Background
Malaria is still a leading cause of morbidity and mortality, with 212 million cases and 429,000 deaths reported in 2016 [
1]. Rapid diagnostic tests (RDTs) are a major milestone in point-of-care malaria diagnosis in an effort to attain universal access to parasite based diagnosis, consistent with WHO strategies and recommendations [
2]. RDTs have several advantages such as ease of use, inexpensive, and real-time diagnosis; but field assessment studies have shown that they can report false results [
3]. During transportation and storage in tropical climates the quality of RDTs deteriorates due to high temperatures and humidity [
4]. Albertini et al. showed that during transport and storage of health commodities, temperatures and humidity can exceed 30 °C and 94%, respectively in Burkina Faso, Senegal, Ethiopia, and the Philippines [
5]. The WHO has set out measures to ensure good manufacturing quality, evaluation programmes for lot testing (pre and post purchase), and provides guidelines for selection of RDTs before purchase [
6]. But, lack of proper quality control and quality assurance mechanisms for malaria RDT during transportation and storage remains a major problem [
7]. Therefore, there is need to develop positive controls for evaluating the quality of RDTs upon transportation and local storage in health facilities.
Positive control wells (PCW) based on recombinant malaria antigens are the most feasible approach to ensuring quality control of RDT in field operational use [
8]. Despite the prospective evaluation of the prototype in a malaria endemic area; they still face challenges such as; validation, long-term storage stability, and technical specifications which are still under development [
8]. While the world waits for the complete development of PCW, dried
Plasmodium falciparum-infected samples were developed as possible cheaper alternatives in monitoring the performance of RDT in routine use [
9]. The dried
P. falciparum-infected sample tubes contain the RDT target antigens; histidine rich protein 2 (HRP2), lactate dehydrogenase (LDH) and aldolase [
9,
10]. The challenge facing their applicability is protein degradation and rapid loss of reactivity during prolonged storage at ambient conditions [
9,
10]. Furthermore, it is challenging to establish well characterized samples for specific RDTs, as well as quantification of target antigens. Consequently, there is need to stabilize these target proteins to ensure their conformational stability during transport and storage in typical field conditions.
Protein stabilization involves the prevention of the irreversible loss of the unique chemical structure of the protein [
11]. Some of the methods used to stabilize proteins for long-term storage include; ligand stabilization, storage as frozen solutions, salted out precipitates, freeze dried solids, and chemical additive stabilization [
12]. Chemical additives prevent the loss of enzymatic activity, prevent denaturation, inhibit irreversible aggregation, and protect proteins against chemical instabilities [
13]. The commonly used chemical additives are; salts such as sodium and potassium; as well as organic solvents such as polyethylene glycol, polyethyleneimine and glycerol; and sugars such as trehalose, sorbitol and sucrose [
14].
Trehalose stabilizes proteins by various mechanisms, such as vitrification, preferential exclusion, water replacement, hydrogen bonding, and non-specific thermodynamic stabilization [
15]. Sucrose stabilizes proteins majorly by preferential exclusion; whereby it increases the chemical potential of the protein which adopts a more stable conformation [
16]. Glycerol has also been shown in previous studies to stabilize proteins by interacting preferentially with the hydrophobic parts of the protein structures [
17]. Alsever’s solution and LDH stabilizer are commercially developed for stabilizing proteins during long term storage [
18,
19]. Biostab enzyme stabilizer has been shown previously to stabilize lysozyme over storage time by preferential hydration on the surface of the protein [
20].
This study hypothesized that addition of commercially available chemical additives to dried P. falciparum-infected samples may potentially improve the long-term storage stability of HRP2, pLDH and aldolase. Therefore, this study aimed to investigate the effect of ten chemical additives on the stability of HRP2, pLDH and aldolase in during 53 weeks of storage.
Methods
Study design and sample collection
The study was conducted at the Walter Reed Project, Kisumu a malaria holo-endemic area where transmission occurs throughout the year [
21]. The study utilized archived whole blood samples collected from the Kombewa Clinical Research Center under a WRAIR/KEMRI approved protocol #1720. Cultured samples were obtained from cryopreserved stocks of
P. falciparum strains (3D7) maintained at the Walter Reed Project-Kisumu and used to initiate a continuous culture according to the WRAIR/KEMRI approved protocol #1919. All the collected samples were characterized by expert microscopy and diluted to three experimental parasite densities i.e. 2000, 500, and 200 p/µL using malaria negative group O+ blood. The Group O+ blood was obtained through a USAMRD-K approved study (Scientific Steering Committee; SSC#1919).
