Association of Thrombocytopenia with Severity of Plasmodium falciparum Malaria: A Study in Chhattisgarh

 

Pragya Gajendra¹* and Mitashree Mitra²

¹UGC-RGNF JRF, School of Studies in Anthropology, Pt. Ravishankar Shukla University, Raipur (C.G.) India.

²Professor, School of Studies in Anthropology, Pt. Ravishankar Shukla University, Raipur (C.G.) India.

 

ABSTRACT:

Introduction: Malaria is still very common problem in world population. It is caused 225 million new cases in each year. Particularly Plasmodium  falciparum malaria contributed to the high mortality and morbidity of death rate in developing countries (WHO, 2010). India shows 15,000 deaths by malaria in each year (WHO, 2008). Hematological changes, which are the most common complication, play a major role in malaria disease. These changes involve in red blood cells, leukocytes and hemostasis.

Present study aimed to determine the association of platelet alternations in patients with different severity of  P. falciparum malaria disease.

Materials and Methods: The study was conducted in two highly affected zones of Chhattisgarh viz. Ambagarh-Chowki Primary Health Center in Rajnandgaon district and Holi Cross Hospital, Kunkuri in Jashpur district. Total 210 patients were included in the study. Plasmodium vivax malaria diagnosed patients were excluded from the study. The patients who were clinically suspicious of P. falciparum malaria and confirmed by peripheral blood film (PBF) examination or by card test or both were included in the study.

3-5ml venous blood from 210 P. falciparum malaria was collected into EDTA filled tubes (with the help of technician) with the individual informed consent of the patients. Tubes were transported in icebox within 24 hours to the Human Genomics Laboratory of School of Studies in Anthropology, Pt.  Ravishankar Shukla University, Raipur for further laboratory analysis. The Cell Counts were performed using ERMA INC fully automatic blood cell counter 210.

Results: Mean age of 210 P. falciparum malaria male and female patients were 35.05±1.53 years and 31.48±1.5 years respectively. The normal platelet count was noted only in 22.85% cases. The mean platelet count was 177.81x10³ /µl (SD) in individuals with mild malaria (with a range of 2980 X10³ /µl  to 14900 X10³ /µl)  whereas  it is 8894.94 x10³ /µl (SD) in individuals with severe malaria and 8494.21 x10³ /µl (SD) in individuals with cerebral malaria. Findings of our study showed the negative correlation (r = - 0.99, p = 0.0285) between platelet count and severity of malaria.

Conclusion: Thrombocytopenia is very common in P. falciparum malaria cases. Its presence in patients, who had acute febrile illness in the tropics, increases the probability of malaria. It can be used as an additional aid for the clinical assessment of this disease.

 

 

 

INTRODUCTION:

Malaria is still very common problem in world population. In 2010, there were an estimated 219 million cases of malaria (range 154–289 million), and 6, 60,000 malaria deaths globally (WHO, 2012). It is caused 225 million new cases in each year. Plasmodium  falciparum malaria (Pf M) contributed to the high mortality and morbidity of death rate in developing countries (WHO, 2010). Approximately 80% of estimated cases in 2010 were found in 17 countries and the Democratic Republic of the Congo, India and Nigeria together accounted for more almost 40% of the global total of estimated malaria cases (WHO, 2012). In 2008 WHO estimates that only about 15,000 deaths each year in India (5,000 children and 10,000 adults) caused by malaria disease, but this too depends indirectly on the low death rates in diagnosed patients. Because the health care facilities are limited, severe malaria in people who die of it without any medical attention is easily mistaken in retrospective enquiries for some other life-threatening fever; conversely, other febrile causes of death can be mistaken retrospectively for malaria. The cases and deaths reported by the Indian Government are concentrated mainly in a few states in east and northeast India, the so-called high-malaria states; mainly Orissa, Chhattisgarh, Jharkhand and the states in the far northeast of India (NVBDCP, 2007).

 

The hematologic changes are well recognized in Plasmodium  falciparum malaria cases (PfM), but specific changes may vary with level of malaria endemicity, background hemoglobinopathy, nutritional status of patients, demographic factors, and malaria immunity (Sitalakshmi et al. 2003, McKenzie et al. 2005). These changes involve red blood cells, hemostasis and leukocytes. Alternation of platelets count is very common in PfM. The severe degree and higher incidence of thrombocytopenia were occurred predominantly in PfM infection (Srichaikul et al. 1988).

