Abstract
Background
Recent reports documented catastrophic sensitivity failures (18%) of Abbott-Bioline™ Malaria Ag Pf/Pv rapid diagnostic tests (RDTs) at the Thailand-Myanmar border, prompting WHO to issue an information notice on quality concerns. We evaluated the same RDT lots under controlled laboratory conditions and in a high-transmission field setting in Madagascar to assess whether performance varied across epidemiological contexts.
Methods
Laboratory evaluation tested four Abbott-Bioline™ Malaria Ag Pf/Pv lots (05DDI018BH, 05DDI020BA, 05DDI041AB, 05DDI040AA) using serial dilutions of cultured Plasmodium falciparum (0–60,784 parasites/µL). Field evaluation enrolled 218 consecutive febrile patients at Ranomafana health centre, southeastern Madagascar. Performance of both RDTs (Abbott-Bioline™ Malaria Ag Pf/Pv and Parascreen® Malaria Ag Pf/Pan) was assessed against microscopy and real-time PCR as reference standards.
Results
In laboratory testing, substantial inter-lot variability was observed, with detection failures occurring between 97 and 373 parasites/µL. Incomplete blood migration and faint test lines were noted at lower densities. In the field, malaria prevalence was 70.2% by PCR and 48.6% by microscopy. Against microscopy, Abbott-Bioline™ Malaria Ag Pf/Pv achieved sensitivity of 99.1% (95% CI 94.9–100) and specificity of 93.7% (95% CI 87.4–97.4). Parascreen® Malaria Ag Pf/Pan showed sensitivity of 100% (95% CI 96.6–100) and specificity of 92.8% (95% CI 86.3–96.8). Against PCR, sensitivity decreased to 73.2% for Abbott-Bioline™ Malaria Ag Pf/Pv and 74.5% for Parascreen® Malaria Ag Pf/Pan, while specificity remained 98.5% for both tests. No significant difference was observed between RDTs (p > 0.05).
Conclusions
Despite using identical lots that showed 18% sensitivity at the Thailand-Myanmar border, Abbott-Bioline™ Malaria Ag Pf/Pv RDTs achieved 99.1% sensitivity in Madagascar. This difference likely reflects higher parasite densities in the Malagasy high-transmission setting (geometric mean 10,006 parasites/µL) compared to low-transmission elimination contexts. Both RDTs met WHO performance thresholds against microscopy but missed approximately 25% of PCR-positive infections. These findings demonstrate that RDT performance is highly context-dependent and underscore the need for enhanced post-deployment surveillance.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12936-026-05869-1.
Keywords: Malaria, Rapid diagnostic test, HRP2, Diagnostic accuracy, Abbott-Bioline™ Malaria Ag Pf/Pv, Quality control, Madagascar
Background
Malaria remains a major public health challenge globally, with an estimated 282 million cases and 610,000 deaths in 2024 [1]. In Madagascar, the malaria burden has escalated sharply, with an estimated 6.2 million cases (range: 4–8.7 million) and 16,000 deaths (range: 6,200–29,900) in 2024. Between 2023 and 2024, the country experienced a dramatic 27.7% increase in incidence, contributing an additional 1.9 million cases and ranking among the three countries (with Ethiopia and Yemen) accounting for 58% of the global case increase. Extreme climate events have disrupted health services and intervention delivery, further exacerbating this surge [1]. Of the 31.2 million population, 88% (27.4 million) live in high-transmission areas (> 1 case per 1,000 population), where Plasmodium falciparum dominates, transmitted by Anopheles funestus s.s., An. gambiae s.l., An. arabiensis, and An. mascarensis [2]. Transmission patterns vary spatially, from perennial transmission in coastal lowlands to epidemic-prone seasonal patterns in highland areas, highlighting the need for context-specific diagnostic tools.
Prompt and accurate diagnosis is essential for reducing malaria morbidity and mortality while preventing inappropriate antimalarial use. Since 2010, WHO has recommended parasitological confirmation of all suspected cases before treatment [3]. While microscopy remains the reference standard when performed by trained personnel, its capacity is limited in peripheral settings due to equipment and staffing constraints. Rapid diagnostic tests (RDTs) have thus substantially improved case management in remote areas due to their simplicity and rapid turnaround time [4, 5]. However, despite widespread deployment and WHO prequalification, recent field investigations have revealed alarming batch-specific performance failures, particularly for Abbott-Bioline™ Malaria Ag Pf/Pv RDTs in the Greater Mekong Subregion.
Aung et al. reported sensitivity of only 18% for P. falciparum and 44% for P. vivax at the Thailand-Myanmar border, with missed infections in febrile patients ranging from 512 to 75,360 parasites/µL [6]. These findings were independently confirmed by Thein et al. during a P. vivax outbreak in Northwest Myanmar, where the Abbott-Bioline™ Malaria Ag Pf/Pv RDT exhibited a similarly poor sensitivity of only 24% overall and 39% at parasite densities ≥ 200/µL, the WHO-recommended detection threshold [7]. This consistency across different epidemiological settings underscores a systemic issue with these RDT lots, which has received broad attention, including a Science news report detailing the accumulating evidence and institutional responses [8]. In response, the WHO Global Malaria Programme issued an internal memorandum (30 April 2025) identifying 11 affected lots, acknowledging that reports from ‘diverse field settings and experienced users’ indicated the issue was unrelated to user error [7, 8]. These findings expose critical gaps in RDT quality assurance, as prequalification testing of selected lots fails to guarantee sustained field performance across all manufacturing batches. This underscores the need for systematic post-market surveillance, combining laboratory assessment with field validation.
