Malaria: Lab Values

Malaria is a protozoal disease caused by infection with parasites of the genus Plasmodium and transmitted to man by certain species of female Anopheline mosquito. Six species of the genus Plasmodium cause nearly all malarial infections in humans. These are Plasmodium falciparum, Plasmodium vivax, two morphologically identical species of Plasmodium ovale (curtisi and wallikeri), Plasmodium malariae and in southeast Asia the monkey malaria parasite Plasmodium knowlesi.^[1] Malaria was first recognized by Romans and Greeks. They observed that intermittent fever, with high incidence during the rainy season which coincides with agriculture, sowing and harvesting. So, they postulated that intermittent fevers were due to the ‘bad odour’ coming from the marshy areas and thus gave the name ‘malaria’ (‘mal’=bad + ‘air’) to the intermittent fevers. Even though today the causative organism is known, the name has stuck to this disease.

Malaria also continues to be a major public health problem in India with socio-economic implications as the disease is more prevalent in rural, tribal and forested underserved areas. ^[2]  In India about 21.98% population lives in malaria high transmission (≥ 1 case/1000 population) areas and about 67% population in low transmission (0 – 1 case/1000 population) areas. About 91% of malaria cases and 99% of malaria deaths due to malaria is reported from North-Eastern states, Chhattisgarh, Jharkhand, Madhya Pradesh, Odisha, Andhra Pradesh, Maharashtra, Gujrat, Rajasthan, West Bengal and Karnataka.Malaria is predominantly characterized by unstable transmission and transmission is seasonal with an increased intensity related to rains. Most of the population has no or little immunity. As result, most of the Indians of all age groups are at risk from malaria prone areas.^[3]

In India, the National Malaria Control Programme (NMCP) was launched in 1953. At that time, there were 75 million cases and 0.8 million deaths due to malaria. Because of the spectacular success achieved in the control of malaria, the NMCP was converted into the National Malaria Eradication Programme (NMEP) in 1958 with the objective of eradication of malaria from the country. Unfortunately, there was a resurgence of malaria in early 1970. Finally, the National Strategic Plan for Malaria Elimination in India 1017 – 2022 has been launched to reduce the disease burden.^[4]

The magnitude of malaria in India during the pre-eradication era was determined by clinically diagnosed cases and the classical parameters are spleen rate, average enlarged spleen, parasite rate etc. Microscopic diagnosis of malaria cases is the main method during the post-eradication era and the parameters are used are annual parasite index (API), annual blood examination rate (ABER), slide positivity rate (SPR) and slide falciparum rate (SFR).

Malaria is a serious public health problem in different parts of the world including India. Malaria can be severe and can lead to death if untreated. Malaria. About 95% population in the country resides in malaria endemic areas and 80% of malaria reported in the country is confined to areas consisting of 20% of the population residing in tribal, hilly, difficult and inaccessible areas. In the year 2017, 0.84 million people out of 1315 million people were affected by malaria. Plasmodium falciparum alone contributes 62.7% of all malaria cases amounting to 0.53 million. The annual parasite incidence (API) is a malariometric index to express malaria cases per thousand populations. As per the National Vector Borne Disease Control Programme (NVBDCP) incidence records the API was 0.64 in 2017.^[1] Severe malaria has high mortality if not treated promptly and adequately^.[3]

Progression to severe and fatal malaria disease is largely but not entirely confined to Plasmodium falciparum infections. Both Plasmodium vivax and Plasmodium knowlesi can also cause severe disease. Severe malaria by definition associated with high morbidity and mortality. Severe manifestation can develop in a Plasmodium falciparum infection in a short period of 12 – 24 hours and may lead to death, if not treated promptly and adequately.

As the target of the malarial parasite is RBC, peripheral blood smear examination is the major diagnostic tool of the disease. Some haematological changes are species-specific. The majority of complications in malaria are due to hyperparasiteamia. Mortality is very high (10-30%) in complicated Plasmodium falciparum infection. Haematological changes play a significant role in these serious complications.^{[3] ,[4], [5]}

The haematological abnormalities that have been reported to consistently with malaria are significantly lower platelets, WBCs, lymphocytes, eosinophils, RBCs and haemoglobin levels, while monocyte and neutrophil counts were significantly higher in comparison to non-malaria infected patients. ^[1]

In recent years, clinicians have recognized thrombocytopenia as a common and early sign of Plasmodium vivax or Plasmodium falciparum malaria infection, whereas disseminated intravascular coagulation (DIC) is rare. Platelet indices were altered during acute and symptomatic infection by Plasmodium vivax.

