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Am. J. Trop. Med. Hyg., 77(1), 2007, pp. 3-13
Copyright © 2007 by The American Society of Tropical Medicine and Hygiene

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Clinico-Pathological Observations on the Pathogenesis of Severe Thrombocytopenia and Anemia Induced by Plasmodium vivax Infections During Antimalarial Drug Efficacy Trials in Aotus Monkeys

Nicanor Obaldía, III*
Malaria Drug and Vaccine Evaluation Center, Tropical Medicine Research, Panama City, Republic of Panama; Gorgas Memorial Institute of Health Studies (ICGES), Panama City, Republic of Panama


ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During routine antimalarial drug efficacy trials, we observed, for the first time, severe thrombocytopenia developing in Aotus monkeys infected with Plasmodium vivax. Data obtained from 26 Aotus infected with the AMRU-1 strain showed that 77% developed severe thrombocytopenia, whereas only 15% had severe anemia, with hemorrhagic diathesis ensuing in 31%. In general, thrombocytopenic monkeys either failed primary treatment with experimental antimalarial drugs or were found to have higher-density parasitemias, longer patency duration, and lower hematocrits. In these monkeys, severe thrombocytopenia inversely correlated to parasitemia (R = –1.0), and animals that received a blood transfusion had significantly higher platelet counts (P < 0.05) by day 38 after inoculation. In conclusion, the AMRU-1 strain of P. vivax, was considered to be highly pathogenic to Aotus monkeys, and thrombocytopenia rather than anemia should be regarded an early indicator of drug treatment failure with this strain.


INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Thrombocytopenia is a classic feature of malaria. However, severe thrombocytopenia is less well described in humans1 with vivax malaria. Human cases of immune-mediated vivax thrombocytopenia have been reported,1–3 and low platelet counts have been found in more Plasmodium vivax cases (74.7%) than Plasmodium falciparum cases (59.9%) in non-immune humans.4 Nevertheless, it was not until recently that human P. vivax malaria cases, exhibiting severe thrombocytopenia,1,5–7 cerebral malaria, disseminated intravascular coagulation (DIC), acute respiratory distress syndrome, renal failure, circulatory collapse, severe anemia, hemoglobinuria, and jaundice, were described.8 In addition, for the first time, a parallel trend between thrombocytopenia and parasitemia was found to be associated with both P. falciparum and P. vivax infection.9 Likewise, an inverse relationship between platelets and parasite levels in humans (P. vivax and P. falciparum) and animal models such as mice infected with Plasmodium berghei berghei10,11 and Saimiri and Aotus monkeys infected with P. falciparum have been observed.12–16 Herein, we report for the first time severe thrombocytopenia (< 50 x 103 platelets/µL) and hemorraghic diathesis occurring in Aotus monkeys infected with the AMRU-1 strain of P. vivax. We also examine its correlation with parasitemia and other blood parameters to understand the pathogenesis of the severe thrombocytopenia observed in this animal model.


MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Data were obtained from spleen intact (male and female) Aotus lemurinus lemurinus monkeys karyotypes VIII and IX,17 with weights of 700–998 g, that had been previously exposed to P. falciparum and cured. Twenty-six were used in two P. vivax drug efficacy trials (DETs; Trial 1 and Trial 2), and six were used as controls in a vaccine study. The animals were housed at Gorgas Memorial Institute in Panama and cared and maintained as described.18 Original protocols were approved by the Gorgas Memorial Institutional Animal Care and Use Committee, under accession numbers 2001/01 and 2002/01, and radical treatment of infection with a curative dose of mefloquine was the designed endpoint of the experiments. The data presented herein is a collateral experimental retrospective data analysis of the original experimental protocols. The experiments reported here were conducted according to the principles set forth in the "Guide for the Care and Use of Laboratory Animals," Institute of Laboratory Animal Resources, National Research Council (1996).