 

Present study aimed to determine the association of platelet alternations in patients with Plasmodium falciparum malaria and severity of the disease from highly affected zones of Chhattisgarh (NVBDCP, 2007).

 

MATERIALS AND METHODS:

The study was conducted in two districts of Chhattisgarh, Rajnandgaon and Jashpur. Samples were collected form Ambagarh-Chowki Primary Health Center in Rajnandgaon district and Holi Cross Hospital, Kunkuri in Jashpur district. Total 210 patients, either hospital treated or hospitalized were included in the study. Plasmodium vivax malaria diagnosed patients were excluded from the study. The patients who were clinically suspicious of PfM and were confirmed by peripheral blood film (PBF) examination or by card test or both were included in the study. 3-5ml venous blood was collected with the help of technician with individual informed consent of the subject in EDTA filled tubes to be used for hematological and other examination. Each tube was assigned a unique patient identification. Tubes were transported in icebox within 24 hours to the Human Genomics Laboratory of School of Studies in Anthropology, Pt.  Ravishankar Shukla University, Raipur for further laboratory analysis. Cell counts (WBC and platelet count (PLAT)) and hemoglobin (Hb) level estimation were performed using ERMA INC fully automatic blood cell counter 210.

 

Thrombocytopenia was defined as platelet count less than 150,000 cells/cmm. Patients were divided into three subgroups based on platelet count. Thrombocytopenia was classified as severe (<50,000 cells/cmm), moderate  (50,000-100,000 cells/cmm) and mild (100,000-150,000 cells/cmm) and for the comparison normal platelet count was used (Patel et al. 2004). Statistical analysis were carried out using SPSS version 16.0 statistical programs.

 

Ethical clearance was obtained from the Institutional Ethical Committee of the Pt. Ravishankar Shukla University, Raipur (C.G) for carrying out research on human subjects.

 

RESULTS:

Table 1 shows the mean age of all patients, including male and female, was 33.36 years. Total 210 PfM patients were included in the present study, out of this 61% (n=128) were male and 39% (n=82) were female having mean age 35.05 and 31.48 years respectively. Most common complication of disease was fever with chills and rigor, sweating and headache. On the basis of symptoms, we have classified three groups of Plasmodium  falciparum malaria viz 50% (n=105) individuals with mild malaria, 33.80% (n=71) individuals with severe malaria and 16.19% (n=34) individuals with cerebral malaria as per the WHO (2007) classification.

 

 

Table no: 1 Gender-wise distribution of sample with their mean age (years)

S.No.	Gender	Number	Percent %	Mean age (year)	SD
1	Male	128	61	35.05	1.53
2	Female	82	39	31.48	1.5
	Total	210	100	33 .36	1.52

 

 

Table no: 2 Hematological profile of P. falciparum malaria patients with respect of severity to disease

Hematological parameters	Severity of PfM	Number of individuals	Mean	SD	Minimum Value	Maximum Value
WBC count/cumm	Mild	105	7260.12	2637.841	2980	14900
	Severe	71	8894.94	3674.916	1090	18000
	Cerebral	34	8494.21	3845.503	2100	19000
	Total	210	8372.25	3453.668	1090	19000
Hb gm/dl	Mild	105	10.083	2.0352	2.0	14.2
	Severe	71	8.990	2.1506	3.0	15.1
	Cerebral	34	8.359	1.9746	3.9	12.6
	Total	210	9.434	2.1662	2.0	15.1
PLAT/µl	Mild	105	177.81	109.410	65	852
	Severe	71	114.27	34.153	33	214
	Cerebral	34	59.29	22.869	31	123
	Total	210	137.14	91.784	31	852

Table no: 3 Results of Least significant difference test for individuals with severity of Plasmodium falciparum malaria

Dependent Variable	Severity of Malaria	Mean Difference	Std. Error	Sig.	95% Confidence Interval
					Lower Bound	Upper Bound
Mild	Severe	63.542*	12.380	.000*	39.13	87.95
	Cerebral	118.515*	15.899	.000*	87.17	149.86
Severe	Mild	63.542*	12.380	.000*	87.95	39.13
	Cerebral	54.973*	16.804	.001*	21.84	88.10
Cerebral	Mild	118.515*	15.899	.000*	149.86	87.17
	Severe	54.973*	16.804	.001*	88.10	21.84

* The mean difference is significant at the 0.05 level.