Selection of malaria rapid diagnostic tests (RDT)
The RDTs were selected based on performance in Round 2 of WHO/FIND/CDC malaria RDT performance evaluations, national guidelines on required performance of RDTs in Kenya, and ability to detect two parasite antigens (WHO/FIND/CDC, 2010). Four different types of RDTs were selected based on panel detection score (PDS) at 200 parasite/μL of ≥ 90 (Table
1).
Table 1
Selected malaria rapid diagnostic tests (RDTs)
CareStart Malaria HRP2 (Pf) | 2 |
Pf
| Access Bio, Inc. | G0141 | 99 |
First Response Malaria Ag Combo (PLDH/HRP2) | 3 | Pf and Pan | Premier Medical Corporation Ltd | II6FRC30 | 100 |
SD BIOLINE Malaria Ag Pf/Pan (HRP2/pLDH) | 3 | Pf and Pan | Standard Diagnostics, Inc. | 05FK60-02-3 | 96 |
BinaxNOW Malaria Ag pf/Pan (HRP2/Aldolase | 3 | Pf and Pan | Binax, Inc., Inverness Medical, ME, USA | 665-025 | 99 |
Selection of chemical additives
The selection was based on the literature review on the ability of the chemicals to improve stability of proteins. Additives chosen for this project were polyethylene glycol (Sigma-Aldrich, MO, USA), sucrose (Sigma-Aldrich, MO, USA), biostab enzyme stabilizer (Sigma-Aldrich, MO, USA), Alsever’s Solution (Fisher Scientific, USA), trehalose (Fisher-Scientific, USA), lactate dehydrogenase stabilizer solution (The Gwent Group UK), polyethyleneimine (Fisher-Scientific, USA), and glycerol (Sigma-Aldrich, MO, USA). Stepwise exploratory tests (36 tests) were conducted to determine which additives, combinations and concentrations gave optimal RDT test outcomes. The tests were conducted by adding the additives to start-up culture samples individually (mono) or in combination (mixed) with other additives at concentrations of 200 and 2000 p/µL.
The ratio of additives used in combination was 1:1 (additive: additive) as in previous studies [
22]. The ratio of additives or combinations to culture was 1:2 (additive: blood), so as to achieve appropriate concentration of the additive in the blood [
23]. A control start-up sample (no additive/s added) was also included. In the event that an additive, combination or concentration was found to interfere with RDT test results, that particular additive or combination was withdrawn. Additives that showed no or little effect on the stability of the proteins were also withdrawn based on weekly retesting results of the samples during 4 weeks of storage on SD Bioline and First Response Malaria Kit. Ten additives/combinations were selected from the 36 combinations (Table
2). The concentration formulations were selected according to extensive previous studies on the effect of different additives on keeping the structure of proteins [
14,
24,
25].
Table 2
Selected chemical additives for stabilization of malaria RDT target antigens
Sucrose | Suc | 0.5 M | Sigma-Aldrich, MO, USA |
Glycerol/sucrose | Gly/Suc | 0.5 M and 10% | Sigma-Aldrich, MO, USA |
Alsever’s/sucrose | Als/Suc | 100% w/v and 0.5 M | Fisher Scientific, USA & Sigma-Aldrich, MO, USA |
Trehalose | Treh | 0.5 M | Fisher-Scientific, USA |
Sucrose/trehalose | Suc/Treh | 0.5 and 0.5 M | Fisher-Scientific, USA & Sigma-Aldrich, MO, USA |
Glycerol/Trehalose | Gly/Treh | 10% and 0.5 M | Fisher-Scientific, USA & Sigma-Aldrich, MO, USA |
Trehalose/biostab | Treh/Bio | 0.5 M and 5% w/v | Fisher-Scientific, USA |
Biostab/sucrose | Bio/Suc | 5% w/v and 0.5 M | Sigma-Aldrich, MO, USA |
LDH stabilizer | LDH Stab | 100% W/v | The Gwent Group, UK |
LDH stabilizer/trehalose | LDH Stab/Treh | 100% w/v and 0.5 M | Fisher-Scientific, USA & The Gwent Group, UK |
Stabilization of Plasmodium proteins
The ten selected additives and their combinations were added to the patient and culture samples (after characterization to respective parasite densities) in a ratio of 1:2, and a control patient and culture sample (without any additive) were also prepared. Baseline data was collected by testing of all the samples to confirm the reactivity of the antigens using SD Bioline, CareStart, First Response and BinaxNOW. The test was conducted according to manufacturer instructions. Reactivity or presence of the HRP2, PLDH, and aldolase was demonstrated by color changes at the test lines. A successful test was confirmed by the presence of a control line. Results were captured as either positive or negative through visual examination of the kit. It was recorded positive if both the control band and the tests bands were visible and it was recorded negative if the control line was visible, but the test lines were absent.