To address these critical quality concerns, we conducted a comprehensive evaluation of Abbott-Bioline™ Malaria Ag Pf/Pv RDT performance using the same lot numbers as those reported by Aung et al. [6], along with a comparative assessment against Parascreen® Malaria Ag Pf/Pan in Madagascar field conditions. The primary objective was to evaluate diagnostic performance of both RDTs against reference standards and determine whether identical Abbott-Bioline™ Malaria Ag Pf/Pv lots could maintain acceptable performance in different epidemiological contexts. Secondary objectives included characterizing inter-lot variability in laboratory conditions, a key factor potentially explaining the discrepancies observed in recent field studies [6, 7].
Materials and methods
Study design
This prospective diagnostic evaluation comprised two complementary phases: controlled laboratory assessment of Abbott-Bioline™ Malaria Ag Pf/Pv RDT lot-specific performance, and field validation comparing two RDT (Abbott-Bioline™ Malaria Ag Pf/Pv RDT and Parascreen® Malaria Ag Pf/Pan) against reference standards in a Madagascar clinical setting. The laboratory evaluation was conducted to assess inter-lot variability in detection thresholds, while the field study evaluated real-world diagnostic performance under operational conditions.
Laboratory evaluation
Setting and materials
Laboratory evaluation was conducted at the University of Strasbourg, France. Four Abbott-Bioline™ Malaria Ag Pf/Pv lots (05DDI018BH, 05DDI020BA, 05DDI041AB, 05DDI040AA) were assessed using cultured Plasmodium falciparum parasites (strain 3601, collected in Pailin, Cambodia, 2010). These lots were identical to those reported by Aung et al. in their Thailand-Myanmar evaluation [6].
Serial dilution protocol
Parasite density was quantified by flow cytometry using SYBR Green I DNA staining [9]. Nine serial dilutions were prepared ranging from 0 to 60,784 parasites/µL (0, 19, 49, 97, 194, 373, 1,621, 5,708, and 60,784 parasites/µL). Five replicate RDTs were tested for each concentration per lot, totaling 180 tests allocated for laboratory evaluation.
Performance assessment
All tests were performed strictly following manufacturer instructions for use, including the use of 5 µL blood (50% hematocrit), addition of four drops of assay diluent, and result interpretation within the specified reading time (15–20 min) [10]. Test line intensity was scored using a standardized 5-point scale: 0 = no visible line (negative), 1 = very faint line (barely visible), 2 = faint line (clearly visible), 3 = moderate intensity (equal to control line), 4 = strong intensity (exceeding control line). Scoring was performed by two independent readers blinded to parasite concentration and lot identification. RDTs were stored at 4 °C according to manufacturer specifications, with continuous temperature monitoring.
Field Evaluation
Setting and study population
Field evaluation was conducted at Ranomafana Health Center, southeastern Madagascar (21°15′S, 47°27′E, altitude 876 m), from February 20–28, 2025 (Fig. 1). This area represents stable, high-intensity malaria transmission with year-round Plasmodium falciparum predominance. Consecutive patients presenting with fever (≥ 38 °C) or fever history within 48 h were enrolled. Exclusion criteria included refusal to participate and previous enrollment during the study period.
Fig. 1.
Geographic location of Ranomafana health center, Madagascar. Map showing the location of Ranomafana Health Center in southeastern Madagascar, where field evaluation of RDTs was conducted. Inset shows Madagascar's position relative to the African continent and Indian Ocean
Sample size and clinical procedures
A total of 218 patients were enrolled during the nine-day study period, determined by logistical constraints corresponding to available Abbott-Bioline™ Malaria Ag Pf/Pv RDT quantities. Capillary blood collection was performed for RDTs testing, microscopy slide preparation, and dried blood spot (DBS) collection for PCR analysis.
RDT testing procedures
RDT specifications. Two RDT were evaluated: Parascreen® Malaria Ag Pf/Pan (Zephyr Biomedicals, India; lots 101,612 and 101,675) used in routine at the health center and Abbott-Bioline™ Malaria Ag Pf/Pv (Abbott Diagnostics, Republic of Korea; lots 05DDI018BH, 05DDI020BA, 05DDI041AB, 05DDI040AA). Parascreen® Malaria Ag Pf/Pan detects HRP2 and pan-specific LDH antigens, while Abbott-Bioline™ Malaria Ag Pf/Pv detects HRP2 and P. vivax-specific LDH antigens.
Testing Protocol. All RDTs were performed according to manufacturer instructions by trained technicians blinded to other diagnostic results. Reading times were 20 min for Parascreen® Malaria Ag Pf/Pan and 15 min for Abbott-Bioline™ Malaria Ag Pf/Pv. RDTs were stored at 4 °C. All tests were used within expiration dates. Technical characteristics of both RDTs are summarized in Supplementary Table S1.
