Platelet profile is associated with clinical complications in patients with vivax and falciparum malaria in Colombia

Edgar Leonardo Martínez-Salazar Alberto Tobón-Castaño About the authors

Abstract

Introduction

Thrombocytopenia is a common complication in malaria patients. The relationship between abnormal platelet profile and clinical status in malaria patients is unclear. In low and unstable endemic regions where vivax malaria predominates, the hematologic profiles of malaria patients and their clinical utility are poorly understood. The aim of this study was to characterize the thrombograms of malaria patients from Colombia, where Plasmodium vivax infection is common, and to explore the relationship between thrombograms and clinical status.

Methods

Eight hundred sixty-two malaria patients were enrolled, including 533 (61.8%) patients infected with Plasmodium falciparum, 311 (36.1%) patients infected with Plasmodium vivax and 18 (2.1%) patients with mixed infections.

Results

The most frequently observed changes were low platelet count (PC) and high platelet distribution width (PDW), which were observed in 65% of patients; thrombocytopenia with <50,000 platelets/µL was identified in 11% of patients. Patients with complications had lower PC and plateletcrit (PT) and higher PDW values. A higher risk of thrombocytopenia was identified in patients with severe anemia, neurologic complications, pulmonary complications, liver dysfunction, renal impairment and severe hypoglycemia. The presence of thrombocytopenia (<150,000 platelets/µL) was associated with a higher probability of liver dysfunction.

Conclusions

Young age, longer duration of illness and higher parasitemia are associated with severe thrombocytopenia. Our study showed that thrombocytopenia is related to malaria complications, especially liver dysfunction. High PDW in patients with severe malaria may explain the mechanisms of thrombocytopenia that is common in this group of patients.

Thrombogram; Thrombocytopenia; Platelet; Plasmodium ; Severe malaria; Colombia


INTRODUCTION

Malaria is a disease caused by infection with Plasmodium parasites11. World Health Organization. The Roll Back Malaria partnership. Geneva: WHO; 2008. and is a major health problem worldwide. According to the World Malaria Report, 219 million malaria cases and 660,000 malaria-related deaths occurred worldwide in 201022. World Health Organization. World malaria report 2012. Geneva:WHO; 2012..

Plasmodium falciparum is the main cause of malaria-related death worldwide; however, other species can also cause serious illness33. Price RN, Douglas NM, Anstey NM. New developments in Plasmodium vivax malaria: severe disease and the rise of chloroquine resistance. Curr Opin Infect Dis 2009; 22:430-435.,44. Picot S, Bienvenu AL. Plasmodium vivax infection: not so benign. Med Sci (Paris) 2009; 25:622-626.. Clinical complications in malaria patients include cerebral malaria, severe anemia (SA), acute kidney failure (AKF), pulmonary edema (PE), severe hypoglycemia, shock, disseminated intravascular coagulation (DIC), acidosis and massive hemolysis. A patient who presents with one or more of these conditions is diagnosed with severe malaria (SM) and has an increased risk of mortality55. World Health Organization. Severe falciparum malaria. World Health Organization, Communicable Diseases Cluster. Trans R Soc Trop Med Hyg 2000; 94 (suppl I):1-90..

Different organs can be affected during a malaria episode, which results in localized or systemic injury. Hematological changes, especially anemia and thrombocytopenia, are common66. Lacerda MV, Mourão MP, Coelho HC, Santos JB. Thrombocytopenia in malaria: who cares? Mem Inst Oswaldo Cruz 2011; 106:52-63.,77. Quintero JP, Siqueira AM, Tobón A, Blair S, Moreno A, Arévalo-Herrera M, et al. Malaria-related anaemia: a Latin American perspective. Mem Inst Oswaldo Cruz 2011; 106:91-104.. Thrombocytopenia has been associated with Plasmodium knowlesi88. Daneshvar C, Davis T, Cox-Singh J, Rafa'ee MZ, Zakaria SK, Divis PCS, et al. Clinical and laboratory features of human Plasmodium knowlesi infection. Clin Infect Dis 2009; 49:852-860., Plasmodium falciparum99. Ladhani S, Lowe B, Cole AO, Kowuondo K, Newton CR. Changes in white blood cells and platelets in children with falciparum malaria: relationship to disease outcome. Br J Haematol 2002; 119:839-847., Plasmodium vivax, Plasmodium ovale1010. Horstmann R, Dietrich M. Haemostatic alterations in malaria correlate to parasitaemia. Ann Hematol 1985; 51:329-335. and Plasmodium malariae infections1111. Hong YJ, Yang SY, Lee K, Kim TS, Kim HB, Park KU, et al. A case of imported Plasmodium malariae malaria. Ann Lab Med 2012; 32:229-233.. The evidence is mixed regarding the existence of a correlation between platelet count (PC) and parasitemia88. Daneshvar C, Davis T, Cox-Singh J, Rafa'ee MZ, Zakaria SK, Divis PCS, et al. Clinical and laboratory features of human Plasmodium knowlesi infection. Clin Infect Dis 2009; 49:852-860.1010. Horstmann R, Dietrich M. Haemostatic alterations in malaria correlate to parasitaemia. Ann Hematol 1985; 51:329-335.,1212. Richards MW, Behrens RH, Doherty JF. Short report: hematologic changes in acute, imported Plasmodium falciparum malaria. Am J Trop Med Hyg 1998; 59:859.1414. Touze JE, Mercier P, Rogier C, Hovette P, Schmoor P, Dabanian C, et al. Platelet antibody activity in malaria thrombocytopenia. Pathol Biol (Paris) 1990; 38:678-681.. In a systematic review, Lacerda et al. found no differences in the frequency of thrombocytopenia across species of Plasmodium; the frequency of thrombocytopenia ranged from 24-94% in all studies66. Lacerda MV, Mourão MP, Coelho HC, Santos JB. Thrombocytopenia in malaria: who cares? Mem Inst Oswaldo Cruz 2011; 106:52-63.. The frequency of thrombocytopenia ranged from 8.0-64.2% in Colombian patients1515. Gonzalez LM, Guzmán M, Carmona J, Lopera T, Blair S. Clinical and epidemiologic characteristics of 291 hospitalized patients for malaria in Medellin (Colombia). Acta Med Colomb 2000; 25:163-170.1717. Tobon A. Danger signs in the malaria patient. Biomedica 2009; 29:320-329..

