Examination of Plasmodium parasites in bone marrow puncture fluid for the diagnosis of imported malaria patients in Yunnan Province, China
Authors: Huang Ying , author) Yan Wu(Co-first , Dong Ying , He Hua , Liang Guiliang , Xu Jun , Luo Yun , Li Yang , Zhang Yanling , author) Hongli Huang(Co-corresponding 
Published: February 2022

Abstract
BackgroundChina has been certified as a malaria-free country by World Health Organization (WHO) in June 2021, yet the pressure of preventing the dissemination of imported malaria persists and thus calling for continued effort of timely detection and management. To compensate for the risk of missed diagnosis of using peripheral blood alone, medical institutions in Yunnan Province launched bone marrow puncture to confirm malaria 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 infected strains from cases ware amplified by conducted 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 Bone marrow puncture was performed on 5 eligible subjects in total. The Plasmodium were found in the bone marrow fluid from all cases, which included two patients with pLDH-negative and three patients with pLDH-positive in peripheral blood detected by malaria rapid tests (RDTs). Two pLDH negative cases under malaria RDTs were diagnosed 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 were proximal to the “fusion region" of antigenic epitopes on the surface of pLDH tetramer. Conclusion Examination of the Plasmodium infection in Bone marrow puncture fluid could make up for the missed diagnosis of malaria that solely relies on peripheral blood examination. For patients who travelled to malaria-endemic areas without common pathogenic microbial infections, yet with hepatosplenomegaly, granulocytic myelosuppression, decreased platelet counts, low level of hemoglobin and suspicious pLDH antigen results, bone marrow puncture can be applied to confirm the diagnosis of malaria more accurately.