Study design. P. vivax AMRU-1. Trial 1 consisted of 14 monkeys that were used to test the efficacy of malaria DHFR inhibitors pyrroloquinazolinediamine derivatives PQZ-4A, with two monkeys per dose level at 0.5, 1, and 3 mg/kg/3 d (Treatment Groups 1, 2, and 3, respectively), and PQZ-BE at 0.5, 1, and 3 mg/kg/3 d (Treatment Groups 4, 5, and 6, respectively); Trial 2 consisted of 12 monkeys to test DHFR inhibitors A, with two monkeys per dose level at 1.25, 2.5, and 5 mg/kg/3 d (Treatment Groups 1, 2, and 3, respectively), B at 2.5 mg/kg/3 d (Treatment Group 4), and pyrimethamine at 5 mg/kg/3 d (Treatment Group 5). All animals of this trial were retreated with PQZ-4A at 1 mg/kg/3 d after primary drug treatment failure on Day 10 post-inoculation (PI). Control monkeys from both Trials 1 and 2 did not receive treatment with pyrroloquinazolinediamine PQZ-4A at 1 mg/kg/3 d until Days 10 and 11, PI, respectively. Drugs were administered by gastric intubation using a 12-Fr urethral catheter (Davol), on a milligram base per kilogram of body weight basis for 3 days when parasitemia reached > 5,000 parasites/µL, which was on Day 6 PI in Trial 1 and on Day 5 PI in Trial 2.

P. vivax Sal-I. Six animals that served as controls for a vaccine study were followed up for parasitemia, complete blood count (CBC), and chemistry profile (0.2 mL EDTA) at different time intervals up to 35 days PI when they received radical treatment with mefloquine at 20 mg/kg to end the experiment.

Parasite inoculation. P. vivax AMRU-1. Trial 1 and 2 animals were inoculated intravenously in the saphenous vein with 5 x 106 P. vivax AMRU-1 parasites on its 60th Aotus passage in Trial 1 and the 61st passage in Trial 2. This strain, a chloroquine- and pyrimethamine-resistant isolate from Papua New Guinea, originally isolated in 199419 (received from the Walter Reed Army Institute of Research in June 24, 1993) and adapted to Panamanian Aotus monkeys,20 was not considered to be highly pathogenic and lethal to Aotus l. lemurinus when these studies were conducted but needed treatment with mefloquine to end the infection.

P. vivax Sal-I. Control vaccine trial animals were inoculated intravenously with 10,000 P. vivax Sal-I parasites (received from CDC in May 6, 1997) that generally self-cure without treatment.

Parasitemia determination. Parasite counts were done by the method of Earle and Perez,21 using daily Giemsa-stained thick blood smears obtained from a prick in the marginal ear vein until parasite clearance occurred, and bi-weekly thereafter, up to day 100 PI when the animals were considered cured. If recrudescence occurred, daily blood smears were resumed.

Groups selection criteria. P. vivax AMRU-1. To analyze the efficacy of primary antimalarial treatment and its effect over parasitemia, CBC, and chemistry profile, the animals used in DET s1 and 2 were divided further into three clinical outcome groups (a, b, and c) using the following criteria: Group a consisted of three Aotus monkeys from Trial 1, MN13073, 13123, and 13079 (Table 1Go), in which primary treatment was effective, and the animals did not receive either mefloquine treatment or a blood transfusion. Group b consisted of 11 monkeys from Trial 1 (MN13022, 12841, 13068, 12857, 12840, 13018, 12856, 13011, 13078, 13122, and 13019), and Group c consisted of 12 monkeys from Trial 2 (MN12953, 12886, 13028, 12848, 12930, 13087, 13024, 12847, 13034, 12918, 13035, and 12997), which failed primary treatment and needed mefloquine treatment, a blood transfusion, or both (Table 1Go).


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TABLE 1
Parasitologic and hematologic findings of clinical outcome groups in P. vivax AMRU-1–infected Aotus monkeys during drug efficacy trials
 
Criteria for thrombocytopenia and anemia. Thrombocytopenia was considered slight when platelet counts were between 149 and 100 x 103/µL; moderate if between 99 and 50 x 103/µL; or severe if < 50 x 103/µL. Anemia was considered severe when hemoglobin concentration was < 6 g/dL.