Various mechanisms have been proposed to explain thrombocytopenia during malaria episodes, including platelet destruction by immune mechanisms1414. Touze JE, Mercier P, Rogier C, Hovette P, Schmoor P, Dabanian C, et al. Platelet antibody activity in malaria thrombocytopenia. Pathol Biol (Paris) 1990; 38:678-681.,1818. Ohtaka M, Ohyashiki K, Iwabuchi H, Iwabuchi A, Lin KY, Toyama K. A case of vivax malaria with thrombocytopenia suggesting immunological mechanisms. Rinsho Ketsueki 1993; 34:490-492.2020. Rios A, Alvarez T, Carmona J, Blair S. Temporal evolution of platelets and anti-platelet antibodies in patients of endemic area with non complicated malaria. An Med Interna 2005; 22:561-568.; low medullar platelet production1414. Touze JE, Mercier P, Rogier C, Hovette P, Schmoor P, Dabanian C, et al. Platelet antibody activity in malaria thrombocytopenia. Pathol Biol (Paris) 1990; 38:678-681.,2121. Abdalla SH. Hematopoiesis in human malaria. Blood Cells 1990; 16:401-416.,2222. Beale PJ, Cormack JD, Oldrey TB. Thrombocytopenia in malaria with immunoglobulin (IgM) changes. Br Med J 1972; 1:345-349.; low thrombopoietin (TPO) synthesis2323. Kreil A, Wenisch C, Brittenham G, Looareesuwan S, Peck-Radosavljevic M. Thrombopoietin in Plasmodium falciparum malaria. Br J Haematol 2000; 109:534-536.; platelet sequestration in organs, such as the spleen2424. Skudowitz RB, Katz J, Lurie A, Levin J, Metz J. Mechanisms of thrombocytopenia in malignant tertian malaria. Br Med J 1973; 2:515-518.,2525. Watier H, Verwaerde C, Landau I, Werner E, Fontaine J, Capron A, et al. T-cell-dependent immunity and thrombocytopenia in rats infected with Plasmodium chabaudi. Infect Immun 1992; 60:136-142. or brain2626. Gall C, Spuler A, Fraunberger P. Subarachnoid hemorrhage in a patient with cerebral malaria. N Engl J Med 1999; 341:611-613.2828. Wassmer SC, Taylor T, Maclennan CA, Kanjala M, Mukaka M. Platelet-induced clumping of Plasmodium falciparum-infected erythrocytes from Malawian patients with cerebral malaria-possible modulation in vivo by thrombocytopenia. J Infect Dis 2008; 197:72-78.; and systemic sequestration2929. de Mast Q, Groot E, Lenting PJ, de Groot PG, McCall M, Sauerwein RW, et al. Thrombocytopenia and release of active von Willebrand factor during early Plasmodium falciparum malaria. J Infect Dis 2007; 196:622-628.3131. Tacchini-Cottier F, Vesin C, Redard M, Buurman W, Piguet PF. Role of TNFR1 and TNFR2 in TNF-induced platelet consumption in mice. J Immunol 1998; 160:6182-6186.. These changes are transient, and in general, patients recover completely after malaria treatment3232. Taylor WR, Widjaja H, Basri H, Ohrt C, Taufik T, Tjitra E, et al. Changes in the total leukocyte and platelet counts in Papuan and non Papuan adults from northeast Papua infected with acute Plasmodium vivax or uncomplicated Plasmodium falciparum malaria. Malar J 2008; 7:259..

