Prozone in malaria rapid diagnostics tests: how many cases are missed?

Abstract

Background: Prozone means false-negative or false-low results in antigen-antibody reactions, due to an excess of either antigen or antibody. The present study prospectively assessed its frequency for malaria rapid diagnostic tests (RDTs) and Plasmodium falciparum samples in an endemic field setting.

Methods: From January to April 2010, blood samples with P. falciparum high parasitaemia (≥ 4% red blood cells infected) were obtained from patients presenting at the Provincial Hospital of Tete (Mozambique). Samples were tested undiluted and 10-fold diluted in saline with a panel of RDTs and results were scored for line intensity (no line visible, faint, weak, medium and strong). Prozone was defined as a sample which showed no visible test line or a faint or weak test line when tested undiluted, and a visible test line of higher intensity when tested 10-fold diluted, as observed by two blinded observers and upon duplicate testing.

Results: A total of 873/7,543 (11.6%) samples showed P. falciparum, 92 (10.5%) had high parasitaemia and 76 were available for prozone testing. None of the two Pf-pLDH RDTs, but all six HRP-2 RDTs showed prozone, at frequencies between 6.7% and 38.2%. Negative and faint HRP-2 lines accounted for four (3.8%) and 15 (14.4%) of the 104 prozone results in two RDT brands. For the most affected brand, the proportions of prozone with no visible or faint HRP-2 lines were 10.9% (CI: 5.34-19.08), 1.2% (CI: 0.55-2.10) and 0.1% (CI: 0.06-0.24) among samples with high parasitaemia, all positive samples and all submitted samples respectively. Prozone occurred mainly, but not exclusively, among young children.

Conclusion: Prozone occurs at different frequency and intensity in HRP-2 RDTs and may decrease diagnostic accuracy in the most affected RDTs.

Figure 1

Flow of patients and samples. Footnotes figure 1: * Prozone results presented for Paracheck-Pf, results for other RDTs are listed in Table 3. For definition of prozone see text.

Figure 2

Example of prozone for Paracheck-Pf. Footnotes figure 2: Paracheck-Pf RDT cassettes run with a blood sample infected with P. falciparum at a parasite density of 8.3%. The sample was assessed in duplicate, undiluted (P25 A and P25 B), 10 × diluted (P25 × 10A and P25 × 10B) and 100 × diluted (P25 × 100A and P25 × 100B). All cassettes show regular control lines, cassettes P25 A and B show no visible test line. Cassettes P25 × 10A and P25 × 10B show weak test lines and the maximum line intensity (strong) was obtained after 100 × dilution (cassettes P25 × 100A and P25 × 100B).