Reference methods
Microscopy. Thick and thin blood films were prepared and stained with 10% Giemsa solution. Microscopic examination was performed by two experienced microscopists blinded to RDTs and PCR results. Parasite density quantification followed WHO standard procedures: parasites/µL = (parasites counted × 8,000)/white blood cells counted for thick smears, and parasites/µL = (parasitized RBCs × 5,000,000)/(20 fields × 250 RBCs) for thin smears. Slides were declared negative after examination of 500 microscopic fields. Discordant results were resolved by a third senior microscopist.
All positive microscopy slides were reviewed by a WHO-certified microscopist from the National Malaria Control Program (NMCP) laboratory. Discordant results underwent examination by a second expert microscopist, with persistent discrepancies resolved by a third impartial reader.
PCR analysis. DNA extraction from DBS was performed using QIAamp 96 DNA Blood Kit (Hilden, Germany) following manufacturer protocols. Molecular detection employed a two-step real-time PCR approach targeting mitochondrial cytochrome b gene for Plasmodium genus screening, followed by species-specific PCR with external amplification for P. falciparum, P. vivax, P. malariae, and P. ovale identification [11]. Primers used for genus screening were RTPCRScreening2_F (5'-tggagtggatggtgttttaga-3') and RTPCRScreening2_R (5'-ttgcaccccaatarctcattt-3'). Nested real-time PCR used RTPCRScreening2_F and RTPCRSreening3_R (5'-accctaaaggatttgtgctacc-3'). Species-specific primers were: P. falciparum: Pf_RTPCR_F (5'-atggatatctggattgattttatttatga-3') and Pf_RTPCR_R (5'-tcctccacatatccaaattactgc-3'); P. vivax: Pv_RTPCR_F (5'-tgctacaggtgcatctcttgtattc-3') and Pv_RTPCR_R (5'-atttgtccccaaggtaaaacg-3'); P. malariae: Pm_RTPCR_F (5'-acaggtgcatcacttgtattttttc-3') and Pm_RTPCR_R (5'-tgctggaattgaagataataaattagtaataact-3'); P. ovale: Po_RTPCR_F (5'-gttatatggttatgtggaggatatactgtt-3') and Po_RTPCR_R (5'-cgaatggaagaataaaatgtagtacg-3'). Melt curve analysis confirmed amplification specificity. Quality controls were integrated throughout all analytical procedures, including negative controls at both extraction and PCR steps, and positive controls for each Plasmodium species sourced from an in-house biobank (WHO quality controls, UKNEQAS).
Statistical analysis
Data were analyzed using RStudio Desktop version 2025.05.0–496. Agreement between diagnostic methods was assessed using Cohen's kappa coefficient (κ). Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and corresponding 95% confidence intervals were calculated using the Wilson score method. McNemar tests were used to compare paired differences between RDTs on the same samples. Chi-square tests compared RDT performance across patient subgroups. Inter-lot variability in laboratory evaluation was assessed using one-way ANOVA. Statistical significance was set at p ≤ 0.05.
Ethical considerations
Participation was voluntary with written informed consent obtained from participants or guardians for minors. Participant data confidentiality was maintained throughout. All malaria-positive cases received immediate treatment according to national guidelines (artesunate-amodiaquine). The study was approved by the National Malagasy Ethical committee (N.44 MSANP/SG/AMM/CERBM, March, 11, 2024).
Results
Laboratory evaluation of Abbott-Bioline™ Malaria Ag Pf/Pv RDT performance.
RDT lot characteristics
Four Abbott-Bioline™ Malaria Ag Pf/Pv RDT lots were evaluated: 05DDI018BH, 05DDI020BA, 05DDI041AB, and 05DDI040AA (Table 1). These lots are identical to those reported by Aung et al. [6]. A total of 180 RDTs were tested using P. falciparum serial dilutions (strain 3601), with 220 additional tests allocated for field evaluation in Madagascar.
Table 1.
Abbott-Bioline™ Malaria Ag Pf/Pv RDT lot specifications
| Lot Number | Manufacturing Date | Expiry Date | Quantity Tested | Storage Conditions | Temperature Range |
|---|---|---|---|---|---|
| 05DDI018BH | 25/05/2023 | 23/05/2025 | 100 tests | 2–30 °C, | 18–25 °C monitored |
| 05DDI020BA | 28/06/2023 | 23/05/2025 | 100 tests | 2–30 °C, | 18–25 °C monitored |
| 05DDI041AB | 19/02/2024 | 17/02/2026 | 100 tests | 2–30 °C, | 18–25 °C monitored |
| 05DDI040AA | 16/02/2024 | 14/02/2026 | 100 tests | 2–30 °C, | 18–25 °C monitored |
Detection threshold performance by lot
Significant inter-lot variability was observed across the four Abbott-Bioline™ Malaria Ag Pf/Pv RDT lots. Line intensity decreased rapidly with declining parasite density across all lots. At 60,784 parasites/µL, all lots achieved maximum intensity (score 4). At 5,708 parasites/µL, all lots scored 3. At 1,621 parasites/µL, lot-specific variations emerged: lot 05DDI041AB showed 2/5 tests scoring 1, lot 05DDI040AA had 1/5 scoring 1, and lot 05DDI018BH had 3/5 scoring 1. At 373 parasites/µL, all lots showed at least one negative: 05DDI020BA (1/5), 05DDI041AB (1/5), 05DDI040AA (1/5), and 05DDI018BH (4/5). At 194 parasites/µL: 05DDI020BA 2/5 positive, 05DDI041AB 3/5 positive, 05DDI040AA 3/5 positive, 05DDI018BH 0/4 positive. At 97 parasites/µL, only lot 05DDI041AB produced one positive result (1/5) (Table 2, Fig. 2).