The 2013 World Health Organization (WHO) criteria that define complicated malaria do not include severe thrombocytopenia3333. World Health Organization. Management of severe malaria - A practical handbook. 3rd edition. Geneva: 2013.. Studies have postulated that malaria should be suspected in travelers who present with fever and thrombocytopenia3434. Eriksson B, Hellgren U, Rombo L. Changes in erythrocyte sedimentation rate, C-reactive protein and hematological parameters in patients with acute malaria. Scand J Infect Dis 1989; 21:434-441.. In malaria patients, thrombocytopenia is generally mild or moderate1313. Kochar DK, Das A, Kochar A, Middha S, Acharya J, Tanwar GS, et al. Thrombocytopenia in Plasmodium falciparum, Plasmodium vivax and mixed infection malaria: a study from Bikaner (Northwestern India). Platelets 2010; 21:623-627.,1717. Tobon A. Danger signs in the malaria patient. Biomedica 2009; 29:320-329., and bleeding is rare3535. Moulin F, Lesage F, Legros A-H, Maroga C, Moussavou A, Guyon P, et al. Thrombocytopenia and Plasmodium falciparum malaria in children with different exposures. Arch Dis Child 2003; 88:540-541.,3636. Kumar A, Shashirekha. Thrombocytopenia--an indicator of acute vivax malaria. Indian J Pathol Microbiol 2006; 49:505-508.. One study found that severe thrombocytopenia in malaria was an independent predictor of death (odds ratio (OR)=13.3, 95%CI=3.2-55.1)3737. Gerardin P, Rogier C, Ka AS, Jouvencel P, Brousse V, Imbert P. Prognostic value of thrombocytopenia in African children with falciparum malaria. Am J Trop Med Hyg 2002; 66:686-691., but this finding has been contested99. Ladhani S, Lowe B, Cole AO, Kowuondo K, Newton CR. Changes in white blood cells and platelets in children with falciparum malaria: relationship to disease outcome. Br J Haematol 2002; 119:839-847.. Thrombocytopenia is more frequent in pregnant women with malaria3838. Tan SO, McGready R, Zwang J, Pimanpanarak M, Sriprawat K, Thwai KL, et al. Thrombocytopaenia in pregnant women with malaria on the Thai-Burmese border. Malar J England 2008; 7:209. and is especially pronounced in primigravidae. Additionally, there is an inverse relationship between parasite density and PC3939. Erhabor O, Jeremiah Z, Adias T. Thrombocytopenia in plasmodium parasitized pregnant women in the Niger Delta of Nigeria. Pathology 2010; 2:1-5..

The relationship between thrombocytopenia and clinical presentation is not sufficiently understood, and platelet changes beyond the PC have been either entirely unstudied or not studied in relation to other aspects of the clinical presentation. Moreover, in low and unstable endemic regions such as Colombia, the hematologic profiles of malaria patients and their clinical utility remain poorly understood. The aim of this descriptive study was to characterize the thrombograms of malaria patients from different regions of Colombia and to explore the relationship between clinical status and platelet alterations in ambulatory and hospitalized patients.

METHODS

Patients and sociodemographic characteristics

A retrospective analysis was conducted using clinical and laboratory data obtained from patients from malaria-endemic regions in Colombia. Patients were treated on an outpatient basis for P. falciparum and P. vivax and were enrolled in clinical and epidemiological studies conducted by the Grupo Malaria (Universidad de Antioquia) between 1997 and 20074040. Blair S, Álvarez G, Campuzano G. Relación entre anemia y malaria en una población rural de Colombia. Bol Dir Malariol Saneam Amb (Venezuela) 1997; 37:7-13.4242. Tobón A, Giraldo C, Blair S. Utilidad de los signos clínicos y parasitológicos en el pronóstico de la malaria grave en Colombia. Biomedica. 2012; 32:79-94.. A second study group consisted of malaria patients hospitalized between 2005 and 2010 (A. Tobón: unpublished data). The reference population consisted of patients from all age groups who presented at first- or second-tier health-care facilities in the municipalities of Turbo and Necoclí (Urabá Region, Antioquia State), El Bagre (Bajo Cauca Region) and Tumaco, Guapi and Timbiquí (Pacific coast), as well as individuals who were treated on an inpatient basis at third-tier hospitals in Medellin with malaria diagnosed microscopically by thick smear (Table 1). For all patients with a complete blood count (CBC), a convenience sample was taken. Eight hundred sixty-two patients were sampled.

TABLE 1 -
Platelet variables: descriptive statistics and categorical classification.

Clinical and laboratory procedures

The thick smear was performed according to the WHO recommendations4343. World Health Organization. New perspectives: malaria diagnosis. Geneva: 2000.. Following a medical assessment, a sample of blood was collected in ethylenediaminetetraacetic acid (EDTA) for cellular and biochemical studies, which included a CBC within 2 hours after sampling. The analysis was conducted in third- and fourth-generation automated analyzers (Cell-Dyn 3200, from Abbott Diagnostics®, Canada; Coulter HmX from Beckman Coulter®, United States of America; Celltac F MEK 8222, from Nihon Kohden®, United States of America and Sysmex KX-21N®, from Sysmex America, Inc. United States of America). Reference values for the platelet variables for the Colombian population were as follows: PC 150-400 x 103 platelet/µL, mean platelet volume (MPV) 6.5-13.5 fl, platelet distribution width (PDW) 15.4-16.8% and plateletcrit (PT) 0.085-0.287%4444. Campuzano G. Del hemograma manual al hemograma de cuarta generación. Medicina & Laboratorio. 2007; 13:511-550..