Criteria for radical treatment or blood transfusions. Radical treatment with mefloquine (a single 20-mg/kg oral dose), was administered to end the experiment if the animals presented any or a combination of the following: 1) severe thrombocytopenia (< 50 x 103/µL), 2) severe anemia (> 50% hematocrit reduction from baseline or hemoglobin < 6 g/dL), or 3) persistent parasitemia beyond day 15. Transfusions with 2–3 mL of citrated whole blood—non–cross-matched—from a healthy donor monkey were carried out if the animals developed severe thromobocytopenia, anemia, or bleed from the gums22 (Tables 1Go and 3Go).


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TABLE 3
Platelet counts in Aotus monkeys infected with P. vivax AMRU-1 and treated with or without transfusion, mefloquine, or both
 
Complete blood counts and chemistry determinations. P. vivax AMRU-1. Blood samples for CBC (0.2 mL EDTA) were evaluated using an automatic cell Counter (Coulter; Beckman, Miami, FL), and manual techniques were used for validation of hematocrit, differential white blood cell count, retyculocyte counts, reticulocyte index adjusted for hematocrit, red cell abnormalities, and exclusion of platelet clumping as a cause for decreased platelet counts. Chemistry profiles for glutamate pyruvate transaminase (GPT), creatinine, and blood urea nitrogen (BUN) were done using a Roche Reflotron System (Basel, Switzerland). In Trial 1 (Groups a and b), blood samples were taken on Days 0, 5, 9, 13, 20, 27, 39, 48, and 62 PI; in Trial 2 (Group c) blood samples were taken on Days 0, 5, 8, 12, 31, 40, and 54 PI.

P. vivax Sal-I. In these animals, blood samples were collected (0.2 mL EDTA) on Days 0, 6, 13, 17, 20, and 28 PI and processed as indicated above.

Data analysis. SigmaStat and SigmaPlot software were used to perform regression and correlation analysis, Mann-Whitney U test and {chi}2 test were used for for non-parametric data, and P ≤ 0.05 was considered statistically significant.


RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Parasitologic and hematologic findings. P. vivax AMRU-1. As shown in Tables 1Go, 2Go, and 3Go, Group a monkeys had a significantly lower mean maximum parasitemia (3; 95% CI: 1, 5; P = 0.010 against Group b; P = 0.004 against Group c), mean day of maximum parasitemia (6; P = 0.038 against Group b; P = 0.004 against Group c), and shorter duration of patency (10; 95% CI: 9, 11; P = 0.005 against Group b; P = 0.004 against Group c) compared with Groups b and c. In this group, moderate to slight thrombocytopenia was observed (mean minimum platelet count = 99; 95% CI: 62, 136), whereas severe thrombocytopenia was observed in Groups b (26; 95% CI: 12, 40) and c (31; 95% CI: 20, 42). When the day of minimum platelet count was determined, no significant difference was found between Groups a and b (P = 0.170), but there was a difference between Groups a and c (P = 0.004) and b and c (P = 0.000015). The mean minimum HCT% was significantly different between Groups a and b (P = 0.038) and a and c (P = 0.008), but was not different between Groups b and c (P = 0.694). In contrast, the day of minimum HCT% was not significantly different between Groups a and b (P = 0.087) and Groups a and c (P = 0.448), but was different between Groups b and c (P = 0.000015). A highly significant difference was found in the day of treatment with mefloquine to end the experiments between Groups b (23; 95% CI: 19, 27) and c (13; P = 0.000031). No statistical significance was found in mean HCT reduction %, and maximum reticulocyte count between groups, but the mean day of maximum reticulocyte count was significantly different between Groups a and c (P = 0.048). Other blood cell parameters such as WBC and the chemistry profile (GPT, creatinine, and BUN; data not shown) were not significantly different between groups. Correlation analysis applied to the mean parasitemia and platelet values showed no correlation in Group a (R = 0.216), but an inverse correlation was detected in Groups b (R = –1.0) and c (R = –0.95; Figure 1Go). When daily parasitemia, erythrocytes, hemoglobin, hematocrit, platelets, reticulocytes, and reticulotcyte index were plotted for each monkey (Figures 2Go, 3Go, and 4Go), a pattern emerged suggesting that, regardless of hemoglobin, hematocrit, or platelets, reticulocytosis did not develop until parasitemia was radically cured with mefloquine as shown for MN12847 (Figure 4A–CGo). An exception to this pattern was observed in Group c monkey MN12997 (Figure 4D–FGo), in which a suppression of reticulocytes with a pattern consistent with transient pancytopenia (indicated by a reticulocyte index < 2) was present until Day 54 PI, even after mefloquine treatment and transfusion. At this time, an elevation in creatinine and BUN was detected in the latter.


