Morphologic features of Plasmodium in bone marrow fluid
Microscopic examination of Plasmodium in the bone marrow aspirate fluid showed that the volume of erythrocytes with Plasmodium parasites in case 1 and case 5 were not distended and slightly reduced, and exhibited a typical stripe-shaped large trophozoite morphology, and that the various stages of Plasmodium, including ring, large trophozoite, schizonts, and round gametophytes could be detected (Figure 1). Therefore, the two cases were identified as Plasmodium malariae infection, with large trophozoites occupying the largest proportion (38.3%, 153/200) (Table 3). The volume of erythrocytes with Plasmodium parasites of case 2 and case 3 was consistently distended. The ring-infected and large trophozoites-infected erythrocytes showed spine-like protrusion. While various stages of Plasmodium parasites were found (Figure 1), the largest and smallest proportions were large trophozoites (44.8%, 179/200) and stage V gametophytes (1.5%, 6/200), respectively. Hence, the two cases could be diagnosed as Plasmodium vivax infection (Table 3). The size of erythrocytes with Plasmodium parasites of case 4 remained normal. The Plasmodium parasites covers various stages, such as ring, large trophozoites, and crescent-shaped gametocytes (Figure 1), and the largest proportion was stage V gametocytes (24.5%, 49/200) (Table 3). Hence, case 4 was identified as Plasmodium falciparum infection.
Table 3 Constituent ratio of different Plasmodium parasites in this group patients
Cases
|
Species
|
Density
(parasites/ul)
|
No.
|
Stages
|
R
|
T
|
S
|
G (Ⅲ-Ⅳ)
No.
(%)
|
G (Ⅴ)
No.
(%)
|
No.
(%)
|
No.
(%)
|
No.
(%)
|
Case 1
|
P. malariae
|
560
|
200
|
38 (19.0)
|
60 (30.0)
|
19 (9.5)
|
83 (41.5)
|
0
|
Case 5
|
P. malariae
|
80
|
200
|
75 (37.5)
|
93 (46.5)
|
0
|
29 (14.5)
|
3 (1.5)
|
Total
|
--
|
600
|
400
|
113 (28.3)
|
153 (38.3)
|
19 (4.8)
|
112 (28.0)
|
3 (0.8)
|
Case 2
|
P. vivax
|
160
|
200
|
31 (15.5)
|
114 (57.0)
|
11 (5.5)
|
40(20.0)
|
4 (2.0)
|
Case 3
|
P. vivax
|
440
|
200
|
45 (22.5)
|
65 (32.5)
|
37 (18.5)
|
41 (21.5)
|
2 (1.0)
|
Total
|
--
|
640
|
400
|
76 (19.0)
|
179 (44.8)
|
48 (12.0)
|
68 (17.0)
|
6 (1.5)
|
Case 4
|
P. falciparum
|
120
|
100
|
0
|
2 (2.0)
|
0
|
9 (9.0)
|
89 (89.0)
|
Note: (1) R, T, S and G are the abbreviations of Ring, Trophozoites, Schizontes, and Gameocytes, respectively, (2) Ⅲ-Ⅳ: Stage Ⅲ-Ⅳ gametocytes, stage Ⅴ gametocytes.
|
A melioration of myelosuppression after antimalarial treatment
As none of the five patients exhibited any of the following unfavorable clinical manifestations, such as coma, severe anemia, acute renal failure, pulmonary edema or acute respiratory distress syndrome, hypoglycemia, circulatory failure or shock, or metabolic acidosis, they were diagnosed as non-severe malaria and received antimalarial treatment (Figure 2). In addition to chloroquine, artemether and artesunate were added to control the clinical malarial episodes of case 1.
The changes of hemogram test results of five patients before, during and after antimalarial treatment are shown in Figure 2. Red blood cell count (×109/L) and hemoglobin concentration (g/L) showed the pattern of parallel changes and remained roughly stable throughout the course, only reaching the lower end of normal range by the end of treatment. Although Red blood cell count increased significantly to 110×109/L and hemoglobin concentration was elevated to 3.81 g/L after antimalarial treatment (Figure 1), the increases were not high enough to reach the normal range, indicating that the indexs of erythrocyte lineage were hindered to return to normal within a short period of time even after the cause of malaria was removed.
