Welcome to the next generation of Malaria Rapid Diagnostic Tests: Comparative Analysis of NxTek Eliminate Malaria P.f, Biocredit Malaria Ag Pf, and SD Bioline Malaria Ag Pf for Plasmodium falciparum Diagnosis in Ghana

Abstract Background : Accurate diagnosis and timely treatment are crucial in combating malaria. Methods : We evaluated the diagnostic performance of three Rapid Diagnostic Tests (RDTs) in diagnosing febrile patients, namely: Abbott NxTek Eliminate Malaria Ag Pf (detecting HRP2), Rapigen Biocredit Malaria Ag Pf (detecting HRP2 and LDH on separate bands), and SD Bioline Malaria Ag Pf (detecting HRP2). Results were compared to qPCR. Results : Among 449 clinical patients, 45.7% (205/449) tested positive by qPCR for P. falciparum with a mean parasite density of 12.5parasites/μL. The sensitivity of the Biocredit RDT was 52.2% (107/205), NxTek RDT was 49.3% (101/205), and Bioline RDT was 40.5% (83/205). When samples with parasite densities lower than 20 parasites/uL were excluded (n=116), a sensitivity of 88.8% (79/89, NxTek), 89.9% (80/89, Biocredit), and 78.7% (70/89, Bioline) was obtained. All three RDTs demonstrated specificity above 95%. The limits of detection was 84 parasites/μL (NxTek), 56 parasites/μL (Biocredit, considering either HRP2 or LDH), and 331 parasites/μL (Bioline). None of the three qPCR-confirmed P. falciparum positive samples, identified solely through the LDH target, carried hrp2 / 3 deletions. Conclusion : The Biocredit and NxTek RDTs demonstrated comparable diagnostic efficacies and both RDTs performed better than Bioline RDT.


Background
Malaria remains a signi cant public health concern in sub-Saharan Africa, including Ghana, where an estimated 5.3 million cases and 12,500 deaths were reported in 2021 [1].Rapid and accurate diagnosis of malaria is crucial for effective treatment and control of the spread of the disease [2].Rapid diagnostic tests (RDTs) are immunochromatographic assays widely used for malaria diagnosis, particularly in resource-limited settings.RDTs are easy to use, require minimal training, and provide rapid results [3].High sensitivity of RDTs is crucial to detect low-density infections.
Numerous studies have investigated the diagnostic performance of RDTs and found varying sensitivities [5][6][7][8][9][14][15][16][17][18]].Variation in sensitivity can be as a result of differences in RDT design, characteristics of the study population (e.g.clinical vs. subclinical infections, or differences in age groups re ecting different levels of acquired immunity and thus different parasite densities), choice of the gold standard (e.g., microscopy or PCR), and differences among sample processing and PCR assays resulting in variation of the limit of detection and parasite quanti cation by qPCR [3,17,19,20,21].As a result, data on sensitivity and LOD of RDTs tested using different protocols are di cult to compare.
Here, we assess the performance of the NxTek Eliminate Malaria Ag Pf, SD Bioline Malaria Ag Pf, and Biocredit Malaria Ag Pf RDTs in diagnosing clinical patients in Ghana.The NxTek and Biocredit test are considered next-generation ultra-sensitive tests.The NxTek and Bioline have one test band for HRP2.The Biocredit Malaria Ag Pf. (LDH/HRP2) has two separate test bands for HRP2 and LDH.Having both targets as separate bands allow diagnosis in the case of hrp2/3 deletion and enables surveillance of deletion status, as samples positive for LDH but negative for HRP2/3 can be selected for molecular con rmation of deletion status.

Ethical approval
Prior to sample collection, informed written consent was obtained from each individual.For minors, assent was obtained in addition to consent obtained from legal guardians.This study was approved by the Committee on Human Research, Publications, and Ethics of the School of Medical Sciences, KNUST (CHRPE/AP/030/20), the University of Notre Dame Institutional Review Board (19-04-5321), and The Ohio State University Institutional Review Board (2020H0539).

Study site and sample collection
Samples were collected from health centers in Mankranso (6.8181° N, 1.8635° W) and Agona (6.9347° N, 1.4870° W) in the Ashanti region of Ghana.The Ashanti region has a reported malaria prevalence of 22% by microscopy [22].The samples were obtained during the rainy seasons, between August and September 2022, known to be periods of high malaria transmission [23].Blood samples (approximately 2 mL) from participants were collected in EDTA tubes, and malaria screening with the three RDTs was performed on-site.Study participants were treated as per the national guidelines by healthcare providers at the hospital.

Rapid diagnostic tests kits and testing
Three different RDT kits were compared, the RDT, NxTek Eliminate Malaria Ag Pf. ((lot no.05LDG008B), manufactured by Abbott, the Bioline Malaria Ag Pf. (lot no.05CDH037C), also manufactured by Abbott, and the Biocredit Malaria Ag Pf. (LDH/HRP2) (lot no.H052BSA002), manufactured by Rapigen.Test were conducted according to manufaturer's instructions.Tests were considered invalid and repeated if the control band was not positive.
DNA extraction, varATS qPCR, and hrp2/3 deletion typing DNA was extracted from 100 μL blood and eluted in 100 μL elution buffer using the Macherey-Nagel NucleoMag extraction kit.qPCR of the P. falciparum varATS multi-copy gene was carried out using a previously described protocol with 4 µL DNA as target resulting in a 95% limit of detection of 0.3 parasties/µL blood [24].hrp2/3 deletion typing for samples that were negative for HRP2 on RDTs but positive for LDH and that were con rmed positive by qPCR was done by multiplexed digital PCR as previously described [25].