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TABLE 2
Chronology of parasitologic and hematologic values in Aotus monkeys infected with Plasmodium vivax AMRU-1 during drug efficacy trials
 

Figure 1
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  		    FIGURE 1. Lineal regression analysis plots between mean density parasitemia and mean platelet counts for Groups a, b, and c, panels A, B, and C, respectively; on the first 8–9 days of infection with Plasmodium vivax AMRU-1. Inset is the correlation coefficient value for each group.

 

Figure 2
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  		    FIGURE 2. Individual plotted values for parasitemia, erythrocytes, hemoglobin, hematocrit %, platelets, reticulocyte index, for Group a Aotus, in which primary treatment was effective (Panels A–F), Group b, treated with mefloquine but had a transfusion (Panels G–L), and Group c treated with mefloquine and a blood transfusion (Panels M–R). Horizontal dotted lines indicates: a) lower hemoglobin limit for severe anemia (< 6 gm/dL) (Panels C, I, and O), b) lower platelet limit for severe thrombocytopenia (< 50,000 platelets x µL) (Panels E, K, and Q) and c) limit for bone marrow suppression (reticulocyte index < 2) (Panels F, L, and R). In the upper right of the first panel of each column is the monkey number.

 

Figure 3
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  		    FIGURE 3. Individual plotted values for white blood cells (WBC), glutamate pyruvate transaminase (GPT); creatinine and blood urea nitrogen (BUN) and weight, for Group a Aotus, in which primary treatment was effective (Panels A–E), Group b, treated with mefloquine but had a transfusion (Panels F–J) and Group c, treated with mefloquine and a blood transfusion (Panels Q–N). In the upper right of the first panel of each column is the monkey number.

 

Figure 4
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  		    FIGURE 4. Plotted values for parasitemia, white blood cells (WBC), platelets, erythrocytes, hematocrit % (HCT %), reticulocytes %, and chemistry profile [glutamate pyruvate transaminase (GPT); creatinine and blood urea nitrogen (BUN)] for Group c monkeys MN12847 panels A–C and MN12997 panels D–F, treated with mefloquine on day 13 post-inoculation (vertical dash line) and transfused on day 19 with citrated whole blood (arrow), are shown for the 54 day followup period after challenge with 5 x 106 Plasmodium vivax AMRU-1 infected Aotus erythrocytes. In the upper right is the monkey number.

 
P. vivax Sal-I. In this group of six animals, as shown in Table 4Go, although the mean parasitemia density peak on Day 17 PI (38.48 ± 37.74 x 103/µL), severe thrombocytopenia and anemia only developed 4 (Day 21 PI) and 11 days (Day 28 PI) later, respectively. Correlation analysis in Table 5Go shows an inverse correlation between parasitemia, erythrocytes, hematocrit, hemoglobin, and platelets but a positive correlation between parasitemia and WBC. When parasitemia, reticulocytes, GPT, creatinine, BUN, and weight were compared, no significant correlation was found. In contrast, an inverse correlation was found between platelets, WBC, and GPT and between creatinine and weight.