The elevation of mean platelet volume (MPV) and platelet distribution width (PDW), and reduction of plateletcrit are related to known potential risk factors for evolution into severe malaria, such as primo infection, longer symptom duration, and the presence of the classical warning signs of severe and complicated Plasmodium falciparum malaria.^{[1] ,[5], [6]}

Levels of procalcitonin, a prohormone of calcitonin are elevated in individuals with severe bacterial infections such as sepsis and peritonitis, and this correlates well with the severity of the disease. Recently, increased levels have been described in Plasmodium falciparum malaria.^{[7], [8] ,[9]}

Communities with high levels of the disease have many chronically ill members, resulting in absenteeism from work and school. Repeated attacks not only result in heavy spending
on treatment but also affect education, the amount of food the family can grow and
the money a family earns. Malaria is a serious risk to pregnant women and infants
and is a common cause of miscarriage. In areas of high transmission, malaria is
responsible for underweight infants at birth and anaemia in the mother (first pregnancies are particularly at risk). Lack of knowledge about malaria, poverty and chronic disease together form a vicious circle, which is difficult to break. ^{[3] ,[10]}

Contrary to the widespread belief that severe malaria is mainly caused by Plasmodium falciparum, malaria caused by Plasmodium vivax infection may also lead to severe clinical manifestations including a plethora of renal, pulmonary, hematologic, neurologic, and multi-organ dysfunction. In highly endemic areas of Plasmodium vivax transmission, early diagnosis is crucial in preventing uncomplicated episodes from progressing into severe and complicated clinical forms. In fact, given the wide geographic distribution of Plasmodium vivax, there is a large burden of disease, often not adequately acknowledged, and resulting from the combined effect of the large numbers of uncomplicated clinical episodes and the increasingly recognized severe and complicated clinical presentations.^{[3], [4]}

Malaria affects almost all blood components and is a true haematological disease. Thrombocytopenia is the most frequently malaria-associated haematological complications. In endemic areas, malaria has been reported as the major cause of low platelet counts. This is so characteristic of malaria, that in some places, it is used as an indicator of malaria in patients presenting with fever. Platelet count of less than 150,000/cu mm increases the likelihood of malaria 12-15 times.^[11] The elevation of mean platelet volume (MPV) and platelet distribution width (PDW), and reduction of plateletcrit are related to known potential risk factors for evolution into severe malaria.^[12]

Anaemia is one of the most common complications in malaria especially in younger children and pregnant women in high transmission areas. It is thought to result from a combination of haemolytic mechanisms and accelerated removal of both parasitized and non-parasitized red blood cells, depressed and ineffective erythropoiesis.^[13]

In severe malaria, findings may include metabolic acidosis, with low plasma concentrations of glucose, sodium, bicarbonate, phosphate and albumin, together with elevations in lactate, BUN, creatinine, urate, muscle and liver enzymes, and conjugated and unconjugated bilirubin. Hypergammaglobulinemia is usual in immune and semi-immune subjects living in malaria-endemic areas. Urinalysis generally gives normal results. In adults and children with cerebral malaria, the mean cerebrospinal fluid (CSF) opening pressure at lumbar puncture is ~160 mm H₂O; usually, the CSF content is normal or there is a slight elevation of total protein level (<1.0 g/L [<100 mg/dL]) and cell count (<20/μL).^[1]

It was found that patients with severe Plasmodium falciparum malaria had significantly higher procalcitonin levels on admission as compared with patients with uncomplicated Plasmodium falciparum disease. Besides, procalcitonin levels in patients with non-falciparum malaria were also higher compared with patients with non-severe falciparum malaria but lower compared with severe Plasmodium falciparum malaria.^[14]

Aims and Objective

1. To find out the prognostic significance of relevant haematological, biochemical parameters including procalcitonin value and their correlation in severe malaria.

• To assess the correlation between procalcitonin value and disease outcome (morbidity & mortality).

Review of Literature

Malaria in man is caused by infection with single-cell parasites of the genus Plasmodium. Malaria parasites are found in all countries, extending from 40^o S to 60^o N.^[5] The tropical zone is endemic for all malarial parasites. Plasmodium falciparum predominates in Africa, New Guinea, and Hispaniola. Plasmodium vivax is more common in Central and South America. The prevalence of these two species is approximately equal to the Indian subcontinent and in eastern Asia and Oceania. Plasmodium malariae is found in most endemic areas especially, throughout sub-Saharan Africa, but is much less common. Plasmodium ovale is relatively unusual outside of Africa and, where it is found, comprises <1% of isolates. Plasmodium knowlesi causes human infections commonly on the island of Borneo and, to a lesser extent, elsewhere in Southeast Asia, where the main hosts, long-tailed and pig-tailed macaques, are found.^[3]

Epidemiology:

Global scenario:

According to the estimates, there were about 228 million cases of malaria occurred worldwide in the year of 2018. Most malaria cases in 2018 were in the World Health Organization (WHO) African Region (213 million or 93%), followed by the WHO South-East Asia Region with 3.4% of the cases and the WHO Eastern Mediterranean Region with 2.1%. Nineteen countries in sub-Saharan Africa and India carried almost 85% of the global malaria burden. The incidence rate of malaria declined globally between 2010 and 2018, from 71 to 57 cases per 1000 population at risk. However, from 2014 to 2018, the rate of change slowed dramatically, reducing to 57 in 2014 and remaining at similar levels through to 2018. The WHO South-East Asia Region continued to see its incidence rate fall – from 17 cases of the disease per 1000 population at risk in 2010 to five cases in 2018 (70% decrease). In the WHO African Region, case incidence levels also declined from 294 in 2010 to 229 in 2018, representing a 22% reduction. All other WHO regions recorded either little progress or an increase in the incidence rate. The WHO Region of the Americas recorded a rise, largely due to increases in malaria transmission in the Bolivarian Republic of Venezuela. Between 2015 and 2018, only 31 countries, where malaria is still endemic, reduced case incidence significantly and was on track to reduce incidence by 40% or more by 2020.^[15]

Plasmodium falciparum is the most prevalent malaria parasite in the WHO African Region, accounting for 99.7% of estimated malaria cases in 2018, as well as in the WHO South-East Asia Region (50%), the WHO Eastern Mediterranean Region (71%) and the WHO Western Pacific Region (65%). Globally, 53% of the Plasmodium vivax burden is in the WHO South-East Asia Region, with the majority being in India (47%). Plasmodium vivax is the predominant parasite in the WHO Region of the Americas, representing 75% of malaria cases.^[15]

In 2018, there were an estimated 4,05,000 deaths from malaria globally, compared with 4,16,000 estimated deaths in 2017, and 5,85,000 in 2010. Children aged under 5 years are the most vulnerable group affected by malaria. In 2018, they accounted for 67% (2,72,000) of all malaria deaths worldwide. The WHO African Region accounted for 94% of all malaria deaths in 2018. Although this region was home to the highest number of malaria deaths in 2018, it also accounted for 85% of the 1,80,000 fewer global malaria deaths reported in 2018 compared with 2010. Nearly 85% of global malaria deaths in 2018 were concentrated in 20 countries in the WHO African Region and India; Nigeria accounted for almost 24% of all global malaria deaths, followed by the Democratic Republic of the Congo (11%), the United Republic of Tanzania (5%), and Angola, Mozambique and Niger (4% each). In 2018, only the WHO African Region and the WHO South-East Asia Region showed reductions in malaria deaths compared with 2010. The WHO African Region had the largest absolute reduction in malaria deaths, from 5,33,000 in 2010 to 3,80,000 in 2018. Despite these gains, the malaria mortality reduction rate has also slowed since 2016.

Map of malaria case incidence rate (cases per 1000 population at risk) by country Source: World Health Organization

Indian scenario:

Malaria is a public health problem in several parts of the country. About 95% population in the country resides in malaria endemic areas and 80% of malaria reported in the country is confined to areas consisting of 20% of the population residing in tribal, hilly, difficult and inaccessible areas. India is predominantly characterized by unstable malaria transmission. Transmission is seasonal with an increased intensity related to rain. Due to low and unstable transmission dynamics, most of the population has no or little immunity towards malaria. As a result, the majority of Indians living in malarious areas are at risk of infection of all age groups affected. The caseload, though steady at around 2 million cases annually in the late nineties, has shown a declining trend since 2002. The number of reported deaths has been levelling around 1000 per year between 1995 to 2010. The mortality peak in 2006 was related to severe malaria epidemics affecting Assam caused by population movements. Annual Parasite Incidence (API) rate has consistently come down from 2.12 per thousand in 2001 to 0.25 per thousand in 2019 but confirmed deaths due to malaria have been continuously down during this period between 1707 and 50.^[2],[3]

Malaria Indian scenario: The figure shows that the cases have consistently declined from 2.08 million to 0.33 million from 2001 to 2019 (Provisional). Similarly, Plasmodium falciparum cases have declined from 1.0 to 0.15 million cases during the same period. Less than 2000 deaths were reported during all the years within this period with a peak in 2006 when an epidemic was reported in north-east states.

The Parasite:

The malaria parasite passes its life cycle in two different hosts. One is the human cycle (asexual cycle) and another one is the mosquito cycle (sexual cycle).

1. Human cycle:

The parasite invades the red blood corpuscle and liver parenchymal cells and begins a period of asexual reproduction (schizogony). So human represents the intermediate hosts.

• Mosquito cycle:

For the initiation of the mosquito cycle, male and female gametocytes (sexual forms) are first developed inside the human host. After that, the gametocytes are transferred to female mosquito, where they developed further and are transformed into sporozoites. These sporozoites are infective to man. Mosquito represents as definitive host as the sexual method of reproduction occurs within its body.