The complications were classified according to the major criteria of the WHO55. World Health Organization. Severe falciparum malaria. World Health Organization, Communicable Diseases Cluster. Trans R Soc Trop Med Hyg 2000; 94 (suppl I):1-90., along with the additional minor criteria proposed for Colombian endemic regions4242. Tobón A, Giraldo C, Blair S. Utilidad de los signos clínicos y parasitológicos en el pronóstico de la malaria grave en Colombia. Biomedica. 2012; 32:79-94.. The complications were classified as follows: 1) Liver failure: severe (total bilirubin >3 mg/dl; AST >120IU/L) or mild (total bilirubin >1.5-3mg/dl; AST >80-120IU/L); 2) Thrombocytopenia: profound (<25,000 platelets/µl) or severe (25-50,000 platelets/µl); 3) Renal impairment: severe (blood urea nitrogen (BUN) >60mg/dl or creatinine >3mg/dl) or mild (BUN 41-60 mg/dl or creatinine 1,5-3mg/dl). 4) Anemia: severe (hemoglobin <5g/dl) or moderate (hemoglobin 5-6.9g/dl); 5) Neurologic complication: cerebral malaria (seizures/coma) or extreme weakness; 6) Pulmonary injury: acute respiratory distress syndrome (ARDS)/pulmonary edema or pleural effusion; 7) Acid-base disturbance: severe acidosis (pH <7.35 and HCO3 <15mEq/L) or acidosis (pH<7.35 and HCO3 15-18mEq/L); and 8) Hypoglycemia: severe (<40mg/dl) or moderate (40-49mg/dl).

Statistical analysis

The Kolmogorov-Smirnov test was used to test quantitative variables for normality; the median values were compared using the Mann-Whitney U test when the variables were not normally distributed. Associations between the qualitative variables were analyzed with the Chi-square test. Spearman's correlation coefficient was applied to analyze specific relationships. The level of statistical significance adopted was 5% (p<0.05). All analyses were conducted using the IBM® SPSS® statistics program, version 19 (licensed for Universidad de Antioquia).

Ethical considerations

All patients provided their informed consent to enroll in this study as required by the ethics committee of the Medical Research Institute of the Medical School of the Universidad de Antioquia (Acta 31, July 2002 and Acta 05, June 2005). The review of patient medical records was approved by the hospital ethics committees from Pablo Tobón Uribe Hospital and San Vicente Foundation Hospital (Letter 5282, Oct. 2009).

RESULTS

We reviewed the clinical records of 862 patients with malaria, including 144 (16.7%) inpatients at the San Vicente Foundation Hospital and Pablo Tobón Uribe Hospital in Medellin. All patients were classified in 4 age groups: less than 1 year (n=8, 0.9%), 1-5 years (n=49, 5.7%), 5.1-15 years (n=187, 21.7%) and >15 years (n=618; 77.7%); the precise age was known in 610 patients (mean 25.9 years; standard deviation (SD) 16.6; median 22.8). In total, 62.9% of the patients were male.

In 592 patients, the mean duration of illness prior to seeking treatment was 6.2 days (SD 6.4; median 5.0; range 1-99). The Plasmodium species diagnosed were P. falciparum, 533 (61.8%) patients; P. vivax, 311 (36.1%) patients; and mixed infection, 18 (2.1%) patients. The clinical presentation was classified as acute malaria in 613 (71.1%) patients and complicated malaria in 248 (28.8%) patients.

Platelet profile

The values of PC, PT, MPV and PDW were not normally distributed (Kolmogorov-Smirnov, p=<0.001). Categorical sorting indicated that the most significant alterations were the low PC and high PDW, which were present in approximately 65% of patients (Table 1).

In the 564 (65.4%) patients with PC values below 150,000 platelets/µL, thrombocytopenia was classified in 4 categories (Table 2). Significant thrombocytopenia (<50,000 platelets/µL) was observed in 93 (11%) patients, which included 2% with <25,000 platelets/µL. Other frequent abnormalities included low values for PT and MPV.

TABLE 2 -
Thrombocytopenia level and platelet profile by species of Plasmodium.

The thrombogram and other variables

We did not identify differences by gender in the median values of PC (p=0.299), PT (p=0.833), MPV (p=0.660) or PDW (p=0.115) or by age (children-adults) in the median values of PC (p=0.914), PT (p=0.972), MPV (p=0.328) or PDW (p=0.640) (Mann-Whitney U test).

The analysis by species is presented in Table 2. The PT, MPV and PDW were significantly lower in the P. vivax cases compared with the P. falciparum cases (Mann-Whitney U test, p<0.05). PC was lower in the P. vivax cases, but this result was not significantly different from that in the P. falciparum cases (p=0.832). The frequency of thrombocytopenia (<150,000 platelets) in the patients with P. falciparum was 66.6% compared with 63.7% in the P. vivax cases. Severe or profound thrombocytopenia (<50,000 platelets) was observed in 55 (10.3%) of the 533 P. falciparum cases, 34 (10.9%) of the 311 P. vivax cases and 4 (22.2%) of the 18 mixed infections (Chi2, p>0.05).

The plateletcrit was higher in mixed infections compared with P. vivax infections (p=0.041), and the PDW was lower in mixed infections compared with P. falciparum infections (p=0.011); additional comparisons by species were not significantly different.

Thrombocytopenia severity and others variables

To explore the relationships between the severity of thrombocytopenia and age, duration of illness and hematological variables, the variables were compared among the patients with no alteration in PC (group A) and the patients with mild (B), severe (C) or profound (D) thrombocytopenia (Table 3). Patient age was not different between the patients with normal or low PCs (p>0.05) but was significantly different between the patients with mild and severe thrombocytopenia (p<0.05), which indicates that younger age could be a risk factor for severe platelet reduction. The median duration of illness was similar between the non-thrombocytopenic patients and the patients with mild changes; however, the duration was significantly higher in the patients with PCs below 50,000/µL (p<0.05). Parasitemia was significantly higher and the erythrocyte count was significantly lower in the patients with PCs below 50,000/µL compared to the other patients groups (Table 3).

