Rapid Diagnostic Tests: It’s all about the Biomarkers

 Recent article in PLoS One on the deficiencies of Rapid Diagnostic Tests for Malaria testing in Peru offers significant lessons for designing effective biosensors. First, the synopsis from this article and few others I discovered while digging a little deeper.

  • Malaria remains a major infectious disease, causing between 250-500 million infections and nearly 1 to 3 million deaths per year.
  • Four species of Plasmodium causes malaria in humans, Plasmodium falciparum, P. vivax, P.malariae and P. ovale. P.falciparum and P. vivax are most prevalent.
  • As few as 10–100 Plasmodium parasites per microlitre of blood can produce significant illness in a naïve person.
  • Microscopic examination of blood smears is the traditional method for detecting malaria parasites and 50-100 parasites/ul of blood can be detected in a good lab. Sensitivity is only 500 parasites/ul in non specialized lab.
  • Rapid diagnostic devices for Malaria are immunochromatographic antigen–antibody capture assays in dipsticks and cassettes format.
  • All existing commercial RDTs target one or more of well characterized Plasmodium protein targets: histidine rich protein 2 (HRP2), parasite lactate dehydrogenase (pLDH), or aldolase
  • HRP2 is present in Plasmodium falciparum only, whereas pLDH and aldolase, are present in all four major species causing human malaria.
  • RDTs have good sensitivity for densities of P. falciparum greater than 500 parasites/μL.
  • World Health Organization recommends a lower sensitivity limit of detection for rapid diagnostic test for P. falciparum of 95% at a parasitaemia of 100P/μL.
  • Over 50 brands of malaria RDTs are manufactured, with over 150 individual products being commercially available.
  • Current tests are essentially qualitative and do not quantitate the risk of developing severe complication. In endemic areas a large proportion of the population will test positive in any qualitative assay, and the clinical relevance of the parasitaemia then needs to be determined.
  • Rapid diagnostic tests in tropical countries have limited shelf life and susceptible to degradation by excessive heat and humidity.

What the current study found was that number of malaria cases in Peru that were positive by microscopy came out negative when tested using RDTs. Extensive study uncovered that a large proportion of P. falciparum parasites in Peruvian Amazon lack HRP2 gene and hence do not express protein biomarker HRP2 detected by majority of RDTs. Study conducted before 2000 showed good sensitivity of RDTs that led authors to conclude that widespread of parasites lacking pfhrp2 gene may be a relatively recent event.

Though RDTs based on biomarkers pLDH and aldolase may be useful for this area a shortcoming for RDTs detecting multiple species is pointed out in another paper “Most RDTs that detect multiple species do not differentiate non-P. falciparum species from each other, nor do they differentiate mixed infections of P. falciparum and non-P. falciparum from P. falciparum monoinfection. This is important, as treatment regimens differ for P. falciparum and the other human species of malaria.”

Another Interesting article: Working without a blindfold: the critical role of diagnostics in malaria control

Lessons for Biosensor design-Stating the obvious: Sensitivity, selectivity, robustness, easy to use, multiplexing and awareness that bugs are smart and can fool methods designed to find them!

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