Table 2.
Abbott-Bioline™ Malaria Ag Pf/Pv RDT Performance by Lot and Parasite Density
| Lot | Diluant | Parasites/µL | RDT1 | RDT2 | RDT3 | RDT4 | RDT5 |
|---|---|---|---|---|---|---|---|
| 05DDI020BA | 05BDDI065 | 60,784 | 4 | 4 | 4 | 4 | 4 |
| 05DDI020BA | 05BDDI065 | 5708 | 3 | 3 | 3 | 3 | 3 |
| 05DDI020BA | 05BDDI065 | 1621 | 2 | 2 | 2 | 2 | 2 |
| 05DDI020BA | 05BDDI065 | 373 | 1 | 1 | 1 | 1 | 0 |
| 05DDI020BA | 05BDDI065 | 194 | 1 | 0 | 0 | 0 | 1 |
| 05DDI020BA | 05BDDI065 | 97 | 0 | 0 | 0 | 0 | 0 |
| 05DDI020BA | 05BDDI065 | 49 | 0 | 0 | 0 | 0 | 0 |
| 05DDI020BA | 05BDDI065 | 19 | 0 | 0 | 0 | 0 | 0 |
| 05DDI020BA | 05BDDI065 | 0 | 0 | 0 | 0 | 0 | 0 |
| 05DDI041AB | 05BDDJ013 | 60,784 | 4 | 4 | 4 | 4 | 4 |
| 05DDI041AB | 05BDDJ013 | 5708 | 3 | 3 | 3 | 3 | 3 |
| 05DDI041AB | 05BDDJ013 | 1621 | 2 | 2 | 1 | 1 | 2 |
| 05DDI041AB | 05BDDJ013 | 373 | 1 | 1 | 1 | 1 | 0 |
| 05DDI041AB | 05BDDJ013 | 194 | 1 | 1 | 0 | 1 | 0 |
| 05DDI041AB | 05BDDJ013 | 97 | 0 | 0 | 0 | 1 | 0 |
| 05DDI041AB | 05BDDJ013 | 49 | 0 | 0 | 0 | 0 | 0 |
| 05DDI041AB | 05BDDJ013 | 19 | 0 | 0 | 0 | 0 | 0 |
| 05DDI041AB | 05BDDJ013 | 0 | 0 | 0 | 0 | 0 | 0 |
| 05DDI040AA | 05BDDJ001 | 60,784 | 4 | 4 | 4 | 4 | 4 |
| 05DDI040AA | 05BDDJ001 | 5708 | 3 | 3 | 3 | 3 | 3 |
| 05DDI040AA | 05BDDJ001 | 1621 | 2 | 2 | 2 | 2 | 1 |
| 05DDI040AA | 05BDDJ001 | 373 | 1 | 1 | 1 | 1 | 0 |
| 05DDI040AA | 05BDDJ001 | 194 | 1 | 1 | 0 | 0 | 1 |
| 05DDI040AA | 05BDDJ001 | 97 | 0 | 0 | 0 | 0 | 0 |
| 05DDI040AA | 05BDDJ001 | 49 | 0 | 0 | 0 | 0 | 0 |
| 05DDI040AA | 05BDDJ001 | 19 | 0 | 0 | 0 | 0 | 0 |
| 05DDI040AA | 05BDDJ001 | 0 | 0 | 0 | 0 | 0 | 0 |
| 05DDI018BH | 05BDDI070 | 60,784 | 4 | 4 | 4 | 4 | 4 |
| 05DDI018BH | 05BDDI070 | 5708 | 3 | 3 | 3 | 3 | 3 |
| 05DDI018BH | 05BDDI070 | 1621 | 2 | 2 | 1 | 1 | 1 |
| 05DDI018BH | 05BDDI070 | 373 | 0 | 0 | 1 | 0 | 0 |
| 05DDI018BH | 05BDDI070 | 194 | 0 | 0 | 0 | 0 | x |
| 05DDI018BH | 05BDDI070 | 97 | 0 | 0 | 0 | 0 | 0 |
| 05DDI018BH | 05BDDI070 | 49 | 0 | 0 | 0 | 0 | 0 |
| 05DDI018BH | 05BDDI070 | 19 | 0 | 0 | 0 | 0 | 0 |
| 05DDI018BH | 05BDDI070 | 0 | 0 | 0 | 0 | 0 | 0 |
Individual RDT performance across four Abbott-Bioline™ Malaria Ag Pf/Pv lots at different parasite concentrations
Scoring system: 4 = Strong intensity line (exceeding control line), 3 = Moderate intensity line (equal to control line), 2 = Faint line (clearly visible), 1 = Very faint line (barely visible), 0 = No visible line (negative), x = Invalid result (migration failure or technical issue)
Fig. 2.