TABLE 3 -
Thrombocytopenia level stratified by quantitative variables (median values).

The comparison between the patients with normal PC and the patients with any degree of thrombocytopenia showed significant differences in the leukocyte count, with the latter group characterized by significantly lower counts of lymphocytes (p<0.001), monocytes (p=0.003), neutrophils (p=0.010) and eosinophils (p=0.029).

In the cases of severe or profound thrombocytopenia, all variables with the exception of age were significantly different compared with the patients with normal counts. Lower leukocyte and erythrocyte counts, higher coagulation times [prothrombin time (PTi) and partial thromboplastin time (PTT)] and higher parasitemia were observed in the patients with severe or profound thrombocytopenia.

Finally, the comparison between the patients with mild, severe and profound thrombocytopenia revealed no leukocyte count differences (p>0.05). Young age, a longer duration of illness and higher parasitemia were related to severe thrombocytopenia, a lower erythrocyte count and a longer coagulation time.

Clinical aspects

Clinical complications were diagnosed in 248 (28.8%) patients, and 206 (23.9%) patients exhibited complications other than severe thrombocytopenia. The complications were classified using major, 105 (12.2%) and minor, 143 (16.6%) criteria in accordance with the guidelines established by the WHO55. World Health Organization. Severe falciparum malaria. World Health Organization, Communicable Diseases Cluster. Trans R Soc Trop Med Hyg 2000; 94 (suppl I):1-90. and the Colombian endemic regions, respectively4242. Tobón A, Giraldo C, Blair S. Utilidad de los signos clínicos y parasitológicos en el pronóstico de la malaria grave en Colombia. Biomedica. 2012; 32:79-94.. The complications were distributed as follows: 1) Liver failure: mild in 96 (11.1%) patients or severe in 74 (8.6%) patients; 2) Thrombocytopenia: severe in 73 (8.5%) patients or profound in 20 (2.3%) patients; 3) Renal impairment: mild in 21 (2.4%) patients or severe in 10 (1.2%) patients; 4) Anemia: moderate in 22 (2.6%) patients or severe in 4 (0.5%) patients; 5) Neurological complications: extreme weakness in 4 (0.5%) patients or seizures/coma in 11 (1.3%) patients; 6) Pulmonary injury: pleural effusion in 5 (0.6%) patients or ARDS/pulmonary edema in 12 (1.4%) patients; 7) Acid-base disturbances: acidosis in 5 (0.6%) patients or severe acidosis in 4 (0.5%) patients; and 8) Hypoglycemia: moderate in 3 (0.4%) patients or severe hypoglycemia in 1 (0.1%).patient.

Patients with complicated malaria (with or without severe thrombocytopenia) exhibited significantly lower median values for PC and PT and higher median values for PDW (Table 4). Median PC values were significantly lower in the patients with complications other than severe anemia or acidosis. In the patients who experienced complications, the median PT was lower and the median PDW was higher compared with the patients who did not experience complications. Furthermore, the PDW was significantly lower in the patients with renal or neurologic complications. The MPV was similar across patients.

TABLE 4 -
Median contrast of platelet features by clinical complications (major or minor).

Severe and profound thrombocytopenia (<50,000 platelets/µL) were related to clinical complications (O.R. 1.6; 95% 95%CI=1.5-1.8; p≤0.001). A higher probability of thrombocytopenia was observed in patients with severe anemia (OR=3.2; 95% CI=1.3-7.9; p=0.007), neurologic complications (OR=12.0; 95%CI=4.0-35.0; p≤0.001), pulmonary complications (OR=8.0; 95%CI=3.0-21.0; p≤0.001), liver dysfunction (OR=3.9; 95%CI=2.2-6.9; p≤0.001), renal impairment (OR=3.7; 95%CI=1.7-8.1; p=0.001) and severe hypoglycemia (OR=38.6; 95%CI 3.9-385; Fisher, p=0.002). The presence of thrombocytopenia (<150,000 platelets/µL) was associated with a higher probability of liver dysfunction (OR=2.4; 95%CI=1.5-3.8; p≤0.001).

Finally, we compared the laboratory data among malaria patients with and without severe thrombocytopenia and stratified by Plasmodium specie (Table 5). The laboratory data were significantly different across categories, with the exception of the white blood cell count, serum creatinine and serum glycemia. In the patients infected with P. falciparum, severe thrombocytopenia was related to abnormal values on all liver-function tests, BUN, PTi and PTT; in the patients with P. vivax, a relationship was identified between severe thrombocytopenia and abnormal values on liver-function tests, BUN, serum glucose, erythrocyte and white cell counts.

TABLE 5 -
Median values of hematological laboratory tests in relation to severe thrombocytopenia.