1. Human cycle:

The human cycle starts when an infected mosquito bites a person and introduces sporozoites from its salivary gland during a blood meal. Sporozoites are motile, minute thread-like curved organisms measuring 9-12μm in length, tapering at both ends with a central elongated nucleus without any pigment. After the establishment of blood infection, sporozoites disappear within 60 minutes from the peripheral circulation. Many of them are destroyed by phagocytes and a few escape to reach parenchyma cells of the liver. Sporozoites undergo a developmental phase inside the hepatic parenchyma cells to form schizonts. This phase of development has been referred to as pre-erythrocytic schizogony.  This duration of development varies on the Plasmodium species. The cycle is about 8 days in Plasmodium vivax, 6 days in Plasmodium falciparum and 9 days in Plasmodium ovale. A single Plasmodium falciparum sporozoite may form as many as 40,000 merozoites. In other species of plasmodium, a single sporozoite produces around 2000 to 15,000 merozoites.^[3],[16] The infected and swollen hepatic parenchyma cells rupture and discharge the motile merozoites into the bloodstream. The liberated merozoites are called cryptozoites. The smaller merozoites enter the circulation and are called micromerozoites. The larger ones re-enter the liver parenchyma cells and are called macromerozoites. In the case of Plasmodium falciparum, intrahepatic schizonts rupture almost simultaneously and there is no persistent tissue phase. Whereas in Plasmodium vivax, Plasmodium ovale and Plasmodium malariae, a proportion of the intrahepatic forms do not divide immediately but remain dormant for considerable periods before they begin to grow and undergo pre-erythrocytic schizogony. This dormant form of the parasite is known as hypnozoite. Hypnozoites are capable of developing into merozoites and the cause of relapses that characterize infection with these species. Plasmodium vivax and Plasmodium ovale may continue to relapse for 2 to 3 years and Plasmodium malariae may persist 10 to 20 years or more. Once the parasite enters the RBC, they do not reinvade the liver.^{[1], [3], [16]}

Many merozoites that are released from the liver parenchyma cells are destroyed quickly. But significant numbers of merozoites invade red blood cells (RBCs) or erythrocytes. Attachment of the merozoites to RBCs is mediated via complex interaction with many specific erythrocyte surface receptors. Plasmodium falciparum merozoites bind to erythrocyte binding antigen 175 and glycophorin A. The other glycophorins also participate. The merozoites reticulocyte-binding protein homologue 5 (PfRh5) plays a critical role in binding to red cell basigin (CD147, EMMPRIN). Plasmodium vivax binds to receptors on young red cells. The Duffy blood-group antigen Fya or Fyb plays an important role in invasion. Most West Africans and people with origins in that region carry the Duffy-negative FyFy phenotype and are generally resistant to Plasmodium vivax malaria. Plasmodium knowlesi also invades Duffy-positive human RBCs preferentially. Inside the erythrocytes, the parasites go through the stages of trophozoite, schizont and merozoite. These asexual forms of parasites can be demonstrated in the thick smear of peripheral blood 3-4 days after the completion of the pre-erythrocytic schizogony. The time is about 12 days in Plasmodium vivax, 9 days in Plasmodium falciparum following exposure. The duration of each cycle of erythrocytic schizogony in Plasmodium vivax, Plasmodium falciparum and Plasmodium ovale are about 48 hours and in Plasmodium malariae it is about 72 hours. During the first few hours of intraerythrocytic development, the small “ring forms” of the different malaria species appear similar under light microscopy. As the trophozoites enlarge, species-specific characteristics become evident, malaria pigment (hemozoin) becomes visible, and the parasite assumes an irregular or ameboid shape. By the end of the intraerythrocytic life cycle, the parasite has consumed two-thirds of the RBC’s haemoglobin and has grown to occupy most of the cell. It is now called a schizont. Multiple nuclear divisions have taken place (schizogony or merogony). The erythrocytic phase ends with the liberation of merozoites. An infected erythrocyte ruptures to release 6–30 daughter merozoites and each potentially capable of invading a new erythrocyte to repeat the cycle. The schizogony cycle may be continued for a considerable period, but in course of time, the infection tends to die out either due to exhaustion of asexual reproductive capacity or the spontaneous destruction by the immune response of the host. Invasion and destruction of erythrocytes by the parasite and host’s immune reactions are responsible for disease manifestation in humans.

After the parasites have undergone erythrocytic schizogony, some of the merozoites, instead of developing into trophozoites and schizonts, give rise to forms that are capable of sexual function after leaving the human body. These forms are called gametocytes and develop in the erythrocytes of the capillaries of internal organs like the spleen, bone marrow. Only mature gametocytes are found in peripheral blood. The maturation is completed in about 96 hours. Gametocytes do not cause any febrile reaction in humans and are produced for the propagation and ultimate continuance of the species. ^{[3], [16], [17]}

• Mosquito cycle:

The mosquito cycle (sporogony) begins when a female Anopheles mosquito ingests blood from an infected person. A mosquito ingests both sexual and asexual forms of the parasite but only the mature sexual forms are capable of further development; the rest die off immediately. A human with Plasmodium must contain at least 12 gametocytes per cubic mm of blood to infect a mosquito and the number of female gametocytes must be more than the numbers of males.

