Limited Level of Accuracy Provided by Available Rapid Diagnosis Tests for Malaria Enhances the Need for PCR-Based Reference Laboratories

Abstract

The rise of imported malaria cases and the high fatality rate in Europe make the search for new and easy diagnostic methods necessary. Rapid diagnosis tests (RDTs) are, in part, developed to cover the lack of diagnosis experience. Unfortunately, our data suggest that the accuracy of RDTs is insufficient and could increase the number of incorrect malaria diagnoses.In recent years, countries in which malaria is not endemic have reported high and increasing numbers of imported malaria cases, with fatalities up from 3.8 to 20% (2). Preventing fatal outcomes in malaria cases requires early recognition of infection, accurate laboratory diagnosis, and prompt therapy (2). Unfortunately, health-care personnel in countries where malaria is not endemic frequently lack experience in the microscopic diagnosis of malaria. In Italy, 80% of cases had less than a 1-week elapse between the onset of malaria symptoms and the microscopic diagnosis, but the average diagnosis took 8.5 days and the range was 1 to 28 days (3). This fact makes the search for new and easy diagnostic methods necessary. Rapid diagnosis tests (RDTs) for malaria might offer a valid alternative to microscopy (5).We studied 206 pre-and posttreatment samples from 169 patients in 1998 and 1999 by microscopic diagnosis and seminested multiplex PCR (4). These samples were also tested using three commercial RDT kits; 189 samples from 149 patients were tested with the ParaSight-F Kit (Becton Dickinson), 197 samples from 126 patients were tested with the OptiMal Kit (Flow Incorporated), and 54 samples from 41 patients were tested with the ICT Pf/Pv kit (Amrad). All patients (with ages of 16 months to 72 years) presented a history of fever and travel in the previous year to an area of malaria endemicity.RDTs were performed according to the manufacturers' instructions. All microscopy-positive samples were confirmed by PCR (4). Furthermore, PCR detected 6 samples with mixed infections (4 Plasmodium falciparum plus P. malariae and 2 P. falciparum plus P. ovale) from samples that were characterized as P. falciparum-only by microscopy and 24 more positive samples (12 P. falciparum, 4 P. ovale, 6 P. malariae, and 2 P. vivax). The three RDT methods showed a high rate of false positives and false negatives (Table 1). Moreover, 29.2% of positive non-P. falciparum samples rendered a positive P. falciparum result when the ParaSight-F test was used, which suggests a high number of cross-reactions, as this test according to the manufacturer detects only P. falciparum infections. In the same way, according to the manufacturers, the OptiMal and ICT Pf/Pv kits are able to detect P. falciparum specifically and the other Plasmodium spp. unspecifically. Our data, however, show that e Average time in which a sample is still positive by RDT (negative by microscopy and PCR) after treatment.

Detection of Four Plasmodium Species in Blood from Humans by 18S rRNA Gene Subunit-Based and Species-Specific Real-Time PCR Assays

“…On 97 EDTA-treated blood samples, no PCR inhibition was observed, and all extraction controls tested negative. Several reports have shown that the DNA-based amplification methods had higher sensitivity (as few as 1 parasite/l of blood) than examination of thin blood smears, especially in cases of low parasitemia or mixed infections (3,4,23) and that they have the ability to differentiate Plasmodium species (31)(32)(33)(35)(36)(37). Other reports have described quantitative real-time PCRs able to monitor the effectiveness of antimalarial therapy but without being able to distinguish between the four species (18,34).…”

“…DNA-based methods were also developed to overcome difficulties in determining the correct species identification faced by laboratories that may not keep appropriate microscopic experts (33). Changing patterns of accepted morphological appearances of malaria species, possibly due to drug pressure or strain variation, cannot be resolved merely by reference to an atlas of parasitology (22).…”

Multiplexed Real-Time PCR Assay for Discrimination of Plasmodium Species with Improved Sensitivity for Mixed Infections

“…For assays that use conserved primers, this is attributed to competition of the primers for multiple templates in the sample (3,12,14,19). Mixed species infections account for 3 to 5% of the malaria infections observed in areas where the disease is not endemic (15,20). They are particularly challenging for microscopists and represent one aspect of malaria diagnosis where molecular methods can clearly support conventional microscopy.…”

Real-Time PCR for Detection and Identification of Plasmodium spp

“…Previous studies have shown that even with experienced microscopists, misdiagnosis occurs, particularly in cases of mixed infection or low parasitemia (12,28). Immunochromatographic assays based on antigen detection have been developed but are also relatively insensitive in cases of low parasitemia (22,24,30). In addition, antigenemia may persist weeks beyond the actual infection, leading to the false diagnosis of malaria parasitemia (6,22).…”