DISCUSSION

In addition to anemia, a reduction in the number of platelets is one of the more well-known hematologic changes observed in patients with malaria. While platelet-count changes are particularly associated with P. falciparum malaria99. Ladhani S, Lowe B, Cole AO, Kowuondo K, Newton CR. Changes in white blood cells and platelets in children with falciparum malaria: relationship to disease outcome. Br J Haematol 2002; 119:839-847.,1212. Richards MW, Behrens RH, Doherty JF. Short report: hematologic changes in acute, imported Plasmodium falciparum malaria. Am J Trop Med Hyg 1998; 59:859.,4545. Wickramasinghe S, Abdalla S. Blood and bone marrow changes in malaria. Best Pract Res ClinHaematol 2000; 13:277-299., this pathology has also been identified in malaria caused by other species, such as P. vivax3636. Kumar A, Shashirekha. Thrombocytopenia--an indicator of acute vivax malaria. Indian J Pathol Microbiol 2006; 49:505-508.,4646. Prasad R, Das BK, Pengoria R, Mishra OP, Shukla J, Singh TB. Coagulation status and platelet functions in children with severe falciparum malaria and their correlation of outcome. J Trop Pediatr England; 2009; 55:374-378.. Changes in other platelet parameters, such as PT, MPV or PDW, have not been well described in malaria patients.

Some studies have found that patients infected with P. vivax or P. falciparum develop anemia, monocytosis, eosinopenia and lymphopenia4545. Wickramasinghe S, Abdalla S. Blood and bone marrow changes in malaria. Best Pract Res ClinHaematol 2000; 13:277-299.. This study found a progressive reduction in hemoglobin levels and white blood cell counts with reduced PCs, which resulted in anemia, monocytopenia, neutropenia and lymphopenia. Thrombocytopenia presented frequently in the patients infected with P. falciparum (67%) and P. vivax (64%). The PC did not differ by species, which suggests that a similar mechanism governs this interaction in P. vivax and P. falciparum infections. Significant differences in PT and MPV were observed among species, but the values remained within the normal range. The PDW was significantly higher in the patients infected with P. falciparum compared with those infected with P. vivax; however, both median values were above the normal range. These findings and the results of various studies that have demonstrated a rapid recovery of PCs following the initiation of antimalarial treatment suggest that thrombocytopenia is related to a systemic endothelial and platelet activation rather than medullary suppression. Systemic endothelial activation likely mediates platelet sequestration and does not depend exclusively on the formation of platelet aggregates and parasitized erythrocytes, as observed in P. falciparum infections4747. Chotivanich K, Sritabal J, Udomsangpetch R, et al. Platelet-induced autoagglutination of Plasmodium falciparum-infected red blood cells and disease severity in Thailand. J Infect Dis 2004; 189:1052-1055..

Interestingly, we found that the patients with P. vivax infection and severe thrombocytopenia had more significant changes in leukocyte and erythrocyte counts compared with the patients without thrombocytopenia. These changes, which were not identified in the patients infected with P. falciparum, favor a stronger inflammatory response in severe P. vivax infections that can affect bone marrow via the production of tumor necrosis factor alpha (TNF-α). This hypothesis is supported by a study that identified increased production of TNF-α in P. vivax malaria and a stronger host immune response in P. vivax malaria compared with P. falciparum malaria4848. Hemmer CJ, Holst FG, Kern P, Chiwakata CB, Dietrich M, Reisinger EC. Stronger host response per parasitized erythrocyte in Plasmodium vivax or ovale than in Plasmodium falciparum malaria. Trop Med Int Health 2006; 11:817-823..

Various mechanisms have been proposed to explain thrombocytopenia in malaria; however, its clinical significance remains uncertain. Thrombocytopenia can be interpreted as evidence of damage and has been associated with clinical complications and high parasitemia99. Ladhani S, Lowe B, Cole AO, Kowuondo K, Newton CR. Changes in white blood cells and platelets in children with falciparum malaria: relationship to disease outcome. Br J Haematol 2002; 119:839-847.,1212. Richards MW, Behrens RH, Doherty JF. Short report: hematologic changes in acute, imported Plasmodium falciparum malaria. Am J Trop Med Hyg 1998; 59:859.,3737. Gerardin P, Rogier C, Ka AS, Jouvencel P, Brousse V, Imbert P. Prognostic value of thrombocytopenia in African children with falciparum malaria. Am J Trop Med Hyg 2002; 66:686-691.. However, some studies have suggested that thrombocytopenia has a protective effect, which reduces the probability of red blood cell aggregation2828. Wassmer SC, Taylor T, Maclennan CA, Kanjala M, Mukaka M. Platelet-induced clumping of Plasmodium falciparum-infected erythrocytes from Malawian patients with cerebral malaria-possible modulation in vivo by thrombocytopenia. J Infect Dis 2008; 197:72-78. or mediates parasite destruction. The latter effect has been observed in vitro in the early stages of erythrocytic infection with P. falciparum and in a mouse model with Plasmodium chabaudi4949. McMorran BJ, Marshall VM, de Graaf C, Drysdale KE, Shabbar M, Smyth GK, et al. Platelets kill intraerythrocytic malarial parasites and mediate survival to infection. Science 2009; 323:797-800.. In this study, we identified a significant relationship between severe thrombocytopenia and other clinical complications, but we were not able to establish causality.

