Examination of Plasmodium parasites in bone marrow puncture fluid for the diagnosis of imported-malaria in patients from the Yunnan Province, China

Abstract

Background China has been certified as a malaria-free country by World Health Organization (WHO) in June 2021, the pressure of continuously preventing the dissemination from imported-malaria urgently calls for timely monitoring and effective management the malaria cases. To compensate for the risk of missed diagnosis of malaria using peripheral blood examination alone, medical institutions in Yunnan Province launched bone marrow puncture examination to confirm Plasmodium infection more accurately. Methods Patients with a recent history of travelling to malaria-endemic areas outside of China, who were excluded from microbial infections such as tuberculosis and exhibited persistent abnormalities, including hepatosplenomegaly and decreased platelet pressure and the negative for Plasmodium lactate dehydrogenase (pLDH) antigen in the peripheral blood, were enrolled in the study. The cases ware conducted the bone marrow aspirate from anterior superior iliac spine to screen for Plasmodium infection in bone marrow fluid. Then the pLDH genes of the Plasmodium isolates from cases ware amplified by nested PCR. The amplification products were sequenced, and the B-cell epitopes and the oligomeric spatial structures of the pLDH peptide chains were analyzed. Results Five patients found Plasmodium infection in bone marrow fluid were included in the study, which included four patients with Plasmodium negative and one patient with Plasmodium falciparum positive in peripheral blood. Two pLDH negative cases in peripheral blood were confirmed as Plasmodium vivax infection, with the proportion of ring stage, large trophozoites, schizonts, and stage III-V gametocytes reaching 28.3%, 38.3%, 4.8%, 11.5%, 16.5% and 0.8%, respectively. The erythrocyte count and hemoglobin concentration of the five cases post-treatment merely increased to the lower end of the normal range. Platelet count returned to the normal range, increasing by 466%, 378%, 252%, 168% and 35%, respectively. There were four to five B-cell antigenic determinants along pLDH peptide chains of the infected strains in the five cases. Of them, the four sequences of 63th ~ 70th aa, 86th ~ 96th aa, 198th ~ 207th aa, 287th ~ 295th were commonly found on pLDH peptide chains from Plasmodium vivax, Plasmodium falciparum and Plasmodium malariea strains. Of note, the sequence of “211-EEVEGIFDR-220" was only detected in Plasmodium vivax strain, whereas the sequence of “207-LISDAE-213” was unique for Plasmodium falciparum strain. The spatial location of both two sequences was proximal to the “fusion region" of antigenic epitopes on the surface of pLDH tetramer. Conclusion Examination of the Plasmodium in bone marrow puncture fluid could make up for the missed diagnosis of malaria that solely relies on peripheral blood.