Malaria in pregnancy (MiP) studies assessing the clinical performance of highly sensitive rapid diagnostic tests (HS-RDT) for Plasmodium falciparum detection

Malaria in pregnancy (MiP) is associated with increased risk of poor maternal and infant health outcomes, including fetal loss, maternal anaemia, pre-term birth, low birthweight and intrauterine growth retardation, which in turn increase the risk of infant morbidity and mortality [1]. Modelled estimates indicate that in 2019, 35% of pregnancies in sub-Saharan Africa (or 11.6 million expectant mothers) were exposed to malaria infections, leading to 0.8 million low birthweight newborns [2, 3]. Malaria prevalence is highest in women who are primigravid and who are in the first or second trimester of pregnancy [4]. To protect against MiP, a strategy of routine intermittent preventative treatment during pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP) is recommended at each scheduled antenatal care visit in the second and third trimester, at a minimum of monthly intervals [5]. However, IPTp with SP is contraindicated in the first trimester of pregnancy, is threatened by parasite resistance to SP, is only recommended in moderate and high transmission areas of sub-Saharan Africa, and the uptake is poor [3], limiting the effectiveness of this intervention.

WHO has reviewed different ‘Test and Treat’ strategies in the context of MiP, such as Intermittent Screening and Treatment (ISTp) at the first antenatal care (ANC) visit [6]. Although wide-scale deployment of rapid diagnostic tests (RDTs) for the detection of malaria has been largely successful in control efforts in the general population, pooled analyses have shown that interventions based on maternal screening with conventional RDTs (co-RDT) did not provide any additional benefit as compared to routine IPTp-SP [6, 7].

IPTp is contra-indicated in the first trimester of pregnancy, yet a high proportion of women are infected in the first trimester [8], which can have devasting consequences for the pregnancy [9‐11]. Early detection and treatment of infections is, therefore, essential. However, detection is difficult as pregnant women infected with Plasmodium falciparum are commonly asymptomatic and have low density infections [12], and infected erythrocytes that sequester in the placenta escape detection in peripheral blood [9]. An important proportion of these low-density malaria infections remain undetected by light microscopy or co-RDTs [13]. A modelling study on malaria screening strategies at ANC visits suggested that more sensitive RDTs could provide incremental improvements in ISTp strategies over IPTp-SP in terms of reducing infection prevalence at delivery, with presumably better outcomes for mother and child [8]. It also highlighted that ISTp during the first trimester could improve pregnancy outcomes [8]. Improved point-of-care diagnostics that can detect more cases of maternal infections and in particular placental malaria may thus contribute to a reduction of adverse clinical outcomes for mothers and newborns in malaria endemic settings.

A highly sensitive RDT (HS-RDT) based on detection of Histidine-Rich Protein 2 (HRP2) was developed (NxTek™ Eliminate malaria Pf, Abbott Diagnostics) to improve the identification of P. falciparum infections below the detection limit of co-RDTs. The HS-RDTs enable point-of-care testing with an analytical sensitivity (limit of detection (LOD)) that is ten-fold lower than that reported for current best-in-class co-RDTs, at 80 pg of HRP2 per mL of blood [14] compared to 800-1000 pg for co-RDTs [15] as tested in vitro in malaria-negative blood. A recent review of data across multiple studies in different settings show that this improvement in analytical performance consistently results in an improvement in sensitivity and prevalence estimates as compared to PCR reference testing. The significance of this improvement varies from setting to setting [16‐18].

In June 2018, the WHO Global Malaria Programme convened a technical consultation to identify the evidence required to develop recommendations on the use of the HS-RDT in different contexts including the detection of MiP [19]. The lack of results from prospective longitudinal studies or trials assessing the clinical impact of the HS-RDT as part of interventions to prevent MiP was noted. It was concluded that diagnostic accuracy studies comparing the HS-RDT to co-RDT are of high priority, and that these should reflect a range of different conditions (e.g. transmission intensities, target populations). This review compiles all existing evidence to date on the performance and use case scenarios of the HS-RDT in the context of pregnancy to inform policy makers and the research community on existing evidence and knowledge gaps.

This evidence review encompassed both published articles in peer-reviewed journals as well as other grey literature such as technical reports or presentations at scientific conferences.

The five studies with available data were heterogenous both geographically and by transmission intensity, aiding generalizability of the results. A key assumption is that molecular testing of peripheral blood can detect placental malaria, making these data comparable between sites [28]. Despite the limitations of using different PCR assays across studies infections in the low transmission sites tended towards lower parasite densities, supporting previous observations that there are more low-density infections in low transmission regions (< 100 parasite/μL [29, 30]). Walker et al. [8] showed that sensitivity decreased with decreasing parasite prevalence and hypothesized that HS-RDTs may have more impact than co-RDTs in low transmission areas with lower parasitaemias. The reviewed studies with the lowest transmission were in Colombia, and HS-RDTs were not significantly more sensitive than co-RDTs. However, looking at low density infections (parasite densities of ~ 10–100 p/μL), the HS-RDT identifies double the number of infections (26/44) as the co-RDT (13/44) in Benin [22], while in Colombia (2) the HS-RDT identifies 9/9 infections yet the co-RDT identifies only 6/9. This suggests an advantage to using HS-RDT to detect lower density infections.