Thrombocytopenia has been associated with disturbances in coagulation and has been implicated in cases of disseminated intravascular coagulation (DIC)5050. Dennis LH, Eichelberger JW, Inman MM, Conrad ME. Depletion of coagulation factors in drug-resistant Plasmodium falciparum malaria. Blood 1967; 29:713-721.. In children with severe malaria, prolonged PTi, PTT and thrombin time (in 47.5, 35.0 and 62.5% of patients, respectively) may lead to platelet hypoaggregation4646. Prasad R, Das BK, Pengoria R, Mishra OP, Shukla J, Singh TB. Coagulation status and platelet functions in children with severe falciparum malaria and their correlation of outcome. J Trop Pediatr England; 2009; 55:374-378.. Activation of coagulation may also be responsible, in part, for thrombocytopenia66. Lacerda MV, Mourão MP, Coelho HC, Santos JB. Thrombocytopenia in malaria: who cares? Mem Inst Oswaldo Cruz 2011; 106:52-63.. Our study showed that the patients infected with P. falciparum, but not P. vivax, had more prolonged PTi and PTT when the thrombocytopenia was more severe. This difference may be the result of increased procoagulant activity in P. vivax compared with P. falciparum infections, which develops in response to increased endothelial activity induced by TNF-α levels and the consequent formation of thrombin-antithrombin III4848. Hemmer CJ, Holst FG, Kern P, Chiwakata CB, Dietrich M, Reisinger EC. Stronger host response per parasitized erythrocyte in Plasmodium vivax or ovale than in Plasmodium falciparum malaria. Trop Med Int Health 2006; 11:817-823..

In this study, patients with severe or profound thrombocytopenia showed higher parasitemia compared with patients without thrombocytopenia, which is in agreement with previous findings. Low PCs have been associated with increased parasite load. This association may result from sensitization induced by the parasitized red blood cells in the platelet, with consequent increased platelet sensitivity to adenosine diphosphate (ADP) and higher dense-granule secretion4545. Wickramasinghe S, Abdalla S. Blood and bone marrow changes in malaria. Best Pract Res ClinHaematol 2000; 13:277-299.,4646. Prasad R, Das BK, Pengoria R, Mishra OP, Shukla J, Singh TB. Coagulation status and platelet functions in children with severe falciparum malaria and their correlation of outcome. J Trop Pediatr England; 2009; 55:374-378.. These changes could ultimately promote platelet aggregation on the endothelium, which has been demonstrated in cerebral malaria2727. Grau GE, Mackenzie CD, Carr RA, Redard M, Pizzolato G, Allasia C, et al. Platelet accumulation in brain microvessels in fatal pediatric cerebral malaria. J Infect Dis 2003; 187:461-466..

Plateletcrit is a measure of platelet mass, the clinical significance of which should be interpreted in terms of the number and size of the platelets. Whitfield et al.5151. Whitfield J, Martin N, Rao D. Genetic and environmental influences on the size and number of cells in the blood. Genetic epidemiology 1985; 2:133-144. demonstrated that in healthy subjects, there was an inverse relationship between the platelet size (MPV) and the platelet number (PC) and proposed that the total platelet mass is closely regulated by changes on MPV or PC. We found that the PT was low in 36% of patients with malaria. This finding may be explained by a combination of low PC with normal MPV (63%), low PC and MPV (35%) and normal PC with low MPV (2%). The reduction in circulating platelet mass is best explained by a reduction in platelet number rather than a reduction in platelet size. It is suspected that in malaria, sequestration leads to pseudothrombocytopenia5252. Pain A, Ferguson DJ, Kai O, Urban BC, Lowe B, Marsh K, et al. Platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes is a common adhesive phenotype and is associated with severe malaria. Proc Natl Acad Sci USA 2001; 98:1805-1810..

Some studies have identified a relationship between the decrease in PC and the appearance of clinical complications, such as anemia, respiratory distress syndrome and cerebral malaria3737. Gerardin P, Rogier C, Ka AS, Jouvencel P, Brousse V, Imbert P. Prognostic value of thrombocytopenia in African children with falciparum malaria. Am J Trop Med Hyg 2002; 66:686-691.. We found that the patients with severe (25,000-49,999 platelets/µL) or profound thrombocytopenia (<25,000 platelets/µL) had more pathological changes in their clinical status compared with the patients without thrombocytopenia. Furthermore, parasitemia, the duration of disease, and TP and TPP increased with an increasing severity of thrombocytopenia (mild→moderate→severe→profound), whereas erythrocyte and leukocyte counts decreased.

In this study, the patients who experienced complications had lower PC and PT values and higher levels of PDW compared with the patients who did not experience such complications. Specifically, the median PCs were significantly lower in the patients with hepatic complications (90,500 vs. 129,000; p≤0.001), renal complications (80,000 vs. 114,000; p=0.017), neurologic complications (42,000 vs. 122,000; p≤0.001), pulmonary complications (55,000 vs. 122,000; p=0.001) and severe hypoglycemia (40,500 vs. 119,000; p=0.004). The low PC may result from the adhesion of platelets to the sites of vascular injuries and the mediation of malaria-parasitized red blood cells (pRBC) sequestration to the endothelium through von Willebrand-factor strings5353. Bridges DJ, Bunn J, van Mourik JA, Georges Grau G, Roger JS. Preston RJS, et al. Rapid activation of endothelial cells enables Plasmodium falciparum adhesion to platelet-decorated von Willebrand factor strings. Blood 2010; 115:1472-1474..

