Study of haematological parameters in malaria: a prospective study

Background: A blood parasites plasmodia is responsible for malaria hence there are haematological alterations in malaria. The haematological changes that have been reported to accompany malaria include anaemia, thrombocytopenia, leucocytosis and leucopenia. Methods: Total 585 smear positive malaria cases were taken and various haematological parameters were studied. Results: Out of 585 smear positive cases, P. vivax was positive in 422 (72%) cases, while P. falciparum was positive in 160 (27%) cases and mixed infection was found in 3 (1%) cases. Anaemia was seen in 420 (71.79%) cases. Normocytic normochromic blood picture was the most common type in anaemic patients 223 (38.11%). Next common finding was Normocytic hypochromic RBCs 185 (31.62%). Microcytic hypochromic RBCs found in 152 (25.98%) cases. Thrombocytopenia was seen in total 490 (83.76%) of the patients. Moderate thrombocytopenia was more common and present in 409 (70%) of patients while Severe thrombocytopenia was seen in 81 (13.84%) of cases. In falciparum malaria thrombocytopenia was present in 90% of the patients while it was present in 81.27% of the patients in vivax malaria. 129 cases (22.05%) shows leucopoenia out of which 81 (13.84%) belong to P. vivax, 45 (7.69%) belong to P. falciparum and 3 (1%) were belong to mixed infection. Total Leucocyte Count was normal in 77.95 % of the patients. Conclusions: Various haematological findings can help in early diagnosis of malaria which is essential for timely and appropriate treatment which can limit the morbidity and prevent further complications.


Introduction
Malaria is a protozoal disease caused by infection with parasites of genus plasmodium. These protozoa comprise 5 species P. falciparum, P. vivax, P. malariae, P. ovale and P. knowlesi (a parasite of long tailed Macaque monkeys may also affect man). Malaria is most important disease in human in terms of geographical distribution, morbidity and mortality. More than 100 countries in world are considered malarious, and more than 2.4 billion of world's population are at risk.
The world annual incidence of malaria is about 300-500 million cases. Malaria kills 1.1 to 2.7 million people annually [1]. In India malaria continue to pose a major public health threat particularly due to Plasmodium falciparum which is prone to complication. In India about 27% population lives in malaria high transmission areas (>1cases/1000 population) and about 58% in low transmission areas. (0-1 case / 1000 population). Malaria surveillance for the period from 1995 to 2011 is shown that Manuscript received: 10 th December 2019 Reviewed: 20 th December 2019 Author Corrected: 28 th December 2019 Accepted for Publication: 2 nd January 2020 API (Annual parasite rate) has been steadily declining in India from 3.29 in 1995 to 1.10 in 2011 [2]. Malaria causing plasmodium are parasites of blood and hence induce haematological alterations [3]. The anaemia is caused by excess removal of non-parasitized erythrocytes in addition to immune destruction of parasitized red cells, and impaired compensation for this loss by bone marrow dysfunction [4,5].
Thrombocytopenia is a common finding in falciparum and vivax malaria. Enhanced splenic uptake or sequestration may contribute to thrombocytopenia.
In immune mediated thrombocytopenia, IgG forms a complex with the malarial antigen, the complex binds to and damages circulating platelets which are then removed from the circulation [6,7,8]. Mild leucopenia has been described in uncomplicated malarias, but a neutrophilic leucocytosis is an important abnormality in patients with severe falciparum malaria and is associated with a bad prognosis. TNF-α may be responsible for this leucocytosis, which may be associated with a complicating bacteraemia.
The other common changes seen are monocytosis found in population living in endemic areas [4,9,10,11].
The aim is to study the changes in haematological parameters in smear positive malaria cases.

Interpretation of Result
Negative: Presence of only one colour band ("C" Control band) within the result window.

Method of preparing Thick and thin smear:
The thin film: Take a single small drop of blood from EDTA sample on the middle of clean glass slide, blood was spread using a spreader slide at angle of 45 O over the length.
The thick film: Place two or three larger drops of blood from EDTA sample on the slide, about 1 cm away from the drop intended for the thin film. Handling the slide by the edges or a corner, make the blood film by using the corner of the spreader to join the drops of blood, and spread them to make an even, thick film. Do not stir the blood. A circular or rectangular film can be made by three to six quick strokes with the corner of the spreader. The circular thick film should be about 1 cm in diameter. The thick film should be dried and be protected from dust, flies, sunlight and extreme heat. Thick smear can be prepared on separate slide [12]. Slides were fixed and stained with Field's Stain.

Results
Total 585 smear positive malaria cases were taken and various haematological parameters were noted. Out of 585 smear positive cases, P. vivax was positive in 422 (72%) cases while P. falciparum was positive in 160 (27%) cases and mixed infection was found in 3 (1%) cases. Out of 585 cases, P. vivax was the most common observed species. Next common was P. falciparum ( Figure 1).

Fig-1: Distribution of plasmodium species in population.
Age distribution: As shown in the Table 1, most of the cases (52.75%) were in the young adults between 21-40 years age group. People of all age groups were seen, youngest one was 3-month-old male child with P. vivax infection along with dengue and oldest was 92 years old male with P. falciparum infection.  As shown in Table 2, both types of Malaria cases were more common in male.
Platelet count: Decreased platelet counts was a constant feature of both types of malaria with 69.91% (409) of cases showing platelets from 50,000 to 1.5 lacs. Severe platelet reduction (<50000) was seen in 13.84% (81) cases (Table 3).

Malaria dengue coinfection:
Total 24 patients were found with co infection of which 16 had P. vivax, and 7 had P. falciparum infection (Table 4).