Another source of variability in sensitivity is likely to be the samples used: although the RDTs are intended for use with fresh blood, the studies in Benin, Colombia (1), and Indonesia all used thawed blood, which may affect RDT performance. The study in Indonesia used thawed red blood cell pellets (stored: ~ 1 year), reconstituted with plasma to 30% haematocrit. This off-label use might account for the very low sensitivity of both the co-RDT and HS-RDT in this study. A further source of variation in sensitivity could be the reference standard used and its associated LOD. LODs ranged from ~ 0.02–6 parasite/μL. Across the five studies included in this analysis however, there was no trend for sensitivity to be associated with reference assay LOD.

Both RDTs tended to have higher sensitivity in febrile women compared with afebrile women, as expected. Prevalence of malaria infection is known to be highest at first ANC visit, and declines with gravidity by both PCR and co-RDT [4, 8]. The included studies did not show a clear relationship between HS-RDT sensitivity and gravidity, nor did this appear to be affected by transmission intensity. A recent modelling-based analysis [8] indicated that the main gain in sensitivity in using HS-RDTs among pregnant women should be expected when infections tend to have lower parasitaemias. When exploring this in the current review of limited studies, the clinical performance of the HS-RDT compared to co-RDTs only suggests a slight increase in analytical sensitivity in the 1st trimester in high-transmission settings, or the 2nd trimester in low transmission settings. One of the reasons behind this discrepancy is that the ‘better performing RDTs’ considered in these models are assumed to provide a 75% to 90% sensitivity, while most of the sensitivities observed in the HS-RDT field evaluations in the context of MiP fall below this range. The only study in which HS-RDT sensitivity significantly outperformed co-RDTs was in the first trimester in a high transmission setting (Benin) with a low proportion of primigravid women (8%). A major limitation of the analyses for febrility, gravidity and trimester are the low numbers of parasite positive women, limiting the statistical power of comparing sensitivities between the tests.

Another reason for minimal differences between HS-RDT and co-RDT could be that co-RDTs have a lower LOD than expected. While the criteria of detection of at least 200 parasite/μL is required for pre-qualification by WHO, the exact LOD is unknown and for some may approach the LOD of HS-RDT (i.e., they may detect lower numbers of parasites/uL). The SD Bioline Pf co-RDT was tested against the HS-RDT by Das et. al [14] and was found to be reactive to cultured parasites at ~ 49 parasite/µL compared to ~ 3.13 parasite/µL by the HS-RDT, i.e., in laboratory tests the LOD of this co-RDT was about four times more sensitive than the minimum required for WHO pre-qualification.

The main limitation of this evidence review is the difficulty of pooling data due to the heterogeneity of the studies, difference in reference assays and reporting formats, and small numbers of studies included. In turn, this heterogeneity is also a benefit to describe the potential advantages of the HS-RDTs in different situations. The main goal of diagnosing MiP is to reduce adverse clinical outcomes for women and their children. The study in Benin [22] identified that those infections in women that were detected with the HS-RDT but not by the co-RDT were more likely to suffer anaemia during pregnancy and suggested a higher risk of LBW, although this interaction is likely underpowered. It may also be due to the study design which treated any microscopy positive patients, which therefore identifies and treats those patients who would also be identified by co-RDT, while lower parasitaemic infections (i.e. those detected by HS-RDT) remain undetected and untreated which may lead to anaemia over time. The ongoing study in Burkino Faso [31] will investigate the impact of screening with HS-RDTs and treating women on placental malaria and LBW. These analyses of clinical outcomes are important as it is still unclear whether identifying more (low density) infections will make a difference to health outcomes. For example Rogerson et al. [9] identify four studies where infections detected by PCR but not by co-RDT were not associated with poorer outcomes in infants [32‐35], and only one study in which infections detected by PCR and missed by microscopy were associated with LBW or prematurity [10], while a meta-analysis linked the presence of infections below co-RDT detection with clinical impacts [7]. More studies linking the use of more sensitive diagnostic tests with clinical outcomes are required.

Using HS-RDT in a similar manner to co-RDTs is supported, for example, using HS-RDT instead of co-RDT at ANC visits to assess community malaria transmission will provide more accurate prevalence estimates. However, other factors to consider in procurement include that HS-RDT has limited temperature stability and shelf-life claims compared to most co-RDT, and the costs of tests. Both tests will be limited in impact by the emergence of hrp2/hrp3 deletion parasites that escape detection [30].

Overall, the studies confirmed that the HS-RDT has a slightly higher analytical sensitivity than co-RDTs in various MiP epidemiological contexts. The use of the HS-RDT could be recommended in all cases where co-RDTs are currently used in ANC settings, although factors other than analytical sensitivity must be weighed in each context.