Dried
P. falciparum-infected samples in tubes were prepared by depositing 40 µL aliquots at the bottom of 2 mL vials, the tubes were left uncapped to air-dry overnight in a bio-safety cabinet, and then sealed tightly by closing the vial cap. The tubes containing dried
P. falciparum-infected sample (stabilized and controls) were stored at room temperature. The temperature of the room was monitored daily, and recorded three times in a day on temperature charts. The average temperature during the study period was 25 °C (range 23–27 °C). Temporal stability data was collected by retesting the dried
P. falciparum-infected samples consecutively after 1, 4, 8, 12, 15, 18, 21, 24, 33, 43 and 53 weeks of storage. The study selected 53 weeks of evaluation because, in developing countries like Kenya, there are existing trends of laboratory stock outs in peripheral health facilities due to poor infrastructure and procurement processes [
26]. Therefore, stability of antigens for up-to 53 weeks will reduce the chances of facilities having stock-outs, reduce associated costs of resupply, and ensure reliability in using the positive controls.
Retesting was completed by rehydrating the dried blood pellets for the control samples and each additive stabilized sample using phosphate buffered saline with Tween-20. The tubes were left for 1 h to ensure complete rehydration. Each dried P. falciparum-infected sample was tested on SD Bioline, CareStart, First Response and BinaxNOW. This study defined loss of reactivity as the number of weeks after which a sample tested negative (complete loss of reactivity; 4 + to 0) on a rapid diagnostic test. Potential degradation of RDTs was done on the batch, if a sample was recorded negative (lost reactivity) for any RDT, the reactivity of that batch of RDT was verified using a positive control sample (frozen at − 80 °C).
Data analysis
Stability was defined as the number of weeks the controls or the stabilized sample remained positive as shown by their reactivity on RDTs. The limit of stabilization of a chemical additive is the duration in which the stabilized sample (both culture and patient sample) tested positive for all the three RDTs. CareStart RDT was not included in the analysis because sample retesting could not be conducted to week 53 of storage; due to lack of similar lot number of the kit in the market. Percentage stability of a sample was calculated against week 53, the endpoint [No. of weeks sample was reactive/53 weeks × 100%]. Average percentage stability was computed by combining both the culture and patient samples percentage stabilities. The Z-test for comparing proportions [
27] was used to compare the percentage stability of the stabilized sample proteins against the non-stabilized sample proteins. To determine the percentage increase in stability of chemically stabilized HRP2, PLDH, and aldolase, [the time taken for a stabilized antigen (in weeks) to lose reactivity on the RDTs was compared to the time taken for non-stabilized antigen to lose reactivity on the RDTs]. The mean percentage increase in stability for each chemical additive was computed from three RDT results for HRP2 or PAN antigens at each parasite density.
Discussion
This study indicates that chemical additives significantly improve the long-term storage stability of HRP2, pLDH, and aldolase as determined by reactivity on the three malaria RDTs (SD Bioline, First Response, and BinaxNOW). Stabilization of dried
P. falciparum-infected samples by sucrose, trehalose, biostab/trehalose, LDH stabilizer/trehalose, and trehalose/sucrose in turn improved the stability of all the three proteins during the 1 year of storage. Previous studies have shown that through preferential interactions these chemical additives improve the long-term stability of various proteins including; ribonuclease A, lysozyme, chymo-trypsinogen recombinant interleukin-1 receptor, monoclonal antibodies, pyro-phosphatase, bovine serum albumin, ribosomal protein S6, cutinase, LDH and lysozyme [
12,
15,
20,
24,
25,
28‐
30]. The findings of the present study show that stabilization of the target proteins ensures prolonged storage of dried
Plasmodium falciparum-infected samples in ambient temperature conditions and can be used as positive controls for validation of malaria RDTs at the point of care.