The initial phase of development occurs inside the mid-gut (stomach) of the mosquito. From one male gametocyte, 4-8 threads like filamentous ‘microgametes’ are developed. This process is called ex-flagellation. The female gametocyte undergoes a process of maturation and becomes a female gamete or ‘macrogamete’. The macrogamete does not show any flagellation. There is only one macrogamete is formed from one female gametocyte. This maturation is achieved by a process of nuclear reduction and extrusion of polar bodies. By a process of chemotaxis, microgametes are attracted to the macrogametes. One of the male gametes attaches to the periphery of the female gamete or macrogamete at the site of a small protrusion and penetrates inside the body. The fusion of both microgamete and macrogamete form zygote. The zygote is at first a motionless body and developed in 20 minutes to 2 hours after a mosquito’s blood meal.

Zygote lengthens and matures into motile oÖkinete in the next 18-24 hours. OÖkinete penetrates the stomach wall of the mosquito and develops into an oÖcyst on the outer surface of the stomach. OÖcyst is a spherical mass surrounded by a structure less capsule and it measures 6-12 μm in diameter, containing a single vesicular nucleus and pigment granules. As the oÖcyst matures, it increases in diameter from 6 to 60 μm. Meiotic and mitotic divisions follow to form a large number of haploid sporozoites. The number of oÖcyst in the stomach wall varies from few to more than a hundred. OÖcyst takes around 10 days to mature after infection.  When mature, the oÖcyst bursts and liberates sporozoites into the body cavity of (haemocele) of the mosquito. The sporozoites are distributed through the circulating fluid into various organs and tissues of the mosquito except for ovaries. Many of the sporozoites migrate to the salivary glands of the mosquito and reach a maximum concentration in the ducts. The mosquito at this stage is capable of transmitting the infection to man. The time required for the development of sporozoite from gametocyte in the body of mosquito is about 10-20 days depending upon the favourable condition of atmospheric temperature and humidity. This period is known as the ‘extrinsic incubation period’. Different species of malarial parasites can develop in the same mosquito and such an infected mosquito can transmit the infection to the man giving rise to cases of ‘mixed infection’. The most common form of mixed infection is Plasmodium vivax with Plasmodium falciparum.   A single bite of a mosquito is sufficient to infect man.

When the parasite develops in the erythrocyte, numerous known and unknown waste substances such as hemozoin pigment and other toxic factors accumulate in the infected red blood cell. These are dumped into the bloodstream when the infected cells lyse and release invasive merozoites. The hemozoin and other toxic factors such as glucose phosphate isomerase (GPI) stimulate macrophages and other cells to produce cytokines and other soluble factors that act to produce fever and rigors and probably influence other severe pathophysiology associated with malaria. Plasmodium falciparum-infected erythrocytes, particularly those with mature trophozoites, adhere to the vascular endothelium of venular blood vessel walls and do not freely circulate in the blood. When this sequestration of infected erythrocytes occurs in the vessels of the brain it is believed to be a factor in causing the severe disease syndrome known as cerebral malaria, which is associated with high mortality. ^{[16], [17]}

      Exo-erythrocytic schizogony:

      The malaria parasite life cycle involves two hosts. During a blood meal, a malaria-infected female Anopheles mosquito inoculates sporozoites into the human host.

• Sporozoites infect liver cells.
• Sporozoites mature into schizonts.

      Schizonts rupture and release merozoites. (Of note, in Plasmodium vivax and Plasmodium ovale a dormant stage [hypnozoites] can persist in the liver (if untreated) and cause relapses by invading the bloodstream weeks, or even years later.)

      Erythrocytic schizogony:

• Merozoites infect red blood cells. The ring stage trophozoites mature into schizonts.

      Schizonts rupture releasing merozoites.

• Some parasites differentiate into sexual erythrocytic stages (gametocytes).

      Sporogonic (Mosquito) cycle:

• The gametocytes, male (microgametocytes) and female (macrogametocytes), are ingested by an Anopheles mosquito during a blood meal.

      Macrogametes generating zygotes.

      The zygotes in turn become motile and elongated (ookinetes).

• Oocysts develop from ookinetes in the midgut wall of the mosquito.

      The oocysts grow, rupture and release sporozoites, which make their way to the mosquito’s salivary glands. Inoculation of the sporozoites into a new human host perpetuates the malaria life cycle.

The life cycle of the malaria parasite

The Vector:

In India there are about 45 species of anopheline mosquitoes are present. Only a few of them regarded as the vectors of primary importance. These are Anopheles culicifacies, Anopheles fluviatilis, Anopheles stephensi, Anopheles minimus, Anopheles philippinensis, Anopheles sundaicus and Anopheles culicifacies. The vector of major importance is Anopheles culicifacies in rural areas and Anopheles stephensi in urban areas. When the vaccine is unavailable, vector control is the only practical approach to control malaria. The majority of Indian mosquitoes bite at night excepting the Aedes mosquitoes.  Malaria is transmitted by the bite of certain species of infected, female, anopheline mosquitoes. A single infected vector during her lifetime may infect several persons. Vector density is one of the major determinants of disease transmission. The key factor in the transmission of malaria is the life span of the vector. The vector mosquito must live for at least 10-12 days after an infective blood meal to become infective. The status of vector resistance to insecticides has a major contribution to disease transmission. ^{[1], [3], [4]}