Detection threshold performance curves by Abbott-Bioline™ Malaria Ag Pf/Pv RDT Lot. Detection performance curves showing mean scoring intensity versus parasite density for four Abbott-Bioline™ Malaria Ag Pf/Pv lots. Each curve represents identical RDT brand and model but different manufacturing lots. Each data point represents the mean score of 5 replicate tests per concentration per lot. X-axis: parasite density concentrations (0, 19, 49, 97, 194, 373, 1,621, 5,708, and 60,784 parasites/µL). Y-axis: Scoring scale: 0 = No visible line (negative), 1 = Very faint line (barely visible), 2 = Faint line (clearly visible), 3 = Moderate intensity (equal to control line), 4 = Strong intensity (exceeding control line). Each curve represents identical RDT brand and model but different manufacturing lots
Migration defects were frequently observed, with incomplete blood migration leaving red background that cleared after 30 min in most cases (Supplementary materials).
Field validation. Clinical performance at health center in Madagascar
Study population characteristics
A total of 218 patients presenting with suspected malaria were enrolled consecutively during the nine-day study period. The median age was 28 years (IQR: 15–45), with pediatric patients (< 15 years) comprising 50.4% (110/218) of the study population. Gender distribution was approximately equal (52.3% female, 47.7% male).
Fever was the predominant symptom, documented in 89.4% (195/218) of patients at enrollment, with 76.1% reporting fever onset within 48 h of presentation. The symptom profile included: headache (84.9%), chills (72.5%), body aches (68.8%), and nausea/vomiting (45.4%) were commonly reported. Treatment history revealed that 15.6% (34/218) of patients had taken antimalarial medication within the previous 30 days, predominantly artemether-lumefantrine. No patients reported severe malaria symptoms.
Parasite densities at enrollment were high overall, with a geometric mean of 10,006 parasites/µL (95% CI: 7,167–13,970), a median of 14,698 parasites/µL, and a wide range from 166 to 348,800 parasites/µL (Table 3).
Table 3.
Patient Demographics and Clinical Characteristics. Baseline characteristics of 218 consecutive patients with suspected malaria enrolled at Ranomafana Health Center, Madagascar, during February 20–28, 2025
| Variables | Number (%) |
|---|---|
| Gender | |
| Male | 117 (53.7) |
| Female | 101 (46.3) |
| Age group (years) | |
| < 5 | 40 (18.3) |
| 5—15 | 70 (32.1) |
| ≥ 15 | 108 (49.6) |
| Clinical presentation | |
| Fever documented | 195 (89.4) |
| Fever onset < 48 h | 166 (76.1) |
| Headache | 185 (84.9) |
| Chills | 158 (72.5) |
| Body aches | 150 (68.8) |
| Nausea/vomiting | 99 (45.4) |
| Treatment history | |
| Recent antimalarials (< 30 days) | 34 (15.6) |
| Parasite density (/µL) | |
| Geometric mean (95%IC) | 10,006 (7,167–13,970) |
| Range | 166–348,800 |
| Median | 14,698 |
Diagnostic performance against reference standards (microscopy and PCR)
Among the 218 samples tested, prevalence varied by diagnostic method. PCR detected the highest proportion of infections at 70.2% (153/218), followed by Parascreen® Malaria Ag Pf/Pan at 52.8% (115/218), Abbott-Bioline™ Malaria Ag Pf/Pv at 51.8% (113/218), and microscopy at 48.6% (106/218). A total of 106 patients (48.6%) tested positive across all four diagnostic methods.
Species distribution analysis revealed P. falciparum predominance across all methods (98%, 150/153 P. falciparum and 2.0%, 3/150 P. vivax). PCR and microscopy detected three cases of P. vivax infections, all of which were also identified by Parascreen® Malaria Ag Pf/Pan, while Abbott-Bioline™ Malaria Ag Pf/Pv detected only two of these P. vivax cases. No discordant cases in species identification were observed between RDTs, microscopy, and PCR.
Performance against microscopy. Concordance analysis showed 96.3% agreement between both RDTs and microscopy. Parascreen® Malaria Ag Pf/Pan achieved sensitivity of 100% (95% CI: 96.6–100) with specificity of 92.8% (95% CI: 86.3–96.8), recording 8 false positives and no false negatives (κ = 0.93). Abbott-Bioline™ Malaria Ag Pf/Pv demonstrated sensitivity of 99.1% (95% CI: 94.9–100) and specificity of 93.7% (95% CI: 87.4–97.4), with 7 false positives and 1 false negative (κ = 0.93). The single missed case by Abbott-Bioline™ Malaria Ag Pf/Pv had parasitemia of 1,847 parasites/µL in the 1,001–5,000 parasites/µL density range. Statistical comparison revealed no significant difference between the two RDTs for any performance metric (p > 0.05) (Table 4).
Table 4.