Leukocytes and neutrophils of the granulocyte lineage also showed parallel changes, showing an upward trend and fluctuation afterwards in Case 3 and case 5. After antimalarial treatment, the leukocyte counts of cases 1, 2 and 3 returned to the normal range (5.27-7.05×109/L), and neutrophil count was within 2.55-4.41 (×109/L). Although leukocyte count and neutrophil count of case 4 were within the normal range throughout the course (leukocytes: 5.79-7.33 ×109/L), neutrophils: 2.38-3.819 ×109/L), both two indicators decreased after antimalarial treatment, with the magnitude of -30.2% and -35.3%, respectively. The decreasing patterns were present in case 5, and the indexes were only close to the low end of normal range after the recovery (Figure 2).
Pre-treatment platelet counts in case 1 and case 4 decreased by 35.1% and 20.0%, respectively, yet recovered to normal after the treatment, with the counts ranging from 146 to 455 (×109/L) (Figure 2). Platelet counts of the five cases rose by 466%, 378%, 252%, 168%, and 35%, respectively, indicating that megakaryocytes’ function of platelet production has been restored swiftly after the treatment of Plasmodium infection
B-cell epitope clustering of pLDH antigen in the Plasmodium strains
Four to five B-cell active antigenic regions were present in the primary peptide chains of Plasmodium pLDH in five cases (Table 3). Among them, the peptide chains of 63th ~70th aa, 86th ~96th aa, 198th ~207th aa and 287th ~295th aa were common, with an activity score of up to 0.43. The sequences of "85-PGKSDKEWNRD-96", "197-IPLQEFINNK-207", and "286-EQVIELQLN-295" were commonly distributed in the pLDH peptide chains of all five strains (Table 3). Moreover, variations at 66th aa and 68th aa were determined in the active region (63th aa to 70th aa) of pLDH peptide chain for different Plasmodium species (Table 4).
Table 4 Polymorphism of pLDH gene and prediction of B cell epitopes
Plasmodium
|
No.
epitopes
|
B-cell epitope
|
Completely homologous sequence
|
aMost homologous sequence
|
Unique sequence
|
Plasmodium vivax
|
5
|
85-PGKSDKEWNRD-96
197-IPLQEFINNK-207
286-EQVIELQLN-295
|
62-GSNSbYDcDL-70
|
211-EEVEGIFDR-220
|
Plasmodium falciparum
|
4
|
62-GSNTbYDcDL-70
|
207-LISDAE-213
|
Plasmodium malariae
|
4
|
62-GSNSbYEcDL-70
|
--
|
Note: a: There are two or less amino acid differences in B-cell epitopes among four species of human Plasmodium. Underline and bold indicate substituted amino acids between non-homologous sequences. b: When the 66thaa of the pLDH peptide chain is S, it belongs to the sequence of Plasmodium vivax and Plasmodium malariae, while when it is T, it belongs to the sequence of Plasmodium falciparum. c: When the 68thaa of the pLDH peptide chain is D, it belongs to the sequence of Plasmodium vivax and Plasmodium falciparum, when it is E, it belongs to the sequence of Plasmodium malariae.
|
pLDH peptide chains of Plasmodium varix and Plasmodium falciparum (pvLDH and pfLDH) exhibited specific B-cell antigenic active regions of "211-EEVEGIFDR-220" and "207-LISDAE-213", respectively (Table 4). pLDH antigen chains of all five malaria cases showed an oligomeric spatial conformation with four subunits (Figure 3A-E). The short peptides of the five epitope clusters in Table 4 were commonly distributed on the oligomeric surface. Among them, the spatial conformation and regional distribution of the active regions of 63th ~70th aa and 86th ~96th aa were mostly the same among Plasmodium malariae, Plasmodium vivax and Plasmodium falciparum. However, in the proximity of the fusion region composed of two active short peptides (198th ~207th aa and 287th ~295th aa), antigenic determinants of “211-EEVEGIFDR-220” and “207-LISDAE-213” were detected in pvLDH and pfLDH, respectively (Figure 3B, C and D).
No additional antigenic determinants were found in pmLDH (Figure 3A, E). Therefore, the antigenic determinants of 211-EEVEGIFDR-220 and 207-LISDAE-213 could be used in the differential diagnosis of Plasmodium vivax and Plasmodium falciparum infection by using RDT.