Malaria in pregnancy (MiP) is a public health concern in sub-Saharan Africa (sSA). In 2023, around 12.4 million pregnant women were exposed to malaria in this region [1]. In addition, MiP due to Plasmodium (P.) falciparum is one of the leading causes of maternal anemia, low birth weight, and fetal growth restriction, which are significant risk factors for neonatal and infant morbidity and mortality [1,2].

Today, intermittent preventive treatment in pregnancy with sulfadoxine/pyrimethamine (IPTp-SP) is a cornerstone of the World Health Organization’s (WHO) recommended regimen for preventing MiP, and pregnant women have the opportunity to benefit from at least three doses of SP during scheduled ANC visits [3]. However, the low IPTp-SP coverage in most sSA countries [4] and the widespread resistance of P. falciparum to SP [5,6] may jeopardize the effectiveness of this strategy. Therefore, new alternative strategies to IPTp-SP are urgently needed.

A possible alternative strategy to IPTp-SP is the intermittent screening of malaria infection and treatment during pregnancy (ISTp) [7,8]. It involves intermittent antenatal screening for malaria with RDTs and treating women with positive RDT results with antimalarials. Although the WHO does not currently recommend ISTp as an alternative strategy to IPTp-SP [4], it is being implemented in settings of low-level malaria transmission [9]. With widespread reductions in malaria transmission [10] and increasing resistance to SP [5,6], ISTp has the potential to offer an alternative to IPTp-SP for the prevention of MiP in malaria-endemic settings, including Burkina Faso. However, there are concerns regarding the ISTp approach because the conventional RDTs (cRDTs) used to screen for malaria infection have a detection limit of 100 parasites/µL and are not designed to detect low-density infections (subpatent infections) in asymptomatic pregnant women [8,9,11]. Therefore, low-cost and field-deployable highly sensitive RDTs (hsRDTs) that may improve the detection of malaria infection in these populations are needed.

Recently, a P. falciparum histidine-rich protein 2 (PfHRP2)-based hsRDT (NxTek™ Eliminate Malaria Ag Pf) was prequalified by the WHO [12] and is a promising diagnosis tool as part of the ISTp strategy. Indeed, the hsRDT has an analytical sensitivity (i.e., detection threshold) ten times higher than cRDTs [13]. Under field conditions, it consistently outperformed the cRDT and microscopy in pregnant women from low, moderate, and high malaria transmission areas [13,14,15,16,17,18]. However, there are limited data on its performance in pregnant women, particularly in SSA [13,17,18].

Malaria is a serious public health problem in Burkina Faso, with pregnant women being one of the most high-risk groups [19]. The prevalence of falciparum malaria among pregnant women attending ANC visits was 18%, and IPTp-SP has been the main strategy used for MiP prevention since 2005 [20]. The first reported case of the quintuple mutation in the country in 2017 [21] suggests an urgent need for alternative strategies to IPTp-SP, such as ISTp. Notwithstanding, knowledge gaps still exist on the diagnostic performance of the hsRDT for detecting P. falciparum malaria infection among pregnant women. This, therefore, provided us with the singular opportunity to design the first study evaluating the performance of the hsRDT in pregnant women from Burkina Faso.

To our knowledge, this is the first study to evaluate the performance of the hsRDT in detecting P. falciparum infection in pregnant women in Burkina Faso. The sensitivity of the hsRDT is similar to that of the cRDT but better than that of microscopy. In our study, the prevalence of falciparum malaria infection in pregnant women using qPCR (59.72%) was higher than that reported in Colombia (4.5%) [15], Ghana (10.54%) [31], Benin (25.49%) [13], Kenya (35.7%) [17], and Democratic Republic of Congo (DRC) (52.2%) [18]. An explanation for this observation could be a difference in study settings and the sensitivity of the molecular reference standards used. In addition, the proportion of women with low-density parasitaemia (40,97%) was higher than that reported in DRC (10%), where 50% of women had benefited from antimalarial drugs, either as IPTp or ISTp [18]. Malaria prevalence was found to be 19.79% by microscopy, which is similar to our previous study conducted in the same site (18.1%) [20] but slightly lower than that recently reported among women attending ANC in Africa (21.5%) [2]. However, in the latter study, diagnostic tests, including microscopy, PCR, and RDTs, were used to detect malaria during pregnancy [2]. Further studies are warranted to identify the factors involved in the persistence of the high prevalence of malaria in pregnant women in this part of Bobo-Dioulasso city.