The Disease:

Malaria is an acute febrile illness and the most common cause of fever in tropical countries. Infection with malaria parasites may result in a wide variety of symptoms, ranging from absent or very mild symptoms to severe disease and even death. The incubation period in most cases of malaria varies from 7 to 30 days. The incubation period is the time interval between the infective bite by the female Anopheles mosquito and the appearance of the first symptoms. The shorter periods are observed most frequently with Plasmodium falciparum and the longer ones with Plasmodium malariae. The period is usually not less than 10 days.  Antimalarial drugs taken for prophylaxis by travellers can delay the appearance of malaria symptoms by weeks or months, long after the traveller has left the malaria-endemic area. This can happen particularly with Plasmodium vivax and Plasmodium ovale, both of which can produce dormant liver stage parasites; the liver stages may reactivate and cause disease months after the infective mosquito bite. Such long delays between exposure and development of symptoms can result in misdiagnosis or delayed diagnosis because of reduced clinical suspicion. ^{[1], [3]}

In general, malaria is a curable disease if diagnosed and treated promptly and correctly. All the clinical symptoms associated with malaria are caused by the asexual erythrocytic or blood-stage parasites. Clinical diagnosis of malaria is notoriously unreliable. The first symptoms of malaria are nonspecific; the lack of a sense of well-being, headache, fatigue, abdominal discomfort, and muscle aches followed by fever are all similar to the symptoms of a minor viral illness. In some instances, a prominence of headache, chest pain, abdominal pain, cough, arthralgia, myalgia, or diarrhoea may suggest another diagnosis. Although headache may be severe in malaria, the neck stiffness and photophobia seen in meningitis do not occur. While myalgia may be prominent, it is not usually as severe as in dengue fever, and the muscles are not as tender as in leptospirosis or typhus. Nausea, vomiting, and orthostatic hypotension are common. The classic malarial paroxysms, in which fever spikes, chills, and rigors occur at regular intervals, are relatively unusual and suggest infection (often relapse) with Plasmodium vivax or Plasmodium ovale. The fever is usually irregular at first (that of falciparum malaria may never become regular). The temperature of nonimmune individuals and children often rises above 40°C (104°F), with accompanying tachycardia and sometimes delirium.

Mild hemolytic jaundice is common in malaria. Severe jaundice is associated with Plasmodium falciparum infections and more common among adults than among children. Jaundice results from hemolysis, hepatocyte injury, and cholestasis. Hepatic dysfunction contributes to hypoglycemia, lactic acidosis, and impaired drug metabolism.^[1]

Severe malaria is a multisystem disease, cerebral involvement is one of the features. Children with severe malaria are present with severe anaemia, hypoglycaemia and coma with convulsions. In Southeast Asia, where transmission is much lower and protective immunity is not acquired, all age groups can get severe malaria, but young adults are the most affected group. Cerebral malaria, renal failure, severe jaundice and adult respiratory distress syndrome are the main complications in this group. Approximately one in ten adult patients develop significant intravascular haemolysis of both infected and uninfected erythrocytes leading to haemoglobinuria (‘black water fever’), causing anaemia and contributing to renal failure. Glucose- 6 phosphate dehydrogenase deficiency is a predisposing factor.^[27] Pregnant women are particularly vulnerable in both high and low transmission settings, with severe anaemia, hypoglycaemia, coma, and pulmonary oedema as common features. In all patients with severe malaria metabolic acidosis is a frequent finding and is important to assess since it has a strong

prognostic significance. Kussmaul type respiration can be a warning symptom for this. Acidosis is mainly, but not entirely, caused by increased lactic acid production as a result of anaerobic glycolysis.^{[28], [29]} In case of renal failure, acid-base homeostasis will be further compromised. Shock is not a common feature of severe malaria and may be result of concomitant presence of septicemia.

Neurological involvement another important finding in severe malaria. Patients are present with diffuse encephalopathy with unarousable coma; focal signs are relatively uncommon. In young children coma can develop rapidly, with a mean onset after only 2 days of fever, but sometimes just a few hours.^[30]

It is often heralded by one or more generalized seizures, which cannot be distinguished clinically from febrile convulsions. In adults the onset in usually more gradual, with high fever (mean duration of 5 days) and increasing drowsiness. Occasionally frankly psychotic behaviour is the first manifestation of cerebral involvement. The level of consciousness may fluctuate over a period of hours. Convulsions are present in about 15% of the cases, whereas half of paediatric patients have convulsions.^{[30], [31], [37], [41]} Tonic-clonic generalized convulsions are commonly faced but there also be Jacksonian type or focal. In small children approximately 25% patients have subtle or subclinical convulsions. These patients often have deviated eyes, excessive salivation and irregular breathing patterns.^[38]