When platelets decrease in number, bone marrow megakaryocytes are stimulated by thrombopoietin, and their nucleus becomes hyperlobulated with a higher deoxyribonucleic acid (DNA) content. These stimulated megakaryocytes produce larger platelets. Thus, platelets with a higher MPV are expected to be present in autoimmune thrombocytopenia when megakaryocytic stimulation is present. Conversely, platelets with a lower MPV are expected in thrombocytopenic states associated with marrow hypoplasia or aplasia5454. Beyan C, Kaptan K, Ifran A. Platelet count, mean platelet volume, platelet distribution width, and plateletcrit do not correlate with optical platelet aggegation responses in healthy volunteers. J Thromb Thrombolysis 2006; 22:161-164.. Interestingly, 83% of the patients studied had normal MPV, and it appears that neither of these two mechanisms associated with thrombocytopenia was significantly activated.

The examination of PDW with MPV provides information regarding thrombocyte volume disturbance. Thrombocyte volume heterogeneity occurs because of the production factors in the bone marrow. PDW is an index of thrombocyte volume heterogeneity similar to erythrocyte distribution, and both MPV and PDW are markers of platelet immaturity and platelet activation5555. Aydogan A, Akkucuk S, Arica S, Motor S, Karakus A, Ozkan OV, et al. The analysis of mean platelet volume and platelet distribution width levels in appendicitis. Indian J Surg 2013:1-6. DOI:10.1007/s12262-013-0891-7.
https://doi.org/10.1007/s12262-013-0891-...
. A high PDW predominated in the patients studied (65%), and we identified significantly higher PDW in the total sample of severe patients; however, the analysis for each complication indicated conflicting results, which could be a result of the differential patterns of inflammation in the spectrum of clinical complications or statistical effects based on the low number of cases when the complications were individually analyzed.

In healthy human subjects, no correlation was identified between PCT, MPV or PDW values and platelet aggregation results5454. Beyan C, Kaptan K, Ifran A. Platelet count, mean platelet volume, platelet distribution width, and plateletcrit do not correlate with optical platelet aggegation responses in healthy volunteers. J Thromb Thrombolysis 2006; 22:161-164.; however, these relationships could be modified in the presence of inflammation.

It has been reported that PDW increases over storage time because of the formation of abnormally small and large platelets5454. Beyan C, Kaptan K, Ifran A. Platelet count, mean platelet volume, platelet distribution width, and plateletcrit do not correlate with optical platelet aggegation responses in healthy volunteers. J Thromb Thrombolysis 2006; 22:161-164., and it is well known that pseudo-thrombocytopenia occurs as a result of exposure to EDTA when used in blood sampling5656. Silvestri F, Virgolini L, Savignano C, Zaja F, Velisig M, Baccarani M. Incidence and Diagnosis of EDTA-Dependent Pseudothrombocytopenia in a Consecutive Outpatient Population Referred for Isolated Thrombocytopenia. Vox Sang 1995; 68:35-39.. There is a need for multi-center and prospective studies with large sample sizes to elucidate the utility and clinical importance of these parameters in malaria.

Our results indicate that thrombocytopenia accompanies malaria complications, especially liver dysfunction. Major changes in total bilirubin, direct bilirubin, aspartate aminotransferase, alanine aminotransferase, prothrombin time and partial thromboplastin time were observed in severe thrombocytopenia (<50,000 platelets/µL). The PCs were significantly lower in the patients who experienced liver, renal, neurologic, and pulmonary complications and severe hypoglycemia.

The liver is a key regulator of platelet production through the synthesis of TPO. Thus, hepatic dysfunction may explain, in part, the thrombocytopenia observed in these patients; however, more data are required because TPO appears to rise during infection by P. falciparum, even when liver function is compromised2323. Kreil A, Wenisch C, Brittenham G, Looareesuwan S, Peck-Radosavljevic M. Thrombopoietin in Plasmodium falciparum malaria. Br J Haematol 2000; 109:534-536..

Differences between P. falciparum and P. vivax infections may arise from the different mechanisms by which the physiopathology of thrombocytopenia is mediated, i.e., via clumping in P. falciparum and via medullar suppression in P. vivax. A better understanding of these mechanisms is still needed.

Despite the inherent limitations of retrospective analyses, these results may contribute to the understanding of severe thrombocytopenia as a negative prognostic marker in patients with malaria. Our findings suggest a need to study hepatic function and to establish close clinical monitoring of malaria patients. Furthermore, there is a substantial need for prospective studies to elucidate the utility and clinical importance of these parameters in malaria.

ACKNOWLEDGMENTS

Our sincere thanks to Grupo Malaria, Universidad de Antioquia, who provided partial data analyzed in this study, and to Felipe Miranda, M.D., Ana del Mar Cortina, M.D., Ana Maria Cadavid, Lia Palacio and Shirley Jolianiz, who are medical students at the School of Medicine at the University of Antioquia, for their help in acquiring hospital medical records. This project was funded by the Universidad de Antioquia, Colombia (Grupo Malaria de la Facultad de Medicina, Programa Jóvenes Investigadores de Vicerrectoría de Investigación y CODI- Estrategia de Sostenibilidad 2014-2015).

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Publication Dates

  • Publication in this collection
    May-Jun 2014

History

  • Received
    14 Apr 2014
  • Accepted
    18 June 2014
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