Circulating antibodies against Plasmodium falciparum histidine-rich proteins 2 interfere with antigen detection by rapid diagnostic tests

Pfhrp2 genes originating from P. falciparum isolate FCQ79 was cloned into the pET8c expression vector (Novagen), expressed in Escherichia coli BL21(DE3) LysS cells and purified using His-trap Ni-IDA column (GE Healthcare) followed by Superdex 75 10/300 column (GE Healthcare) (Lee et al., unpublished).

The concentrations of the purified rPfHRP2 were determined using the Biruet assay [23]. Briefly, a working reagent was prepared by mixing an equal volume of stock reagent (5% sodium citrate, 3% sodium carbonate and 0.5% copper sulphate) with 150 mM NaOH. In each well, 40 μl of rPfHRP2 sample was mixed with 160 μl of working reagent in triplicate and incubated at room temperature for 25 min. The absorbance of each well was read at 540 nm and sample concentrations were determined from the standard curve of bovine serum albumin (BSA).

A total of 121 plasma samples were collected from malaria-endemic regions of four countries at different times. Some were collected as part of the parasite collection for the Specimen Bank used in the WHO Product Testing for malaria RDTs [24, 25]. Samples from acute malaria patients (symptomatic and microscopy positive) were collected at time of presentation from Cambodia (KH, n = 15), Nigeria (NG, n = 23) and the Philippines (PH, n = 37) by Pasteur Institute of Cambodia, University of Lagos and Research Institute for Tropical Medicine, the Philippines [24, 25]. An additional ten and eight samples were collected from microscopy-negative subjects in Nigeria and the Philippines (uninfected endemic controls), respectively. The collection and use of the samples was approved by the ethics committees of the collection institutions. Methods used for blood collection, parasite speciation and quantitation were described in detail in a WHO Methods Manual [26]. Plasma was separated from each patient blood and stored at −20°C. A total of 28 plasma samples were collected in 1987 [27] in Solomon Islands (SB) with informed consent from asymptomatic villagers (13–66 years old) living in Guadalcanal, Solomon Islands [27]. Eleven of these villagers were microscopy positive for P. falciparum with low densities while the remaining 17 villagers were microscopy negative at the time of collection. All plasma samples were stored at −80°C. Plasma samples were also collected from six Brisbane residents who had no exposure to Plasmodium infection for use as non-exposed negative controls.

Patient blood sample collection was coordinated by WHO, TDR and FIND and conducted by investigators and institutions within the WHO-FIND Malaria RDT Quality Assurance Programme.

Purified rPfHRP2 (50 ng/well) was coated on flat-bottomed, 96-well microtitre plates (Nunc, Denmark) at 4°C overnight or 37°C for two hours. Plates were blocked with 1% skim milk at 37°C for one hour. Plasma samples were diluted to 1:200, added in duplicate at 50 μL/well and incubated at 37°C for one hour. Plates were washed five times with wash buffer and air-dried. Anti-human IgG conjugated with horseradish peroxidase (Sigma) was diluted 1:5,000 and added into the wells (50 μL/well). Plates were incubated for one hour at 37°C and then washed five times. OPD substrate buffer (Sigma Aldrich) was added to each well (100 μL/well), and incubated in the dark for 20 minutes at room temperature. Absorbance was read at 450 nm using a microplate reader (VeraMax, Molecular Devices, USA).

To establish relative antibody concentrations against rPfHRP2, five human plasma that contained high anti-PfHRP2 antibody levels were identified and pooled for use as a reference standard. This standard plasma pool was two-fold diluted in PBS from 1:100 to 1:1,600, and applied, in duplicate, to each assay plate. ELISA units were arbitrarily assigned to each dilution. Two wells that contained PBS were used as a negative control and were also used to blank all samples and standard plasma.

The OD₄₅₀ readings of the serial dilutions of the standard plasma pool were plotted on a four-parameter hyperbolic curve (SOFTmax Pro ver 3.1; Molecular Devices). If R² was less than 0.9, the assay was deemed invalid and was repeated. ELISA units in test plasma were interpolated from their mean OD₄₅₀ values from the standard curve. The positive threshold (13.1) was defined as two standard deviations above the mean ELISA units (8.4) of a pool of six human plasma samples from individuals never exposed to malaria.

To quantify the plasma PfHRP2 level, a PfHRP2 antigen standard concentration curve was established using a set of doubling dilutions of a P. falciparum 3D7 culture supernatant. The concentration of PfHRP2 in the culture supernatant (ng/mL) was determined by comparing its OD₄₅₀ value to a known protein concentration standard curve that was prepared by serial dilutions of rPfHRP2 (FCQ79). These standard dilutions were used as a reference standard for each assay plate. The PfHRP2 concentration (ng/mL) in each plasma sample was determined by reference its OD₄₅₀ value to the standard curve assayed on the same plate. The limit of detection of this ELISA was determined to be 0.5 ng/mL.

