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

Abstract

Background: Rapid diagnostic tests (RDTs) for detection of Plasmodium falciparum infection that target P. falciparum histidine-rich protein 2 (PfHRP2), a protein that circulates in the blood of patients infected with this species of malaria, are widely used to guide case management. Understanding determinants of PfHRP2 availability in circulation is therefore essential to understanding the performance of PfHRP2-detecting RDTs.

Methods: The possibility that pre-formed host anti-PfHRP2 antibodies may block target antigen detection, thereby causing false negative test results was investigated in this study.

Results: Anti-PfHRP2 antibodies were detected in 19/75 (25%) of plasma samples collected from patients with acute malaria from Cambodia, Nigeria and the Philippines, as well as in 3/28 (10.7%) asymptomatic Solomon Islands residents. Pre-incubation of plasma samples from subjects with high-titre anti-PfHRP2 antibodies with soluble PfHRP2 blocked the detection of the target antigen on two of the three brands of RDTs tested, leading to false negative results. Pre-incubation of the plasma with intact parasitized erythrocytes resulted in a reduction of band intensity at the highest parasite density, and a reduction of lower detection threshold by ten-fold on all three brands of RDTs tested.

Conclusions: These observations indicate possible reduced sensitivity for diagnosis of P. falciparum malaria using PfHRP2-detecting RDTs among people with high levels of specific antibodies and low density infection, as well as possible interference with tests configured to detect soluble PfHRP2 in saliva or urine samples. Further investigations are required to assess the impact of pre-formed anti-PfHRP2 antibodies on RDT performance in different transmission settings.

Figure 1

Anti-PfHRP2 antibody levels in plasma samples. Levels of PfHRP2-specific antibodies in the plasma of subjects were measured by ELISA. Cut-off is shown by the dotted line, and Error Bars represent 95% Confidence Intervals. Country of origin is represented using ISO 3166 country code: Cambodia (KH), Nigeria (NG), the Philippines (PH), and Solomon Islands (SB). BNE represents Brisbane. Samples from subjects who were microscopy negative for P. falciparum are shown as Neg.

Figure 2

PfHRP2 antigen levels in plasma samples. PfHRP2 antigen levels were measured in a commercial ELISA test (Standard Diagnostics, Korea). Country of origin is represented using ISO 3166 country code: Cambodia (KH), Nigeria (NG), the Philippines (PH), and Solomon Islands (SB). Shown are the results from subjects who tested positive. Error Bars represent SEM.

Figure 3

Relationship between parasitaemia and plasma PfHRP2 level in samples from symptomatic patients.

Figure 4

Relationship between plasma anti-PfHRP2 antibody and plasma PfHRP2 antigen level. Country of origin is represented using ISO 3166 country code: Cambodia (KH), Nigeria (NG), the Philippines (PH), and Solomon Islands (SB). Ab represents antibody. PF + and PF – represents microscopy positive and negative, respectively, for P. falciparum.

Figure 5

Anti-PfHRP2 antibody inhibits ELISA detection of soluble PfHRP2 antigen. Culture supernatant from an in vitro culture of P. falciparum was pre–incubated in serial dilution with a plasma sample with high-titre anti-PfHRP2 antibody and tested in a commercial HRP2 antigen-capture ELISA. Four-fold dilutions are shown.

Figure 6

Anti-PfHRP2 antibody impairs the ability of three PfHRP2-detecting RDTs to detect cultured P. falciparum . Three brands of RDT (SD, ICT and First Response) were tested with P. falciparum-parasitized red cells pre-incubated with normal plasma (NP) or plasma from a subject with high titre anti-PfHRP2 antibody level (S14). Blood was serially 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 (1–8). The experiment was undertaken before (A) and after (B) the blood was washed to remove any residual PfHRP2 present in the culture medium.