In areas of high transmission (Sub-Saharan Africa) a high background prevalence of peripheral parasitemia can hamper the diagnosis of ‘cerebral malaria’. A positive blood slide in a febrile comatose child does not always adequately exclude other possible diagnoses in this setting. The presence of retinal haemorrhages can sometimes be useful here because of its specificity for malaria.^{[32], [40]} Cranial nerve involvement in patients with cerebral malaria is rare.^{[38], [39]}

In children neurological residual abnormalities are more common, with approximately 12% still having symptoms at the moment of discharge, including hemiplegia, cortical blindness, aphasia and cerebellar ataxia.^[33] These symptoms will completely resolve over a period from 1 till 6 months in over half of the children, but a quarter will be left with a major residual neurological deficits.^[41]

Malaria in early pregnancy causes fetal loss. In areas of high malaria transmission, falciparum malaria in primi- and second gravida women is associated with low birth weight (average reduction, ~170 g) and consequently increased infant mortality rates. In general, infected mothers in areas of stable transmission remain asymptomatic despite intense accumulation of parasitized erythrocytes in the placental microcirculation. In areas with unstable transmission of malaria, pregnant women are prone to severe infections and are particularly likely to develop high parasitemias with anemia, hypoglycemia, and acute pulmonary edema. Fetal distress, premature labor, and stillbirth or low birth weight are common results. Fetal death is usual in severe malaria. Congenital malaria occurs in fewer than 5% of newborns whose moth­ers are infected.

In a study by B Al-Nawas and et al. in Germany in 1997 determined procalcitonin (PCT) in 38 hospitalized patients with suspected malaria. All of them had signs of infection and had recently returned from Africa. Plasmodium vivax was proven in 15, Plasmodium falciparum in one and infection with both species were found in another case (n = 17). PCT was determined on admission and the days thereafter. In one patient PCT was determined every 4 hours on the first day. The maxima of the PCT concentration on day 0 and 1 were 5.3 ng/ml with proven Malaria and 0.43 ng/ml without. In the following days, we found a decrease to normal values (<0.5 ng/ml) which correlated with the general condition of the patient. At a cut-off point of 2 ng/ml, we found a sensitivity of 52%, a positive predictive value of 74%, a specificity of 86%, a negative predictive value of 71%. procalcitonin, malaria.

They concluded that serum procalcitonin level is increased in proven cases of malaria on day 0 and 1. Then decreases to normal values (<0.5 ng/ml) which correlated with the general condition of the patient ^{[34], [35]}

U. Hollenstein and et al. in Thailand in 1998 showed Levels of procalcitonin (PCT) be elevated in individuals with Plasmodium falciparum malaria. In this study PCT levels were measured in 27 Thai patients with complicated malaria before and during/after treatment with artesunate and mefloquine. Initial parasite counts averaged 290,680/ μl (range = 533-1,147,040). On admission, all but one patient had elevated PCT levels. The level in untreated patients was 40 ng/ml (range = 0.04–662). The one patient with normal admission PCT levels had a very low parasite count of only 533 despite a temperature up to 39°C before treatment. On day 7, parasite counts in all patients were 0. Levels of PCT at this time point were 1.3 ng/ml (range = 0.01-6.5). With the exception of patient 24, PCT levels were within the normal range thereafter: day 14, 0.08 ng/ml (range = 0.01-20); day 21, 0.06 ng/ml (range = 0.01-1.5); day 28, 0.05 ng/ml (range = 0.01–1.4). Mean admission PCT levels in eight patients with recrudescence were 185.3 ng/ml compared with 52.4 ng/ml in the remaining patients.

The median PCT level in healthy controls was 0.08 ng/ml (range = 0.04-0.9). There was a significant correlation between initial parasite count and PCT levels before treatment (r = 0.43, P < 0.05;).

Procalcitonin levels on day 1 (5 days of admission, before treatment) and on day 7

Correlation of procalcitonin levels on admission with initial parasite count.

They concluded that high levels of serum procalcitonin in Plasmodium falciparum malaria that correlated with parasite density and can serve as markers of disease severity. Procalcitonin might be of value in predicting disease course and outcome. However, the small number of patients with recrudescent infections does not allow for definitive conclusions to be formed.^[7]

In a study by Collins Batsirai Chiwakata and et al. in Germany in 2000 showed that among 66 nonimmune and semi-immune patients, the serum levels of procalcitonin (PCT) in Plasmodium falciparum malaria were evaluated for clinical significance. Out of the 66 patients, 36 had uncomplicated malaria, 24 had severe and complicated malaria, and 6 had fatal malaria (5 from previous studies). Pre-treatment procalcitonin concentrations were closely correlated with parasitemia. Concentrations were lowest in semi-immune patients with uncomplicated malaria, compared with those in nonimmune patients (geometric mean concentrations [GMCs], 1.07 and 2.37 ng/mL, respectively), and were highest in severe and complicated cases (GMC, 10.67 ng/mL; P < .001 among all subgroups). Six of 7 patients with procalcitonin concentrations 125 ng/mL

died. procalcitonin concentrations decreased on day 2 of treatment in survivors but not in patients with fatal outcome. Thus, repeated procalcitonin measurements may provide useful prognostic information, especially in medical centers that are not experienced in parasite density determination.