Two high-titre plasma samples from Solomon Islands, S14 and S53, were identified and used as sources of anti-PfHRP2 antibody; culture supernatant from an in vitro culture of the P. falciparum strain 3D7 was used as source of parasite PfHRP2 antigen. The culture supernatant was subject to two-fold doubling dilutions with RPMI media from 1:2 to 1:126; 50 μL of each dilution was mixed with 50 μL of plasma, undiluted or diluted 1:5. Culture supernatant was used as the ‘no plasma’ control; pooled plasma from non-exposed negative controls was used as the negative plasma control. Antibody-antigen mixtures were incubated at 37°C for one hour, transferred to the SD Malaria Antigen Pf ELISA kit, and processed as described above. The OD₄₅₀ in mixtures with and without human plasma was compared. Concurrently, 5 μl of each mixture was transferred onto three different malaria RDTs: ICT Malaria Combo Cassette Test (ML02, ICT Diagnostics, South Africa), First Response Malaria Antigen pLDH/HRP2 Combo (116FRC30, Premier Medical Corp, India) and SD Malaria Antigen Pf (05FK50, Standard Diagnostics, Korea). Results were read by two independent readers and recorded as levels 0–4 based on the test band intensity following the WHO colour chart [24].

Three culture-adapted P. falciparum lines originating from Solomon Islands (S55, SJ44 and SJ15) were cultured in vitro using standard culture conditions [28]. Parasite cultures were repeatedly synchronized using 5% sorbitol [29], then maintained and harvested when parasitaemia reached 2% with majority at ring stage. An aliquot of the harvested parasites was concentrated to 50% haematocrit by centrifuging and discarding excess supernatant. A second aliquot of the culture was pelleted, washed and resuspended in fresh media to a 50% haematocrit. Both aliquots were diluted using normal human blood maintaining a 50% haematocrit to parasite densities of 100,000, 30,000, 10,000, 3,000, 1,000, 300, 100, and 30 parasites/μL. An aliquot of each dilution was incubated with the immune human plasma (S14 or S53) or negative control plasma for one hour at room temperature. These samples were then tested on a RDT as described above.

The ages, parasite densities and PfHPR2 antigen levels in the patient samples were compared between countries of origin using the Kruskal Wallis test. Proportions were compared between countries using either Fisher’s exact test (for comparison of two categories) or Chi-square Test (>two categories). Correlation between parasitaemia and antibody or antigen level was assessed using Spearman’s Rank Correlation.

Blood samples were collected from patients with acute malaria in Cambodia (n = 15), Nigeria (n = 23) and the Philippines (n = 37). The age of these individuals and levels of parasitaemia are presented in Table 1 and Additional file 1. There were no significant differences in the ages of the patients recruited at each site (P > 0.50). The geometric mean parasite density of samples differed by country of origin (P < 0.001); densities from Nigeria (18,528/μL, 95% CI: 12,686-27,059/μL) and Cambodia (20,940/μL, 95% CI: 9,766-44,897/μL) were not significantly different (P > 0.05), but were significantly higher than in samples from the Philippines (1,767/μL, 95% CI: 811–3,850/μL; P < 0.01). The Solomon Islands samples were collected in 1987 from a high transmission area. All subjects were asymptomatic at the time of collection. Eleven of the 28 individuals were microscopy-positive for P. falciparum, with low-density infection (exact parasite density was not available). The remaining 17 individuals, nine of whom reported a history of malaria were blood smear negative at the time of collection.

Plasma samples were analysed for anti-rPfHRP2 antibody levels using ELISA. The mean ELISA unit among the non-exposed negative controls was 8.4 ± 2.3, while the corresponding level for the ten parasite-negative Nigerian samples was 2.7 ± 1.1. Nineteen of the 75 patients with acute malaria (25.3%) had anti-PfHRP2 antibody levels above the cut-off value (13.0 units), with the prevalence of antibodies in samples from Cambodia, Nigeria and the Philippines not being significantly different (20.0, 34.7 and 21.6%, respectively; P > 0.40). The prevalence of anti-PfHRP2 antibodies among the asymptomatic Solomon Islanders was lower at 10.7%, although not significantly different from prevalence in patients with acute malaria (P > 0.15).