Discussion
Malarial parasite enters human body through bite of anopheles mosquito. The various factors which are the determinants of rate of transmission include temperature and humidity. Optimum temperature for sporogony is 25° C to 30° C and it ceases below 16°C. Rain is related to humidity and saturation. The deficit of both affects mosquito survival. Adult vector longevity increases with humidity over 60%.
Manmade environmental changes and agricultural pattern with construction of dams, formation of reservoirs and irrigation systems increase the risk to human settlements by providing a breeding space for larvae of the vector. After entering to human malarial parasite targeting liver cells and red blood cells. It then multiplies and develops morphologically in infected cells ultimately resulting in necrosis and rupture of infected cells. Haemolysis and liver cell necrosis result in anaemia and jaundice. The pathophysiology of malaria results from  Destruction of erythrocyte  Liberation of parasite and  Invasion of erythrocyte by merozoites. The binding of parasitized red blood cell (RBC) to uninfected red blood cell (Rosetting).  Binding of parasitized red blood cells to endothelial cells in critical organ (cytoadherence)  The induction of pro-inflammatory response cytokines most notably tumor necrosis factor (TNF-α)  The host reaction to these events  Sequestration of parasites in microcirculation of vital organs interfering with microcirculatory flow and host metabolism.
Malaria is the third most common of these diseases in India after diarrhoea and typhoid. Gujarat ranks 5th in the total number of malaria cases in the country [13]. Severe malaria is a disorder that affects several tissues and organs. Metabolic acidosis is recognized as a principal pathophysiological feature that is seen in the classical clinical syndromes of cerebral malaria and severe malarial anaemia. Individuals affected with malaria are dehydrated and relatively hypovolaemic which exacerbates microvascular obstruction by reducing perfusion pressure. The destruction of RBCs also part of malaria, and anaemia further compromises oxygen delivery. In case of P. falciparum other organs like brain and kidney are also affected. In Present study the incidence was 5.33% of which 72.13% were P. vivax and 27.35% were P. falciparum, which was comparable to study by Paltial Palat et al [14] done in Ahmedabad, Gujarat in year 2011-12 showing 69% and 31% respectively for both the species.
In study conducted by Shraddha M. Kevadiya et al [15] overall slide positivity rate was 15.01%, slide P. falciparum rate was 38.32%, while P. vivax comprised of 69.68% cases of malaria & mixed infection of P. vivax and P. falciparum was seen in 1% of cases. Study done by Pankti D. Panchal et al [16] shows 64% of cases are due to P. vivax and 35% of total cases are due to P. falciparum, which was also comparable to the present study.
The most common age group affected in present study was 21 to 40 years (52.75%). This was comparable to study by Sunita et al [17] (60%). In Paltial Palat et al [14] Infection was commonest in both vivax and falciparum (63.13%) between 16 and 40 years of age which was also comparable to the present study. Young adults were more prone to infection due to their increased mobility. 71.79% (420) patients were having low Haemoglobin below 11 gm/dl in present study, which was comparable to Bashwasri et al [18] & Sharma et al [5] showing 94.4% and 86.7% respectively.
In study conducted by Richard MW et al [19] in London only 15% cases of malaria show anaemia which was vary less in comparison to the present study. Decreased HCT found in 59.68% (225) in male patients & 40.38% (84) in female patients which was less than found in Bashwari et al [18] (71.50%).
Leucopenia was found in 22.05% (129) patient which was higher than found in Bashawri LAM et al [18] which shows leucopenia in 13.3% of the total malaria cases. Study done by Echieverri M et al [20] had 29% cases of leucopenia in vivax malaria cases. Thrombocytopenia was found in 83.76% (490) patients which was comparable to Horstman et al [6] & which was more than found in Bashwari et al [18] with 55.6% The prevalence of thrombocytopenia was  [21] which was also comparable to the present study. In present study most common finding was Normocytic normochromic RBCS 38.11% comparable to 47.3% found in Shamim Akhtar et al [22] study. Next common finding was normocytic hypochromic RBCS 31.62% (185), which was comparable to Shamim Akhtar et al [22] study. In the present study, Microcytic hypochromic RBCS found in 25.98% (152). Microcytic hypochromic anaemia found in Bashwari et al [18] was 17.7%, which was less than that found in the present study. Incidence of anaemia and severity of anaemia were more observed in P. falciparum cases compared to P. vivax cases. In present study, it was observed that co-infection with dengue 4.10% (24 patients) of which one was youngest, one 3 months old male child showing marked thrombocytopenia. One patient died due to coinfection with dengue and malaria with multiorgan failure.

Limitation of study:
The main limitation of this study is a confounding factor that may affect hematological parameters such as bacterial, virus, and helminth infections, micronutrient deficiencies, and genetic backgrounds of patients.

Conclusion
Malaria is one of the most common infections in Indian Subcontinent. In the present study incidence of malaria is 5.33%. Malaria affects mostly adults with male predominance in the present study. Most common finding of peripheral smear in the present study was normocytic normochromic RBCs found in 38.11%. In the present study, though a greater number of cases are due to P. vivax but more death occurred due to P. falciparum. In a patient with febrile illness, observation of thrombocytopenia warrants careful search for malaria parasite. Coinfection with dengue can increase the morbidity and mortality so prompt treatment should be given.

What the study adds to the existing knowledge?
Though the present study is not adding novel information to the existing literature, yet it confirms to the existing information about correlation of various haematological finding with peripheral smear for early diagnosis of malaria which is essential for timely and appropriate treatment which can limit the morbidity and prevent further complications.