Concordance between RDT results and microscopy reference standard
| Microscopic examination | |||
|---|---|---|---|
| Parascreen® Malaria Ag Pf/Pan | Positive n (%) | Negative n (%) | Total |
| Positive | 107 (49.1) | 8 (3.7) | 115 |
| Negative | 0 (0) | 103 (47.2) | 103 |
| Total | 107 | 111 | 218 |
| Abbott-Bioline™ Malaria Ag Pf/Pv | Positive n (%) | Negative n (%) | Total |
| Positive | 106 (48.6) | 7 (3.2) | 113 |
| Negative | 1 (0.5) | 104 (47.7) | 105 |
| Total | 107 | 111 | 218 |
Cross-tabulation of RDT results against microscopy findings for 218 patients with suspected malaria at Ranomafana Health Center, Madagascar (February 2025)
Numbers represent absolute counts with percentages of total study population (n = 218) in parentheses. Parascreen® Malaria Ag Pf/Pan κ = 0.93, Abbott-Bioline™ Malaria Ag Pf/Pv κ = 0.93
Performance against PCR. Parascreen® Malaria Ag Pf/Pan showed 81.7% concordance.
(178/218, κ = 0.63) with PCR, achieving sensitivity of 74.5% (95% CI: 66.8–81.2) and specificity of 98.5% (95% CI: 91.7–100), with 1 false positive and 39 false negatives (all P. falciparum). Abbott-Bioline™ Malaria Ag Pf/Pv demonstrated 80.7% concordance (176/218, κ = 0.61) with PCR, with sensitivity of 73.2% (95% CI: 65.5–80) and specificity of 98.5% (95% CI: 91.7–100), recording 1 false positive and 41 false negatives (40 P. falciparum, 1 P. vivax). No statistically significant differences were observed between RDTs (p > 0.05) (Table 5).
Table 5.
Concordance between RDT results and PCR reference standard
| PCR | |||
|---|---|---|---|
| Parascreen® Malaria Ag Pf/Pan | Positive n (%) | Negative n (%) | Total |
| Positive | 114 (52.3) | 1 (0.5) | 115 |
| Negative | 39 (17.9) | 64 (29.4) | 103 |
| Total | 153 | 65 | 218 |
| Abbott-Bioline™ Malaria Ag Pf/Pv | Positive n (%) | Negative n (%) | Total |
| Positive | 112 (51.4) | 1 (0.5) | 113 |
| Negative | 41 (18.8) | 64 (29.4) | 105 |
| Total | 153 | 65 | 218 |
Cross-tabulation of RDT results against PCR findings for 218 patients with suspected malaria at Ranomafana Health Center, Madagascar (February 2025)
Numbers represent absolute counts with percentages of total study population (n = 218) in parentheses
Parasite density stratification. Against microscopy, Parascreen® Malaria Ag Pf/Pan maintained 100% sensitivity across all parasite density categories. Abbott-Bioline™ Malaria Ag Pf/Pv achieved 100% sensitivity in most categories except the 1,001–5,000 parasites/µL range (95.5% sensitivity). For submicroscopic infections (PCR-positive, microscopy-negative), both RDTs showed reduced sensitivity: 15.2% for Parascreen® Malaria Ag Pf/Pan and 13.0% for Abbott-Bioline™ Malaria Ag Pf/Pv.
Predictive values. Against microscopy, positive predictive values were 93% for Parascreen® Malaria Ag Pf/Pan and 93.8% for Abbott-Bioline™ Malaria Ag Pf/Pv, with negative predictive values of 100% and 99% respectively. Against PCR, positive predictive values were 99.1% for both RDTs, while negative predictive values were 62.1% (Parascreen® Malaria Ag Pf/Pan) and 61% (Abbott-Bioline™ Malaria Ag Pf/Pv) (Table 6).
Table 6.
Diagnostic performance of RDT against microscopy and PCR reference standards
| RDT | Sensitivity [95% CI] |
Specificity [95% CI] | PPV [95% CI] |
NPV [95% CI] |
|---|---|---|---|---|
| Versus Microscopy | ||||
| Parascreen® Malaria Ag Pf/Pan |
100% [96.6–100] |
92.8% [86.3–96.8] |
93% [86.8–96.9] |
100% [96.5–100] |
| Abbott-Bioline™ Malaria Ag Pf/Pv |
99.1% [94.9–100] |
93.7% [87.4–97.4] |
93.8% [87.7–97.4] |
99% [94.8–100] |
| Versus PCR | ||||
| Parascreen® Malaria Ag Pf/Pan |
74.5% [66.8–81.2] |
98.5% [91.7–100] |
99.1% [95.2–100] |
62.1% [52–71.5] |
| Abbott-Bioline™ Malaria Ag Pf/Pv |
73.2% [65.5–80] |
98.5% [91.7–100] |
99.1% [95.1–100] |
61% [50.9–70.3] |
Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of Parascreen® Malaria Ag Pf/Pan and Abbott-Bioline™ Malaria Ag Pf/Pv evaluated against microscopy and PCR reference standards (n = 218). Statistical comparisons between RDTs showed no significant differences for any performance metric (McNemar test, p > 0.05)
Discussion
This study demonstrates that both Parascreen® Malaria Ag Pf/Pan and Abbott-Bioline™ Malaria Ag Pf/Pv RDTs exhibited excellent diagnostic performance in a high-transmission setting in Madagascar. The evaluation compared these RDTs to microscopy and PCR in febrile patients from a stable transmission area characterized by a semi-immune population presenting with variable parasitemia levels (range from 166 to 348,800 parasites/µL). With sensitivities of 100% for Parascreen® Malaria Ag Pf/Pan and 99.1% for Abbott-Bioline™ Malaria Ag Pf/Pv against microscopy, both diagnostic tools substantially exceeded WHO-recommended thresholds (≥ 95% sensitivity and specificity) [12]. These performances align with many African studies, including in Madagascar, that consistently report high sensitivities for HRP2-based RDTs in stable endemic areas [13–19]. The current findings fall within the upper range of these reported values, confirming the reliability of both RDTs in similar epidemiological contexts.