Data analysis
We assumed a test positivity of 50% and obtained a minimum sample size of 384 required to detect a 5% difference in sensitivity with a 95% con dence level (α = 0.05, power 1 -β = 0.20) using a previously described method [26].A total of 449 clinical samples were obtained, exceeding the minimum requirement.Performance characteristics were determined for all three RDTs, and for the Biocredit RDT, the HRP2 and LDH targets were considered separately and in combination [3].
Sensitivity was calculated as the number of infections detected by an RDT divided by the number of infections detected by qPCR, and against thresholds of 2000, 200, and 20 parasites/µL (by qPCR).This was done to comparability with other studies, as different methods for sample collection, DNA extraction, and qPCR result in different sensitivities and, thus, different numbers of positive samples [21].Speci city was calculated as the proportion of negative RDTs among individuals that tested negative by qPCR.The positive predictive value (PPV) was calculated as the probability that the infection is present when the RDT is positive and parasite density is >20 parasites/ µL [27].Samples with densities of >0 to 20 parasites/µL were exluded from the calculation of NPV and PPV.This threshold was set as lowdensity infections might be incidental, i.e. likely are not the cause of febrile illness.The negative predictive value (NPV) was calculated as the probability that qPCR is negative when the RDT is negative [27].The limit of detection (LoD) was de ned as the lowest parasite density where a qPCR-positive infection would be detected with 95% probability and logistic regression analysis was conducted to determine the LoD of each RDT target.
The area under the receiver operating characteristic curve (AUC) was calculated with a nonparametric analysis using 1000 bootstrap replications.As for PPV, a threshold of 20 parasites/µL by qPCR was set to count a sample as true positive, and samples with >0 to 20 parasites/µL were exluded from the calculation.As parasite density distributions were skewed, geometric mean densities are given whenever densities are reported.CI95 stands for the 95% con dence interval.The p values to compare groups for qPCR test positivity and RDT sensitivity were calculated by Chi-square test, while Welch's ANOVA was used for parasite density.

Study population demographics
A total of 449 clinical samples were collected and analyzed.Table 1 provides the demographic information of the study participants.
Among the participants, only 7.8% were below 5 years of age, while the majority (67.5%) were above 15 years of age.The majority of participants were female (71.7%).
RDT diagnostic performance 205/449 (45.7%) clinical samples tested positive for P. falciparum by varATS qPCR, with a mean parasite density of 12.5 parasites/μL.There were no statistically signi cant differences in positivity by qPCR based on participant's age or gender (Table 1).Of the 205 qPCR-positive samples, 142 (69.3%) had densities <200 parasites/μL blood.There was no signi cant difference in densities between male and female participants (p=0.44,Table 1).Parasite density was highest in participants aged 0 to 5 (Table 1).  1 For the RDT data, the results from all three RDTs were combined, with either RDT and either target (HRP2 or LDH) positive counting as a positive test.

Discussion
This study showed similarly high sensitivities of the NxTek and the Biocredit RDT.These tests are considered ultra-sensitive, i.e.
substantially more sensitive than previous tests such as the SB Bioline.They detected around 50% of all qPCR-positive infections, compared to around 40% by the SD Bioline.At densities >200 parasites/µL, the NxTek and Biocredit sensitivity was >93%, and 89% for the SD Bioline.The LOD of the NxTek and Biocredit is approximately four-fold lower than the LOD for the SD Bioline, and the LOD of the Biocredit con rmed values obtained from a previous study in Burundi applying the same methodology [3].RDT sensitivity reached 73% in children aged 6-15 years.Older participants (> 15 years) had lower parasite densities, resulting in lower RDT sensitivity.
None of the three P. falciparum malaria-positive samples detected via the LDH target alone carried hrp2/3 deletions.The current data thus corroborated recent ndings of very low frequency of hrp2/3 deletions in Ghana [28,29].The diagnostic performance of the LDH target in the Biocredit RDT was found to be comparable to that of the conventional Bioline HRP2 RDT.This suggests that the Biocredit RDT, with its LDH target, can be a suitable alternative to the Bioline RDT in regions where hrp2 deletion is prevalent.While to-date in Ghana HRP2-based RDTs remain effective, the Biocredit RDT will be a reliable option for malaria diagnosis shall deletions ever spread in the country.
In conclusion, the NxTek and Biocredit RDTs showed higher sensitivity and a lower LOD compared to the SD Bioline RDT, which has been in use for clinical diagnosis by the Ghana National Malaria Control Programme [30].Such more sensitive RDTs are crucial to administer proper treatment and to accelerate malaria elimination efforts.

Declarations Ethics approval and consent to participate
Prior to sample collection, informed written consent was obtained from each individual.For minors, assent was obtained in addition to consent obtained from legal guardians.

Table 1 .
Demographics of the study population, parasite density, test positivity (by qPCR) and RDT sensitivity by age group and gender.

Table 2 .
Measure of diagnostic performance of Bioline, NxTek and Biocredit RDTs This study was approved by the Committee on Human Research, Publications, and Ethics of the School of Medical Sciences, KNUST (CHRPE/AP/030/20), the University of Notre Dame Institutional Review Board (19-04-5321), and The Ohio State University Institutional Review Board (2020H0539).