The sensitivity of the hsRDT in our study (51.16%) was lower than that reported in studies that recruited both febrile and afebrile pregnant women ranging from 54.7 to 88% [18,32]. However, sensitivities of 19.6% and 53.8% were reported for the hsRDT in afebrile pregnant women from Indonesia [16] and Ghana [31], respectively. This difference could be attributed to variations in study settings (with malaria transmission intensities ranging from low to high), disease status (febrile versus afebrile), source of the samples used (fresh blood versus thawed blood), parasite density, and the reference test used [18,32].

The sensitivity of the hsRDT is similar to that of the cRDT in our study. This finding is consistent with that of Acquah et al., who showed that the hsRDT and cRDT had the same sensitivity of 53.8% in pregnant women from Ghana [31]. A plausible explanation for this observation could be that the cRDTs used had a lower detection limit than expected [32]. However, the hsRDT showed a slightly higher sensitivity than the cRDT in most studies conducted among pregnant women. Still, this difference was only statistically significant in the study conducted in Benin [32].

Both the hsRDT and the cRDT did detect high proportions of false negatives (48.84% versus 49.42%) when compared to qPCR. False negatives may be caused by certain factors, including low PfHRP2 concentrations [13] and the prozone effect [33]. Low levels of PfHRP2 may be due to low parasitaemia, the degradation of PfHRP2 during storage, or deletions of the Pfhrp2 and Pfhrp3 genes [1,16,34]. Thus, the high proportions of false negatives observed in our study could be attributed to either low parasitemia or the circulation of malaria parasites carrying the Pfhrp2 and Pfhrp3 gene deletions. Indeed, for parasite density ˂ 100 p/µL, the two RDTs had the same sensitivity of 28.81% but lower than those obtained for parasite density ≥ 100 p/µL. In addition, our results highlight the need to update the prevalence of Pfhrp2 and Pfhrp3 gene deletions, as malaria parasites harboring these mutations are circulating in regions neighboring Bobo-Dioulasso city [35].

The sensitivity of the hsRDT and cRDT was higher in afebrile-infected participants who carried high-density infections (median = 7.51 p/μL) compared to febrile women. This is likely because high-density infections among afebrile individuals may result in a high concentration of PfHRP2 detected by both RDTs [12]. However, a recent review reported a high sensitivity of both tests among febrile women compared to afebrile ones, as was expected [32]. Indeed, those previous studies included a higher proportion of febrile women (ranging from 8.6 to 18.7%) [32] compared to our study (3.83%).

Our study did not find any trend of decreasing the hsRDT and cRDT sensitivity with increasing gravidity, as previously reported in Colombia [15], DRC [18], and Kenya [17]. Indeed, parasite densities tended to be higher in primigravida participants than in women with one or more previous pregnancies, although the difference was not significant.

The hsRDT and cRDT performed better among second-trimester women than first-trimester participants. This is consistent with the findings from high-transmission settings where both RDTs were more sensitive in later trimesters, although this difference was not significant [13,17]. However, in low-transmission settings, the highest sensitivity was observed in first-trimester women, but with no statistical difference [14,15]. This difference could be attributable to the lack of a clear pattern in the distribution of parasite densities by trimester [17].

The hsRDT, cRDT, and microscopy were highly specific and showed comparable specificity greater than 98%. The specificity of the hsRDT in our study (98.28%) was similar to that reported in Colombia and Indonesia, ranging from 98.2 to 99.9% [14,15,16], but higher than that found in Benin (93.5%) [13], DRC (94.4%) [18], and Kenya (95.8%) [17]. The lower specificity of the hsRDT observed in the latter studies could be attributed to false positive results that can be caused by several factors, including the persistence of PfHRP2 in the blood for a while after the clearance of parasitaemia [36], cross-reactivity with rheumatoid factors [37], or inflammatory syndrome [38].

We acknowledge some limitations of this study. Firstly, we could not measure the concentration of PfHRP2 in the blood samples collected due to a limited budget. Second, the small number of women in the third trimester did not allow us to estimate the performance of the tests in this group. Finally, the ultrasensitive qPCR used in our study may have overestimated parasite density due to variable numbers of copies of the amplification target and persistence of nucleic acid from non-viable parasites.

The sensitivity of the hsRDT is similar to that of the cRDT but better than that of microscopy. The hsRDT, cRDT, and microscopy are highly specific with similar values. There was a moderate agreement between RDTs and qPCR, whereas microscopy showed a fair agreement with the qPCR results. Further studies on the performance of the hsRDT, including the measurement of PfHRP2 concentration and assessment of the Pfhrp2 and Pfhrp3 gene deletions, particularly among false negatives, are warranted to better guide recommendations on its use for malaria control and elimination.