The slightly reduced specificity (92.8% and 93.7% for Parascreen® Malaria Ag Pf/Pan and Abbott-Bioline™ Malaria Ag Pf/Pv respectively) resulted from false positive results (8 for Parascreen® Malaria Ag Pf/Pan and 7 for Abbott-Bioline™ Malaria Ag Pf/Pv) among microscopy-negative patients. These false positives likely reflect HRP2 antigen persistence following treatment, a well-documented phenomenon [20–24]. Given that 15.6% of the study cohort had received anti-malarial treatment within the preceding 30 days, this factor potentially contributed to the observed false positives. Specificities vary according to epidemiological context, typically showing very high values in low-transmission areas (98.8–99.8%) but reduced values in high-transmission areas for HRP2 tests (79.7–80.7%) due to antigen persistence after recovery [25]. The moderate proportion of recently treated patients in this study may explain the relatively higher specificity observed for both tests compared to other high-transmission settings.
Statistical analysis revealed no significant differences between the performances of the two RDTs, suggesting that either option could be selected based on logistical and economic considerations. This performance equivalence has been observed in other African studies comparing these RDTs [13]. The single case missed by Abbott-Bioline™ Malaria Ag Pf/Pv (parasitemia of 1,847 parasites/µL) remains within the range of occasional detection failures reported for this parasite density range [26].
It should be noted that RDTs are not designed to achieve the same level of detection as PCR. They are intended to perform equivalently to expert microscopy, which remains the WHO-recommended reference for clinical diagnosis. The comparison with PCR was included to characterize the full spectrum of infections present in the study population, including submicroscopic parasitemia, and should be interpreted in this context. When PCR served as the reference standard, RDT sensitivity predictably decreased (74.5% for Parascreen® Malaria Ag Pf/Pan and 73.2% for Abbott-Bioline™ Malaria Ag Pf/Pv), while specificity remained high (98.5% for both tests). This discrepancy reveals the primary limitation of current RDTs: their inability to detect submicroscopic infections [27–31]. Previous studies have similarly observed lower and variable sensitivities (37–88%) when comparing RDTs to PCR, particularly in asymptomatic individuals with submicroscopic infections [32, 33]. The dramatic performance decline for low-density infections becomes particularly critical in malaria elimination contexts [34]. However, in high-transmission areas like Madagascar, this limitation has less impact on clinical management of symptomatic cases, where the primary diagnostic need focuses on detecting clinically relevant parasitemia [35].
An often overlooked aspect of WHO diagnostic guidance is that when malaria remains clinically suspected despite a negative RDT result and no alternative diagnosis can be identified, referral for confirmatory microscopy is explicitly recommended [36]. This recommendation is directly relevant to the context described in this manuscript and should be reinforced in settings where RDT performance may be suboptimal. The performance gap between RDTs and PCR observed in this study raises broader questions about the appropriate use of conventional RDTs in very low prevalence settings, particularly for screening asymptomatic individuals. In such contexts, where parasite densities are typically low and submicroscopic infections predominate, conventional RDTs have limited utility and their negative predictive value becomes unreliable. For malaria elimination programs, more sensitive diagnostic tools are clearly needed. The development of ultrasensitive RDTs, particularly for P. vivax where HRP2-based detection is not applicable and pLDH-based tests show reduced sensitivity at low densities, represents an unmet priority. Until such tools become available and affordable at scale, conventional RDTs should be complemented by microscopy or molecular methods in elimination settings.
This study has several limitations that should be considered when interpreting the results. The study population consisted predominantly of symptomatic patients (89.4% with documented fever), which may have biased sensitivity estimates upward as RDTs more reliably detect higher parasitemia cases. The single-site design in a high-transmission area limits generalizability to other epidemiological contexts. Evaluation of P. vivax performance was constrained by the low prevalence of this species in the sample. While storage conditions were controlled, potential variations in field handling that might affect RDT performance cannot be entirely excluded.
Laboratory evaluation identified concerning inter-lot variability for Abbott-Bioline™ Malaria Ag Pf/Pv RDTs (lots 05DDI018BH, 05DDI020BA, 05DDI041AB, and 05DDI040AA). Inter-lot differences became evident at parasitemia below 1,621 parasites/µL, with lot 05DDI018BH showing the lowest sensitivity (0/4 detection at 194 parasites/µL) and lot 05DDI041AB the highest (1/5 detection at 97 parasites/µL). These lots originated from the same 2024 production batch supplied to the Shoklo Malaria Research Unit (SMRU) that was independently evaluated by Aung et al. during their investigation of diagnostic failures at the Thai-Myanmar border [6]. Concurrently, Thein et al. evaluated a different lot (05DDI027A, expiry September 2025) during a P. vivax outbreak in northwest Myanmar (December 2024), reporting a sensitivity of only 24% among febrile patients (11/46 microscopy-confirmed cases) and 39% for parasitemia ≥ 200/µL [7]. The Madagascar cohort exhibited a geometric mean parasite density of 10,006 parasites/µL (range: 166–348,800) among microscopy-confirmed cases, with only one false-negative result at 1,847 parasites/µL. In contrast, Aung et al. conducted their evaluation in both symptomatic clinic attendees (geometric mean density: 2,498 parasites/µL) and asymptomatic survey participants (geometric mean density: 157 parasites/µL) [6]. Their findings demonstrated that these same RDT lots failed to detect 59 of 64 P. falciparum cases in asymptomatic surveys (density range: 2–12,416 parasites/µL) and 11 of 20 cases in febrile patients (density range: 512–42,320 parasites/µL), resulting in an overall sensitivity of only 18%.