The results on percentage increase in stability clearly indicate that the parasite density was not a factor in conferring stability to the proteins, but loss of reactivity was probably due low levels of the antigens in the samples. As observed, stabilization of the target proteins at 200 p/µL did not prevent their loss of reactivity quicker (1 + to 0) than samples at 2000 p/µL (4 + to 0). Consistent with this findings, Aidoo et al. documented the loss of reactivity in the samples standardized at 200 p/µL as shown by ten different malaria kits was quicker as compared to samples at 2000 p/µL [
9]. The present study shows that
Plasmodium LDH and aldolase were detectable at baseline by all the four RDTs, but the reactivity of antigens declined quickly with decrease in parasite density during storage. A study by Versteg and Mens indicated that none of the samples containing a parasite density of 300 p/μL gave a signal throughout their study while the samples containing 3000 p/μL were positive for 4 weeks and samples at 30,000 p/μL yielded a signal that remained visible for some time [
31].
These results indicate that stability of HRP2, LDH and aldolase increased as concentration of the chemical additives decreased. The percentage temporal stability of all the proteins at 2000 p/µL was lower than 500 p/µL and it increased further at 200 p/µL on SD Bioline, First Response, and BinaxNOW malaria. This can be due to the dilution effect and surface area to volume ratio [
32] whereby there is a specific distribution of the additives around the proteins at a particular concentration and size of the protein. In theory, if the protein concentration is reduced and the same amount of the additive is maintained the proteins achieve more stability because all the binding sites of the protein will be saturated depending on the molecular weight. A previous study on effect of sucrose concentration on protein stability showed that; a 1:1 weight ratio provided a sixfold stabilization toward aggregation for hGH (22 kD), a fourfold stabilization for rHSA (66 kD), and a 20-fold stabilization for IgG1 antibody (150 kD) protein [
32].
Glycerol/trehalose and glycerol/sucrose are the only additives shown to destabilize or decrease stability of HRP2, LDH, or aldolase. A previous study has shown that glycerol is also preferentially excluded from the surface of the protein, but the exclusion by glycerol is thermodynamically unsuitable, as it favours the unstructured form of proteins [
33].
The main challenges observed in this study include; differences in the stabilization of HRP2 antigen versus the PLDH and aldolase antigen, low parasite concentration affecting possible outcomes, and noticeable dissimilarity in test performances between the three malaria kits. The differences between stabilization of the proteins can be due levels of the antigens in the sample. This is because different patients have varying expression profiles of the parasite antigens [
34] and LDH/aldolase levels can be very low in patient sample as compared to HRP2. Martin et al. showed that the levels of HRP2 are higher in individuals (approximately sevenfold), and can persist in the bloodstream for more than 2 weeks as compared to the PLDH which cannot be detected in smaller volumes of blood and the clearance time rate is less than 5 days [
35].
The noticeable dissimilarity between different RDTs can be due to manufacturer differences, and lot to lot or batch to batch variation. Previous studies on patient samples and WHO panel detection scores have shown that the performance of malaria RDTs varies between different manufacturer brands and between different lots from the same manufacturer [
2,
3,
6,
7,
9]. Manufacturer factors, such as the type of immunoglobulin used for antigen capture, the type of strip used, type of buffer used, the quality of fluorescence particles, and the overall kit development, play a critical role in the variability of results among kits [
7]. In field use (health facilities), the RDTs have several limitations, such as decreased and variable sensitivity at lower parasite densities, false negative results, and false positive results [
3,
36].
Conclusion
This study demonstrated that the presence of chemical additives in dried P. falciparum-infected samples significantly improves the long-term stability (~ 53 weeks) of HRP2, PLDH, and aldolase. Specifically, sucrose, trehalose, sucrose/trehalose, biostab/trehalose demonstrated the ability to significantly improve the stability of the RDT target antigens. This study also recommends that future exploration in the field be carried out on the use of biostab/trehalose stabilized dried P. falciparum-infected samples. Stabilized dried P. falciparum-infected samples as RDT positive controls should be sent to off-site facilities in different climatic zones both in Kenya and other countries for testing under ambient temperature conditions in-order to determine their field operational viability.
Authors’ contributions
CM contributed in concept development, study design, execution of the project, data analysis, data interpretation and drafting the manuscript. CA was the University supervisor responsible for intellectual content development, study design, interpretation of data and manuscript drafting. GA, RA, and CM participated in the coordination of the study, performing the assays, data collection, and drafting the manuscript. BO, JW and EW substantially contributed to the conception, study design, supervision, data interpretation and drafting of the manuscript. All authors participated in drafting, review manuscript for publication. All authors read and approved the final manuscript.