False-negative rapid diagnostic tests for malaria and deletion of the histidine-rich repeat region of the hrp2 gene

Abstract

We identified 480 persons with positive thick smears for asexual Plasmodium falciparum parasites, of whom 454 had positive rapid diagnostic tests (RDTs) for the histidine-rich protein 2 (HRP2) product of the hrp2 gene and 26 had negative tests. Polymerase chain reaction (PCR) amplification for the histidine-rich repeat region of that gene was negative in one-half (10/22) of false-negative specimens available, consistent with spontaneous deletion. False-negative RDTs were found only in persons with asymptomatic infections, and multiplicities of infection (MOIs) were lower in persons with false-negative RDTs (both P < 0.001). These results show that parasites that fail to produce HRP2 can cause patent bloodstream infections and false-negative RDT results. The importance of these observations is likely to increase as malaria control improves, because lower MOIs are associated with false-negative RDTs and false-negative RDTs are more frequent in persons with asymptomatic infections. These findings suggest that the use of HRP2-based RDTs should be reconsidered.

Figure 1.

hrp2 PCR in subjects with positive and negative HRP2 RDTs. Lane 1: DNA molecular weight markers VI (Roche, Indianapolis, IN). Lanes 2–6: results of PCR with DNA from a thick smear-positive subject with a negative HRP2 RDT using allotype-specific primers for the block 2 region of msp1 (lanes 2–4 show one K1 amplicon and no MAD20 or Ro33 amplicons), forward and reverse primers for hrp2 (lane 5 shows the absence of hrp2 amplicons), and species-specific primers for P. falciparum ribosomal DNA (lane 6 shows an amplicon of the expected size of 206 bp). In contrast, lane 7 provides a positive control for the hrp2 PCR based on DNA from a thick smear-positive subject with a positive HRP2 RDT (same forward and reverse primers as in lane 5 and Table 1).