All three studies documented consistent physical defects in test line appearance. The current laboratory evaluations confirmed faint/absent test lines and incomplete blood migration previously documented by Aung et al. during external quality control assessments [6]. Thein et al. used ImageJ analysis to objectively confirm that visually negative RDTs produced no detectable signal despite parasitemia ranging from 200–5,200/µL, well above the WHO-recommended detection threshold of 200 parasites/µL [7]. While storage conditions were controlled in all settings, Aung et al. noted striking inter-lot variability: 88% (52/59) of positive Abbott-Bioline™ Malaria Ag Pf/Pv RDT results originated from a single lot (05DDI040AA). Controlled testing ruled out storage conditions as the explanatory factor, pointing instead to manufacturing inconsistencies [6]. The fact that Thein et al. observed nearly identical performance issues with an entirely different lot (05DDI027A) against P. vivax underscores the systemic nature of the manufacturing problem across both Plasmodium species and production batches [7]. The marked difference in field performance between the different epidemiological contexts, despite using the same RDT product, underscores how operational factors and parasite density distributions influence diagnostic accuracy.
Beyond diagnostic failure rates, Thein et al. documented severe clinical consequences in remote Myanmar areas, including five pediatric deaths from P. falciparum malaria (2022–2023) following repeatedly negative RDT results, and one medical assistant who developed severe malaria after four consecutive negative tests [7]. These findings illustrate the life-threatening implications of low-sensitivity RDTs in settings where microscopy is unavailable and referral is prohibitively difficult.
These complementary findings demonstrate that Abbott-Bioline™ Malaria Ag Pf/Pv RDT performance is highly context-dependent, maintaining acceptable sensitivity (99.1%) in high-transmission settings like Madagascar but showing dramatically reduced performance (18%−24%) in low-transmission elimination contexts as reported by Aung et al. and Thein et al. [6, 7]. The substantial inter-lot variability observed across all three studies, combined with identical lots producing vastly different field outcomes, reveals critical gaps in current WHO pre-qualification systems that evaluate selected lots under controlled conditions but fail to capture real-world performance variations [37]. This evidence underscores the urgent need for improve post-deployment monitoring and context-specific diagnostic strategies, with supplementary confirmation by microscopy or PCR warranted in elimination settings where missed diagnoses could undermine malaria control progress.
Supplementary Information
Acknowledgements
We thank all study participants for their cooperation. We acknowledge the healthcare workers and staff at Ranomafana health centre for their assistance during field data collection.
Author contributions
Study design and conceptualization: ARat and DM. Field data collection: JF and AR. Laboratory evaluation of Abbott-Bioline™ Malaria Ag Pf/Pv RDT performance: LP, AW, BC, YNGT, AM, LT, EC, P-ES. Molecular biology work: LT, EC, P-ES. Data analysis and interpretation: JF, AR, DM and AR. Manuscript drafting: AR, Arat and DM. Critical review and manuscript revision: All authors. Project supervision and coordination: DM and Arat. Funding acquisition: DM. All authors read and approved the final manuscript.
Funding
This work was supported by funding from the Université de Strasbourg (R42PHAV), the Institut Universitaire de France (IUF) under the Senior Chair program (2024–2029) and the French Government (Agence Nationale de la Recherche) Investissement d’Avenir programme, Laboratoire d’Excellence (LabEx) “French Parasitology Alliance For Health Care” (ANR-11-LABX-0024-PARAFRAP). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Availability of data and materials
Aggregated data supporting the main findings are provided in the article and supplementary materials. Individual participant data are not publicly available due to patient confidentiality and ethical committee restrictions but may be made available upon reasonable request with appropriate institutional approval.
Declarations
Ethics approval and consent to participate
This study was conducted in accordance with the Declaration of Helsinki and received approval from the National Malagasy Ethical Committee (N.44 MSANP/SG/AMM/CERBM, March, 11, 2024). Written informed consent was obtained from all adult participants prior to enrollment. For children, written informed consent was obtained from parents or legal guardians. All participants were informed that participation was voluntary and that refusal would not affect their access to healthcare. All malaria-positive cases received immediate treatment with artesunate-amodiaquine according to Madagascar national treatment guidelines, regardless of study participation.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher's Note
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Contributor Information
Didier Ménard, Email: dmenard@unistra.fr.
Arsène Ratsimbasoa, Email: a.ratsimbasoa@gmail.com.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
Aggregated data supporting the main findings are provided in the article and supplementary materials. Individual participant data are not publicly available due to patient confidentiality and ethical committee restrictions but may be made available upon reasonable request with appropriate institutional approval.