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TABLE 4
Chronology of parasitologic, hematologic values and chemistry profile of Aotus monkeys infected with P. vivax Sal-I
 

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TABLE 5
Correlation matrix between parasitemia, hematologic values, chemistry profile, and weight during infection with P. vivax Sal-I in Aotus monkeys
 
Blood transfusions. P. vivax AMRU-1. A striking finding was that 6 (60%) of 10 transfused animals from Groups b and c (Group 3; Table 3Go) that eventually recovered had significantly different platelet counts (geometric mean: 936 ± 234 x 103/µL) compared with Group 1 (P < 0.05) and Group 2 (P < 0.01). An exception was for MN12997 shown in Figure 4D–FGo, which was removed from the calculations because of a pancytopenic pattern. Some monkeys, such as MN12847 shown in Figure 4A–CGo, reached platelet levels after transfusion > 1.2 x 106/µL on Day 40 PI and returned to normal by Day 54 PI. In Group c, 62% (5/8) of the monkeys that received a blood transfusion responded favorably (P < 0.05, {chi}2 = 4.285; Table 1Go).

Clinical and pathologic findings. P. vivax AMRU-1. Of the 26 monkeys examined from DETs 1 and 2 Groups a, b, and c, all developed thrombocytopenia. Twenty (77%) developed severe thrombocytopenia, five (19%) developed moderate thrombocytopenia, and only one (4%) developed slight thrombocytopenia. In contrast, only 4 of 26 (15%) presented with severe anemia (P < 0.001, {chi}2 = 19.809). Hemorrhagic diathesis manifesting as bleeding from the gums was found in 8 of 26 (31%). Internal bleeding, characterized by profuse parlor and petechial and/or ecchymotic hemorraghes in all serosal surfaces, was present at necropsy.


DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Historically since 1976 at Gorgas Memorial Institute, experimental P. vivax infections in Aotus monkeys used in DETs have been considered benign and self-curable. In retrospect, however, when the AMRU-1 strain of P. vivax was used, it was found that 20 (77%) of 26 monkeys that presented severe thrombocytopenia, 8 (31%) developed hemorraghic diathesis and 4 (15%) had severe anemia. In general, severe thrombocytopenic monkeys failed treatment, had a faster decrease in platelet count, higher density parasitemia, longer patency duration, and lowest HCT compared those successfully treated, that developed a slight to moderate thrombocytopenia and anemia (Table 1Go). These findings contrast with those in semi-immune Aotus monkeys infected with P. falciparum, where anemia developed in 30%, and thrombocytopenia, although present, was not considered severe.16 Generally, severe thrombocytopenia occurred on Day 12 PI in the P. vivax AMRU-1–inoculated Aotus monkeys (Figures 2K and QGo and 4AGo), 2 and 4 days before that reported for Aotus monkeys infected with P. falciparum15,16 and 9 days before the P. vivax Sal-I inoculated group of this study (Table 4Go). In both instances, an inverse correlation between parasitemia and platelets was observed (Figure 1Go; Table 5Go). This correlation has also been observed in Saimiri and Aotus monkeys infected with P. falciparum12–16 and mice infected with P. b. berghei.10,11

Although a number of factors other than density of parasitemia could have contributed to the shortened life span of platelets observed in these studies, such as platelet hypersensitivity,10 direct parasitization of platelets,23 antiplatelet auto-antibodies,3,11,24,25 platelet-mediated clumping,26 platelet dysfunction, aggregation and sequestration,27,28 platelet oxidative stress,29 DIC,30 and macrophage colony-stimulating factor activation of the reticuloendothelial removal system,13 it is evident from the results obtained in the correlation analysis performed in these studies, that parasite density could be implicated as a direct cause of the severe thrombocytopenia observed in these animals (Figures 1Go, 2K and QGo, and 4AGo; Tables 4Go and 5Go). For instance, it is known that P. falciparum–infected red blood cells (IRBCs) bind to platelets through the CD36 surface receptor31 and that human megakaryocytes and platelets express CD36 on their surface.32 In addition, activation of human platelets by IRBCs is triggered by parasite-derived substances shed from the erythrocyte membrane.33 However, this mechanism has not been characterized in Aotus monkeys, and a homologous receptor in Aotus platelets or megakaryocytes has yet to be identified. Moreover, unlike cells infected with P. falciparum, those infected with P. vivax do not adhere to platelets or purified CD36 receptors, suggesting that thrombocytopenia in vivax malaria is not related to platelet–red cell attachment, although P. vivax–infected red cells can form rosettes.34

The fact that control monkeys from both Trials 1 and 2 developed severe thrombocytopenia 1–3 days after receiving treatment with pyrroloquinazolinediamine PQZ-4A on Days 10 and 11 PI, too early to be associated with drug-associated bone marrow toxicity, which have been reported for other DHFR inhibitors when used for prolonged periods of time,35–38 supports our findings that an inverse correlation exists between parasitemia and thrombocytopenia (Figure 1Go), as was also observed with the monkeys from the vaccine study (Table 5Go).

