BackgroundThe prozone effect (or high doses-hook phenomenon) consists of false-negative or false-low results in immunological tests, due to an excess of either antigens or antibodies. Although frequently cited as a cause of false-negative results in malaria rapid diagnostic tests (RDTs), especially at high parasite densities of Plasmodium falciparum, it has been poorly documented. In this study, a panel of malaria RDTs was challenged with clinical samples with P. falciparum hyperparasitaemia (> 5% infected red blood cells).MethodsTwenty-two RDT brands were tested with seven samples, both undiluted and upon 10 ×, 50 × and 100 × dilutions in NaCl 0.9%. The P. falciparum targets included histidine-rich protein-2 (HRP-2, n = 17) and P. falciparum-specific parasite lactate dehydrogenase (Pf-pLDH, n = 5). Test lines intensities were recorded in the following categories: negative, faint, weak, medium or strong. The prozone effect was defined as an increase in test line intensity of at least one category after dilution, if observed upon duplicate testing and by two readers.ResultsSixteen of the 17 HRP-2 based RDTs were affected by prozone: the prozone effect was observed in at least one RDT sample/brand combination for 16/17 HRP-2 based RDTs in 6/7 samples, but not for any of the Pf-pLDH tests. The HRP-2 line intensities of the undiluted sample/brand combinations with prozone effect (n = 51) included a single negative (1.9%) and 29 faint and weak readings (56.9%). The other target lens (P. vivax-pLDH, pan-specific pLDH and aldolase) did not show a prozone effect.ConclusionThis study confirms the prozone effect as a cause of false-negative HRP-2 RDTs in samples with hyperparasitaemia.
Malaria rapid diagnostic tests (RDTs) are instrument-free tests that provide results within 20 min and can be used by community health workers. RDTs detect antigens produced by the Plasmodium parasite such as Plasmodium falciparum histidine-rich protein-2 (PfHPR2) and Plasmodium lactate dehydrogenase (pLDH). The accuracy of RDTs for the diagnosis of uncomplicated P. falciparum infection is equal or superior to routine microscopy (but inferior to expert microscopy). Sensitivity for Plasmodium vivax is 75-100%; for Plasmodium ovale and Plasmodium malariae, diagnostic performance is poor. Design limitations of RDTs include poor sensitivity at low parasite densities, susceptibility to the prozone effect (PfHRP2-detecting RDTs), false-negative results due to PfHRP2 deficiency in the case of pfhrp2 gene deletions (PfHRP2-detecting RDTs), cross-reactions between Plasmodium antigens and detection antibodies, false-positive results by other infections and susceptibility to heat and humidity. End-user's errors relate to safety, procedure (delayed reading, incorrect sample and buffer volumes) and interpretation (not recognizing invalid test results, disregarding faint test lines). Withholding antimalarial treatment in the case of negative RDT results tends to be infrequent and tendencies towards over-prescription of antibiotics have been noted. Numerous shortcomings in RDT kits' labelling, instructions for use (correctness and readability) and contents have been observed. The World Health Organization and partners actively address quality assurance of RDTs by comparative testing of RDTs, inspections of manufacturing sites, lot testing and training tools but no formal external quality assessment programme of end-user performance exists. Elimination of malaria requires RDTs with lower detection limits, for which nucleic acid amplification tests are under development.
BackgroundMalaria Rapid Diagnostic Tests (RDTs) are widely used for diagnosing malaria. The present retrospective study evaluated the CareStart™ Malaria HRP-2/pLDH (Pf/pan) Combo Test targeting the Plasmodium falciparum specific antigen histidine-rich protein (HRP-2) and the pan-Plasmodium antigen lactate dehydrogenase (pLDH) in a reference setting.MethodsThe CareStart™ Malaria HRP-2/pLDH (Pf/pan) Combo Test was evaluated on a collection of samples obtained in returned international travellers using microscopy corrected by PCR as the reference method. Included were P. falciparum (n = 320), Plasmodium vivax (n = 76), Plasmodium ovale (n = 76), Plasmodium malariae (n = 23) and Plasmodium negative samples (n = 95).ResultsOverall sensitivity for the detection of P. falciparum was 88.8%, increasing to 94.3% and 99.3% at parasite densities above 100 and 1,000/μl respectively. For P. vivax, P. ovale and P. malariae, overall sensitivities were 77.6%, 18.4% and 30.4% respectively. For P. vivax sensitivity reached 90.2% for parasite densities above 500/μl. Incorrect species identification occurred in 11/495 samples (2.2%), including 8/320 (2.5%) P. falciparum samples which generated only the pan-pLDH line. For P. falciparum samples, 205/284 (72.2%) HRP-2 test lines had strong or medium line intensities, while for all species the pan-pLDH lines were less intense, especially in the case of P. ovale. Agreement between observers was excellent (kappa values > 0.81 for positive and negative readings) and test results were reproducible. The test was easy to perform with good clearing of the background.ConclusionThe CareStart™ Malaria HRP-2/pLDH (Pf/pan) Combo Test performed well for the detection of P. falciparum and P. vivax, but sensitivities for P. ovale and P. malariae were poor.
BackgroundIn the Peruvian Amazon, Plasmodium falciparum and Plasmodium vivax malaria are endemic in rural areas, where microscopy is not available. Malaria rapid diagnostic tests (RDTs) provide quick and accurate diagnosis. However, pfhrp2 gene deletions may limit the use of histidine-rich protein-2 (PfHRP2) detecting RDTs. Further, cross-reactions of P. falciparum with P. vivax-specific test lines and vice versa may impair diagnostic specificity.MethodsThirteen RDT products were evaluated on 179 prospectively collected malaria positive samples. Species diagnosis was performed by microscopy and confirmed by PCR. Pfhrp2 gene deletions were assessed by PCR.ResultsSensitivity for P. falciparum diagnosis was lower for PfHRP2 compared to P. falciparum-specific Plasmodium lactate dehydrogenase (Pf-pLDH)- detecting RDTs (71.6% vs. 98.7%, p<0.001). Most (19/21) false negative PfHRP2 results were associated with pfhrp2 gene deletions (25.7% of 74 P. falciparum samples). Diagnostic sensitivity for P. vivax (101 samples) was excellent, except for two products. In 10/12 P. vivax-detecting RDT products, cross-reactions with the PfHRP2 or Pf-pLDH line occurred at a median frequency of 2.5% (range 0%–10.9%) of P. vivax samples assessed. In two RDT products, two and one P. falciparum samples respectively cross-reacted with the Pv-pLDH line. Two Pf-pLDH/pan-pLDH-detecting RDTs showed excellent sensitivity with few (1.0%) cross-reactions but showed faint Pf-pLDH lines in 24.7% and 38.9% of P. falciparum samples.ConclusionPfHRP2-detecting RDTs are not suitable in the Peruvian Amazon due to pfhrp2 gene deletions. Two Pf-pLDH-detecting RDTs performed excellently and are promising RDTs for this region although faint test lines are of concern.
Background: Two malaria rapid diagnostic tests were evaluated in a travel clinic setting: the SD FK50 Malaria Ag Plasmodium falciparum test (a two-band test) and the SD FK60 Malaria Ag P. falciparum/Pan test (a three-band test).
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