Mixed malaria: the paradox of visual diagnosis in the tropics.
Authors: Rondón Carvajal Julián ,   Faculty of Medicine, University of Antioquia. Department of Internal Medicine, Pontificia Universidad Javeriana. Clinical Professor, Faculty of Medicine, CES University. Medellín, Colombia.
Published: July 2024

Malaria is the parasitic disease with the greatest impact on humans. It is an infectious disease caused by protozoa of the genus Plasmodium spp. and transmitted to humans by the bite of the mosquito Anopheles spp. Its incidence has increased in the tropical regions of Africa and Southeast Asia, areas already defined by the World Health Organization (WHO) as malarial zones [1]. In these places, malaria generally disappears at altitudes above 2.000 meters above sea level. The most frequently circulating parasites are P. vivax and P. falciparum; P. malarie is also widely distributed, but less frequent. In West Africa, P. ovale replaces P. vivax. The most widespread species in the world is P. vivax, which has been found from sea level to 2.770 meters above sea level. The endemic areas, however, are generally in the tropics. By definition, the number of parasites found in the peripheral blood depends on the species: the highest number corresponds to P. falciparum which infects 10 to 40% of all red blood cells; it is worth mentioning that when parasitaemia of this species reaches more than 25%, it is usually fatal [2]. Multiple invasion of red blood cells is frequent with P. falciparum, rare with P. vivax (which has a predominance of reticulocytes) and very rare with P. malariae [1- 3]. Without treatment, maximum parasite multiplication is reached in 2 weeks in P. vivax, and in 10 days in the case of P. falciparum. The simultaneous presence of erythrocytic parasitic forms (asexual and/or sexual) of two or more species of Plasmodium spp. is called mixed plasmodial infection. If, in addition, there are symptoms such as fever, chills, headache, sweating, among others, it corresponds to the disease called mixed malaria. In the world, the prevalence of this last entity is uncertain, depending on the diagnostic method used: 2% by light microscopy (e.g., thick blood smear [TBS] or peripheral blood smear) and up to 65% by polymerase chain reaction (PCR) [4]. In Latin America, prevalence is estimated at 0.46% by TBS and 12.8% by PCR. In terms of performance, the thick smear is 20-30 times more sensitive than the thin smear (peripheral blood smear), although less specific for the identification of the erythrocytic asexual (young rings or trophozoites, mature trophozoites, schizonts) and sexual (gametocytes) forms of the five species of Plasmodium spp. that parasitize humans (P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi), which explains an underdiagnosis of both clinical scenarios (both plasmodial infection and mixed malaria) by light microscopy, with serious implications for diagnostic and therapeutic guidance. Nevertheless, some methods have been developed to improve the diagnostic capability of TBS [2,3]: • Fluorescent stains (acridine orange): used to increase sensitivity of point-of-care diagnosis, without improving specificity of species identification; additionally, some stains may be toxic. • Fluorescence staining + microcentrifuge: increases diagnostic speed and sensitivity for P. falciparum; reduces sensitivity for other species. • Magnetic deposition: takes advantage of the magnetic characteristics of hemozoin to precipitate the parasites to the plate with a magnet. It is an inexpensive method. It increases sensitivity; however, it is not species specific, without good representation of ring stage parasites.