please update to: “The risk of Plasmodium vivax parasitaemia after Plasmodium falciparum malaria: an individual patient data meta-analysis from the WorldWide Antimalarial Resistance Network”

2. Short Title: Please update to: “Risk of P. vivax after P. falciparum malaria”

3. Funding Statement: Please replace the last sentence with our standard text, if applicable: “The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

4. Competing Interests Statement: All authors must declare their relevant competing interests per the PLOS policy, which can be seen here:

https://journals.plos.org/plosmedicine/s/competing-interests For authors with ties to industry, please indicate whether any of the interests has a financial stake in the results of the current study. Please therefore include employment by author(s) to AstraZeneca. Please also add this statement to the manuscript's Competing Interests: "EAA and NJW are Academic Editors on PLOS Medicine's editorial board."

5. Abstract:

a. Please report your abstract according to PRISMA for abstracts: http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.1001419 . Please include the databases searched, the method used to assess quality/risk of bias, a summary of the quality/risk of bias assessment, and the key funding source(s).

b. Please summarise the numbers of each study type included, eg. RCTs, cohort studies.

c. Please summarise the number of participants by drug combination (from around line 340), and by region (from Table 1).

d. Please add the following summary data for the patients from the main Results section: “Overall 14,146 (92.2%) patients had P. falciparum mono-infection and 1,195 (7.8%) mixed infection with P. falciparum and P. vivax confirmed by blood film microscopy. The median age was 17.0 years (inter-quartile-range (IQR) = 9.0 – 29.0 years; range = 0 – 80 years), with 1,584 (10.3%) patients younger than 5 years.”

e. Please quantify all results with 95% CIs and p values.

f. Line 138: Please start with "In this study, we found that ..." or similar

g. Line 140 (and 169): Please replace “effective” with “recommended” or similar, to avoid implying causality from your findings.

6. Author Summary:

a. Line 149: Please clarify “parasitaemia” for a lay reader.

b. Lines 152, 168, 169: Please clarify “hypnozoite” for a lay reader, or replace with a simpler term.

c. Lines 161-162: Please clarify “short relapse periodicity” for a lay reader.

d. Line 164: Please give species names here as elsewhere, for clarity.

e. Line 165: Perhaps replace “is … heterogeneity” with “are … differences”, for simplicity.

f. Line 171: Perhaps replace “complementary” with “further”, for simplicity.

7. Please move your citation callout to before punctuation, with a space after the text, eg.: “associated with 405,000 deaths [1].”

8. The terms gender and sex are not interchangeable (as discussed in http://www.who.int/gender/whatisgender/en/ ); please use the appropriate term.

9. Lines 609-648: Please remove this information (Contributions, COIs, Funding, and Data) from the main text and instead include it in the online submission form.

10. Lines 651-655: Please move the ethics information to the Methods.

11. Please provide full access details (eg. DOI or URL) for references 1, 2, 10, and 68. Please remove this from reference 63: “following competing interests: NJW is a member of the Editorial Board of PLOS Medicine.”

12. When completing the PRISMA checklist, please use section and paragraph numbers, rather than page numbers.

---------------

Comments from Reviewers:

Reviewer #1: Author responded to all issues I raised. I would recommend publish this manuscript in PLOS Medicine.

Reviewer #2: The authors have addressed my points.

Michael Dewey

Reviewer #3: The authors did a very nice job in addressing the reviewers comments. The manuscript is now suitable for publication.

Any attachments provided with reviews can be seen via the following link:

[LINK]

25 Sep 2020

Dear Prof Price,

On behalf of my colleagues and the academic editor, Dr. James G. Beeson, I am delighted to inform you that your manuscript entitled "The risk of Plasmodium vivax parasitaemia after Plasmodium falciparum malaria: an individual patient data meta-analysis from the WorldWide Antimalarial Resistance Network" (PMEDICINE-D-20-02368R3) has been accepted for publication in PLOS Medicine.

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Thank you again for submitting the manuscript to PLOS Medicine. We look forward to publishing it.

Best wishes,

Artur Arikainen,

Senior Editor

PLOS Medicine

plosmedicine.org