 

Table no 4: Distribution of platelet counts according to severity of  PfM                                                                       

Thrombocytopenia	Mild	Severe	Cerebral	Total
Severe Thrombocytopenia (<50)	0	2 (2.81%)	17 (50%)	19 (9.04%)
Moderate Thrombocytopenia (50-100)	16 (15.23%)	27 (38.02%)	15 (44.11%)	58 (27.61%)
Mild Thrombocytopenia (100-150)	51 (48.57%)	32 (45.07%)	2 (5.88%)	85 (40.47%)
Normal Platelet (>150)	38 (36.19%)	10 (14.08%)	0	48 (22.84%)
Total	105 (100%)	71 (100%)	34 (100%)	210 (100%)

 

 

Table 2 shows the distribution of mean White Blood Cell Count (WBC) in patients. In individuals with mild PfM, the mean WBC was 7260.12 /cumm (SD 2637.841) whereas in severe and cerebral PfM, it was 8894.94/cumm (SD 3674.91) and 8494.21/cumm (SD 3845.5) respectively. The mean difference of WBCs in between the group and within the group was statistically significant (F= 16.118, p=< 0.0001). The mean hemoglobin (Hb) level was 10.0gm/dl in individuals with mild malaria, 8.9gm/dl in individuals with severe malaria and 8.35gm/dl in individuals with cerebral malaria. The lowest hemoglobin level was 2.0gm/dl in mild malaria, 3.0gm/dl in severe malaria and 3.9gm/dl in cerebral malaria cases. Mean Hb value was statistically significant between the groups and within the groups (F= 11.4, p=< 0.0001). The mean platelet count was 177.81 X 10³ /µl(SD 177.81 ) in individuals with mild malaria (with a range of  2980 X 10³ /µl -14900 X 10³ /µl),  8894.94 X 10³ /µl (SD 34.15) in individuals with severe malaria and 8494.21 X 10³ /µl (SD 22.86) in individuals with cerebral malaria.

 

Table 3 shows the results of least significant difference test for individuals with different level of severity of Plasmodium falciparum malaria. Mean difference revealed that the differences are higher between all three groups. The statistical significant difference persisted between the Platelet counts individuals with different levels of severity of Plasmodium falciparum malaria disease and platelet counts.

 

Table 4 shows different gradation of thrombocytopenia with different level of severity of malaria. A total of 19 (9.04%) PfM cases were found with severe thrombocytopenia, in which 2.81% were suffering from severe and 50% cases were suffering from cerebral type PfM.  58 (27.61%) cases had moderate thrombocytopenia. Further analysis revealed that 15.23% mild, 38.02% severe and 44.11%  cerebral PfM cases had moderate thrombocytopenia. Mild thrombocytopenia was found in 85% patients. It was observed that 46.5% individuals with mild and 45.07% individuals with severe type of PfM had mild thrombocytopenia whereas only 5.88% cerebral PfM cases falls under this category. Normal platelet count was found in 79.16% individuals with mild malaria and 20.83% individuals with severe malaria. No individual with cerebral malaria had normal platelet count.

 

DISCUSSIONS:

Severe falciparum malaria accompanied by serious secondary complications, which often prove fatal and include cerebral malaria, severe anemia, lactic acidosis, renal impairment, non cardiogenic pulmonary edema and hypoglycemia ( WHO, 1990 and March et al. 1995). The hematological abnormality is considered a hallmark of P. falciparum malaria and reported to be most pronounced in falciparum infection. It might be due to high parasitemia in blood of these patients (Facer, 1994). The severe degree and higher incidence of thrombocytopenia were observed predominantly in complicated P. falciparum infection (Srichaikul et al. 1988). One of the common but important features of  falciparum malaria is thrombocytopenia. In our study 77.14%  patients having falciparum malaria developed thrombocytopenia, which is higher than reported by other investigators (Robinsons et al 2001, Rodriguez et al 2005 and Bashwari et al 2002). The platelets survival is reduced in individuals with severe P. falciparum malaria and the thrombocytopenia seems to be mainly due to a reduced platelet life span and splenic pooling.  The reduced platelet life span may be caused by binding of malaria antigen onto platelets followed by activation in vivo (Horstmann et al. 1981). Macrophage activation and hyperplasia especially in the spleen may also play a role (Horstmann et al. 1981). The release of platelet contents can activate the coagulation cascade and contributes to DLC and consequently further thrombocytopenia.  Due to enhance splenic uptake or sequestration (clumping of endothelial cells to RBCs) may contribute to thrombocytopenia. In patients with Disseminated Intravascular Coagulation (DIC) platelet may removed from the circulation of sites of fibrin deposition and accompanied clinical bleeding (Essien, 1989). Thrombocytopenia is rarely accompanied by clinical bleeding or biochemical evidence of DIC. The platelet count fall down below 25,000/ µl, but it’s uncommon in malaria infection. In the present study platelet count fall down to 31,000/ µl, so we did not observed any bleeding predisposition and DIC.

 

Findings of our study show that 100% cerebral malaria cases had either severe or moderate type of thrombocytopenia whereas in mild malaria cases 44.66% and in severe malaria cases 85.91% had thrombocytopenia. It revealed the fact that normal platelet count is uncommon in the laboratory findings of falciparum malaria. Some studies reported that  thrombocytopenia is a classical feature of malaria and a low platelet is usually seen in about 85% of patients with uncomplicated (mild) malaria and all patients with severe falciparum malaria (Facer, 1994). Thrombocytopenia was seen in 40-90 percent of patients infected with P. falciparum malaria in Indian context (Krishna et al. 1994, Sharma et al. 1992, Akhtar et al. 2012). Severe thrombocytopenia occurred on the 5-6 days of infection, and gradually returned to normal within 5-7 days after parasitemia ceased (Srichaikut et al. 1988). The ultimate mechanism of thrombocytopenia in malaria is remain unclear but some researchers have recommended the facts that in thrombocytopenia bone marrow examination usually shows normal or increased megakaryocytes  (Large bone marrow cell)  ( Jandle, 1996), Peripheral destruction, induced by P. falciparum, in which immune complexes generated by malarial antigens lead to sequestration of the injured platelets by macrophages in the spleen, although this mechanism has not been properly evaluated in P. vivax malaria.

 

This could be explained by platelet activation and an enhanced aggregability (Essien, 2006). Present study in concordance with other reported studies showed negative correlation (r = -0.99, p = 0.0285) between platelet count and severity of malaria. Some studies depicted thrombocytopenia was neither associated with the severity of the disease or death in malaria (Casals-Pascual et al. 2006, Ladhani and Newton, 2003).

 

It has been reported that anaemia is the best parameter which increases the probability of severity of malaria (Lathia and Joshi, 2004) and our study also support it. Anaemia was observed in 72.42% of the falciparum malarial cases. Both leucocytosis and leucopaenia have been described in malaria. Leukocytosis was commonly observed in our study (56.19%). Rates of leukocytosis (>10x 10^{9/ L)}  in adults range  between 1 and ~7% was also reported in many studies (Martelo et al. 1969, Goldstein, 1968, Eriksson et al. 1989, Sharma et al. 1992).

 

CONCLUSION:

In the present study thrombocytopenia has emerged as the strongest predictor of severity of falciparum malaria. It is statistically significantly associated with P. falciparum malaria. Its presence in patients, who had acute febrile illness in the tropics, increases the probability of malaria. It can be used as an additional aid for the clinical assessment of this disease. In an endemic area, all patients who have acute febrile illness, the platelet count should be checked as an indicator of Plasmodium falciparum malaria. 

 

ACKNOWLEDGMENTS:

Authors would like to thank Dr. N.L. Bhuarya and staff of  Ambagarh-Chowki Primary Health Center and all staff of Holy Cross Hospital Kunkuri for their  help and support during field work. Authors are also grateful to all the volunteer donors who have participated in this study. First author is supported by a grant from UGC, New Delhi in the form of UGC-RGNF JRF, which is duly acknowledged.

 

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Received on 01.08.2014                Modified on 25.08.2014

Accepted on 03.09.2014                © RJPT All right reserved