Levels of anti-PfHRP2 antibodies are shown in Figure 1 (with raw Optical Density results shown in Additional file 2). The majority of the antibody-positive samples had antibody levels between one to three-fold higher than the cut-off value, while two plasma samples had antibody levels that were five-fold (NG02F22) and 16.7-fold (SB014) higher than the cut-off value. Overall there was a positive correlation between antibody level and age of infected subjects. However, this was not statistically significant for the combined set of parasite positive samples (r = 0.20, P = 0.08, n = 79), nor within individual countries (KH: r = 0.23, P = 0.39; PH: r = 0.05, P = 0.75; NG: r = 0.16, P = 0.47, SB (Pf positive): r = −0.32, P = 0.50). There was also no significant correlation between parasitaemia and antibody level in the symptomatic patient samples (r = −0.06, P = 0.62.)

PfHRP2 antigen was detected by ELISA in 100, 91.3 and 62.2% plasma of patients with symptomatic malaria in Cambodia, Nigeria and the Philippines, respectively, while only 6/28 (21.43%) of plasma from asymptomatic subjects from the Solomon Islands had detectable PfHRP2 antigen (Table 2). Five of these six subjects were positive for P. falciparum by microscopy. None of the parasite-negative subjects from Nigeria (n = 10), the Philippines (n = 8) or Brisbane (n = 6) had detectable PfHRP2 in their plasma samples (Table 2).The level of PfHRP2 antigen present in individual plasma samples is shown in Figure 2. For samples from symptomatic patients with detectable antigen, there was no significant difference in the distribution of antigen level according to country of origin (P = 0.27). The mean antigen levels were 9.45 ng/mL (95% CI: 7.42-11.49 ng/mL), 11.62 ng/mL (95% CI: 9.14-14.11 ng/mL) and 11.14 ng/mL (95% CI: 6.60-15.69 ng/mL), for Cambodia, Nigeria and the Philippines, respectively. The antigen levels among the Solomon Islands asymptomatic subjects (for which antigen was detected) were significantly lower (mean = 0.70 ng/mL, 95% CI: −0.09-1.48 ng/mL) than for the symptomatic patients in Cambodia, Nigeria and the Philippines (P < 0.01). There was a significant positive correlation between parasite density as determined by blood smear and plasma PfHRP2 level for the symptomatic patient samples (r = 0.45, P < 0.0001, n = 74, Figure 3).

Data on anti-PfHRP2 antibody level and PfHRP2 antigen level were available for 74 samples. Of the 18 samples from patients with acute malaria who had anti-PfHRP2 antibodies above the cut-off, 14 (77%) were PfFHRP2 antigenaemic. There was no significant association between antibody presence and presence of antigen (P > 0.90). Logistic regression indicated that antigen level was not a significant factor for predicting the presence of anti-PfHRP2 antibody (P = 0.44). Although samples with high antigen levels tended to have lower antibody levels, and samples with the highest antibody level tended to have low antigen levels (Figure 4), this relationship was not statistically significant.

When two plasma samples with high-titre anti-PfHRP2 antibody were pre-incubated with serial dilutions of culture supernatant from an in vitro culture of the P. falciparum, a significant reduction in OD values in the HRP2 antigen-capture ELISA was observed at all antigen dilutions tested. Figure 5 demonstrates the effect of a serum from one of these two subjects (S14). The same effect was also observed when the plasma was diluted five-fold, except at the highest antigen concentration tested (55.5 ng/mL), or when undiluted plasma from another subject with high-titre antibody (S53) was used. The plasma pool from Brisbane subjects had no or minimal effect on the detection of antigens by ELISA.

When the pre-mixed plasma-antigen mixtures were tested on three commercially available malaria RDTs, complete blocking of the detection was observed with two brands of RDT (First Response and SD) resulting in negative RDT results (Table 3). The blocking effect diminished when the plasma was diluted five-fold. Interestingly, the performance of the third brand of RDT (ICT combo) was not as markedly affected by pre-incubation with plasma with high-titre, anti-PfHRP2 antibody, and tested positive in all situations (Table 3).

Suspensions of three separate P. falciparum lines were tested at seven different dilutions (100,000 P/μL to 30 P/μL) by incubating each with either a human plasma sample of high anti-PfHRP2 antibody (S14) or normal human plasma for one hour at room temperature, then tested in the antigen ELISA and the three RDTs named above. When the three brands of RDT were tested, a varying level of blocking effect was observed for all three brands tested against the N70 parasite line that originated from the Solomon Islands (Figure 6). The effect was apparent as a reduction of signal intensity from four to three or two at 100,000 P/μl and/or a reduction of lower detection threshold by ten-fold (from 300 to 3,000 P/μl, or from 1,000 to 10,000/μl). A similar blocking effect was alsoobserved when the three RDTs were tested on S55 line (Additional file 3). However, a markedly lesser blocking effect was seen when RDTs were tested on SJ15 line (Additional file 4).