Morphological atypia and molecular profile of Plasmodium vivax: Findings from an outbreak in the Brazilian Amazon

Abstract

This study aimed to perform morphological and molecular analyses of parasites isolated from the blood of malaria-infected individuals during an outbreak in the Microregion of Cametá, State of Pará, Brazilian Amazon. A total of 260 positive samples were identified by microscopy as Plasmodium vivax; however, in three samples, forms considered unusual for the species were found and defined as morphological atypia of P. vivax. Single P. vivax infection was confirmed by qPCR in all samples. Among 256 genotyped samples, the VK247 genotype alone was identified in 255 samples, and the VK210 genotype was found in only one. The study showed that this malaria outbreak was caused by the etiological agent P. vivax, and for the first time, morphological atypia was described in isolates circulating in Brazil. Likewise, for the first time, the VK247 genotype was detected predominantly in single infections in an area of the State of Pará, which may suggest a greater circulation of the genotype in the region.

Keywords: Genotyping; Morphological atypia; Outbreak; Plasmodium vivax.

Figure 1

Atypical P. vivax morphologies found in sample CAM-185. Slides were prepared by the blood distension technique, stained by the Giemsa method, and visualized at 100× magnification under an optical microscope. (A) basket-form trophozoite; (B–C) gametocytes in oval red blood cells; (D) band-form gametocyte; (E) schizont in oval red blood cell; (F) simultaneous infection of schizont (with about 20 merozoites) and gametocyte in a single red blood cell. Bar = 10 μm.

Figure 2

Atypical P. vivax morphologies found in the sample CAM-190. Slides were prepared by the blood distension technique, stained by the Giemsa method, and visualized at 100× magnification under an optical microscope. (A) multi-infected red blood cell with trophozoites; (B) binucleated trophozoite; (C) band-form gametocyte; (D–F) elongated gametocytes resembling a sausage shape. Bar = 10 μm.

Figure 3

Atypical P. vivax morphologies found in the sample CAM-466. Slides were prepared by the blood distension technique, stained by the Giemsa method, and visualized at 100× magnification under an optical microscope. (A–B) multi-infected red blood cells with trophozoites; (C) gametocyte in oval red blood cell; (D) atypical gametocyte with extra nuclei similar to trophozoites nuclei; (E–F) simultaneous infection of schizont (with 19–21 merozoites) and gametocyte in a single red blood cell. Bar = 10 μm.

Figure 4

Samples showing a banding pattern compatible with the VK210 and VK247 genotypes after digestion with the AluI enzyme of the amplified product. Electrophoresis in 2.5% agarose gel visualized under ultraviolet light. (1) 50 bp molecular weight marker; (2–4) positive controls of the VK210, VK247 and P. vivax-like genotypes, respectively; (5) negative control; (6) single sample positive for the VK210 genotype; (7–12) samples positive for the VK247 genotype, with samples 7, 8 and 9 showing atypical morphology in the microscopic analysis.