Based on these observations, we postulate that P. vivax–infected cells release parasitic-derived substances39,40 or induce the release of pathologic amounts of inflammatory cytokines.41,42 That in turn inhibits platelet production at the bone marrow level or depletes a plasma factor needed for thrombo and/or erythropoiesis,43,44 as observed in the depressed reticulocyte index (< 2) before radical treatment with mefloquine (Figure 2L–RGo). This hypothesis is supported by the fact that, once a blood transfusion was administered to the monkeys in Groups b and c (Table 3Go), platelets—that are known to have a half-life of 7–9 days45—recovered in great numbers (P < 0.05) by Day 38 PI (19 days after transfusion; Figure 2QGo). Observation, that cannot be only explained by the numbers of transfused cells; but indicated that a plasma factor, depleted during infection, could have played an important role in this remarkable recovery. The proposed model is supported by the findings in this study, and those of others,16,46 that reticulocytosis did not develop until parasitemia density had fallen below detectable levels or the animals are radically cured with mefloquine (Figure 4A–CGo). An exception to this pattern was observed in one animal (MN12997) from Group c (Figure 4D–FGo), which presented a reticulocyte suppression until Day 54 PI. In this animal, platelets recovered to baseline by Day 31 PI (12 days after transfusion), but the animal became thrombocytopenic again by Day 54 PI (75 x 103/µL), indicating an immune-mediated thrombocytopenia, a transient drug-induced bone marrow suppression, or an impaired erythro- and/or thrombopoiesis caused by renal disease, as indicated by the increase in creatinine and BUN (Figure 4FGo). This observation could help explain why 38% of the monkeys in Group c did not respond to a blood transfusion, although they had been treated with mefloquine on average 10 days earlier than Group b (Day 13 PI; P = 0.0000031).

In conclusion, P. vivax AMRU-1 induced severe thrombocytopenia in Aotus monkeys, which was inversely correlated to density of parasitemia. Severe thrombocytopenia should be considered an early indicator of drug failure during DETs in Aotus infected with P. vivax. Aotus monkeys infected with P. vivax are good models for studies of severe thrombocytopenia and anemia mechanisms in vivax malaria, which could help elucidate the pathogenesis of the severe complicated P. vivax malaria cases detected recently in humans.

Received December 22, 2005. Accepted for publication March 20, 2007.

Acknowledgments: The author thanks William Otero and Jorge Aparicio for microscopy and technical assistance, Camilo Marin and the animal care takers for assistance with animal handling and care, Maritza Brewer for secretarial assistance, Gladyz Calviño for administrative assistance, and the directors of Tropical Medicine Research (TMR), Malaria Drug and Vaccine Evaluation Center/Gorgas Memorial Institute (ICGES), in Panama City, Panama for their constant encouragement and support.

Financial support: This work was supported by USAMRDC Contract DAMD17-01-C0039.

Disclaimer: The opinions and assertions contained herein are the private ones of the author and are not to be construed as official or reflecting the views of the U.S. Army.

* Address correspondence to Nicanor Obaldía III, ICGES, Apdo 00816-02593, Panama, Republic of Panama. E-mail: nobaldia{at}gorgas.gob.pa Back

Author’s address: Nicanor Obaldía III, ICGES, Apdo 00816-02593, Panama, Republic of Panama. E-mail: nobaldia{at}gorgas.gob.pa.


REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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N. Obaldia III, W. Milhous, and D. Kyle
Adaptation of a Thai Multidrug-Resistant C2A Clone of Plasmodium falciparum to Aotus Monkeys and Its Preliminary in vivo Antimalarial Drug Efficacy-Resistance Profile
Am J Trop Med Hyg, October 1, 2009; 81(4): 587 - 594.
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