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.

BackgroundChina 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.MethodsPatients 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.ResultsFive 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.ConclusionExamination of the Plasmodium in bone marrow puncture fluid could make up for the missed diagnosis of malaria that solely relies on peripheral blood.

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.

Background More than 85% of the malaria burden is caused by imported vivax malaria in Yunnan Province and Yunnan is also where the majority of vivax malaria cases are diagnosed across China. Timely removal of the source of Plasmodium vivax and its breeding environment remains the key to eliminating the secondary transmission of imported malaria. To compensate for the uncertainty of epidemiological surveys in tracing vivax malaria recurrence, this study attempted to use molecular markers for identification. Materials and methods To do so, blood samples were collected from cases diagnosed and revalidated as single infections of Plasmodium vivax in Yunnan Province from 2013 to 2020. Specifically, samples from suspected relapses with recurrent episodes were subjected to PCR amplification, product sequencing, and analysis of the Plasmodium vivax circumsporozoite protein (pvcsp) gene. Results Seventy-eight suspected recurrent cases were retrieved from 2484 vivax malaria cases, with a total of 81 recurrent episodes. A total of 159 blood samples from primary infection Plasmodium vivax and recurrences were subjected to PCR amplification and sequencing to obtain 156 CDS sequences of pvcsp gene, 121 of which can be matched into the paired sequences of 59 cases. There were 475 polymorphic loci and 84 haplotypes (H01-H84) in the 121 sequences. Also, there were 79 and 5 haplotypes with CRR repeat units (PRM) of VK210 and VK247 structure, respectively. Of the 59 pairs of pvcsp gene sequences, every one of 31 pairs showed only one haplotype and no variant sites, meaning the every paired sequences were completely homologous and the paired Plasmodium vivax strains were homologous single clone. Every one of the remaining 28 paired sequences had two haplotypes but no length polymorphism, and except for 2 polymorphic loci (39 and 1027), all single nucleotide polymorphisms were double-equivalent bases differentially transferred between paired sequences, indicating that the paired sequences are "weakly heterologous" with no fragment insertions (or deletions) and only individual site polymorphisms. All 59 vivax malaria recurrences were respectively caused by the activation of Plasmodium vivax hypnozoites from the same population as the primary infection. Conclusions The paired analysis of the similarity of Plasmodium high variant genes allowed the identification of recurrent episodes caused by Plasmodium vivax homologous hypnozoites, and also demonstrated pvcsp gene as a good molecular marker. Moreover, the study showed most of the hypnozoites causing vivax malaria recurrence in Yunnan Province belonged to homologous single clone or sibling strains comparison with the original infection strains.