Pre-treatment procalcitonin serum concentrations in falciparum malaria patients. A, uncomplicated malaria in semi-immune patients (n = 20); B, uncomplicated malaria in nonimmune patients (n = 16); C, severe malaria (n = 30). Horizontal bars and nos., geometric mean concentrations; m, 6 patients who died. Differences between all subgroups are significant (P < .001).

Follow-up values of procalcitonin in 3 patient groups: uncomplicated falciparum malaria (n = 36); severe falciparum malaria, survivors (n = 24); and severe falciparum malaria, patients who died (n = 6). A pattern of declining procalcitonin values on day 2 was seen in all surviving patients.

In conclusion, they demonstrated that serum procalcitonin concentrations are highly correlated with the absence of semi-immunity, the degree of disease severity, and mortality. Procalcitonin concentrations > 5 mg/mL appear to indicate a highly increased risk of mortality. Procalcitonin may therefore serve as a suitable biochemical parameter in falciparum malaria, particularly in settings in which correct parasite counting is not easily done.^[40]

In a study by Shamez Ladhani and et al. in Kenya in 2001 measured the white cell count (WCC) and platelets of 230 healthy children from the community, 1369 children admitted to the hospital with symptomatic malaria, and 1461 children with other medical conditions. Children with malaria had a higher WCC compared with community controls, and leucocytosis was strongly associated with younger age, deep breathing, severe anaemia, thrombocytopenia and death. The WCC was not associated with a positive blood culture. In children with malaria, high lymphocyte and low monocyte counts were independently associated with mortality. A platelet count of less than 150 × 10⁹/l was found in 56.7% of children with malaria, and was associated with age, prostration and parasite density, but not with bleeding problems or mortality. The mean platelet volume was also higher in children with malaria compared with other medical conditions.

They concluded that leucocytosis was associated with both severity and mortality in children with falciparum malaria, irrespective of bacteremia, whereas thrombocytopenia, although very common, was not associated with adverse outcomes.^[41]

Dennis A Hesselink and et al. in the Netherlands in 2009 performed a study over 100 consecutive travellers with various imported malaria species. In this study, twenty-nine travellers acquired a non-Plasmodium falciparum malaria infection (Plasmodium vivax n = 23; Plasmodium ovale n = 4; Plasmodium malariae n = 2), 65 travellers had an uncomplicated Plasmodium falciparum infection, whereas another six patients had severe Plasmodium falciparum disease. Thirty-one patients with uncomplicated Plasmodium falciparum disease, one patient with severe Plasmodium falciparum disease and eight patients with non-Plasmodium falciparum disease were considered to be semi-immune or partially immune, respectively. Patients with a severe Plasmodium falciparum infection had the highest serum CRP, lactate, creatinine, lactate dehydrogenase and bilirubin concentrations and the lowest haemoglobin concentrations and platelet counts on admission. The median procalcitonin concentrations differed significantly between the three groups (Chi-square 17.8; P < 0.001). The lowest median PCT concentration was observed in the group of patients with non-severe Plasmodium falciparum infections, whereas patients with severe Plasmodium falciparum infections had the highest median procalcitonin concentration.

When the groups of patients with Plasmodium falciparum infections were combined and analyzed together, a significant correlation between parasitemia and procalcitonin concentrations was observed. Patients with a high procalcitonin concentration had significantly higher parasitemia compared with patients who had a low or moderate procalcitonin concentration. For all patients with severe Plasmodium falciparum malaria the elevated procalcitonin levels on admission were classified as moderate (n = 2) or high (n = 4), respectively. Depending on the selected cut-off point, procalcitonin had excellent sensitivity for severe P. falciparum disease, whereas specificity was poor. At a cut-off point of 10 ng/mL, the test characteristics became clinically relevant with a sensitivity of 0,67 and a specificity of 0,94 for severe Plasmodium falciparum disease.

They concluded that severe Plasmodium falciparum disease and non-falciparum malaria are associated with elevated procalcitonin levels on admission. Potential drawbacks in the interpretation of elevated procalcitonin levels on admission may be caused by infections with non-falciparum Plasmodium species and by concomitant bacterial infections. ^[36]

Dinesh Yadav and et al. performed a study in India in 2011 to look for a profile of severe malaria and the contribution of Plasmodium vivax infection to malarial morbidity in North Indian children. A total of 342 patients were admitted over 3 y with a clinical diagnosis of severe malaria. Out of these, 237 cases had malaria confirmed by peripheral smear examination and/or rapid malaria antigen test, while the remaining 105 had clinical suspicion of malaria where both peripheral smear and rapid antigen test were negative. Among confirmed cases, 131 (55.3%) had Plasmodium vivax, 79 (33.3%) had Plasmodium falciparum and 27 (11.4%) had mixed infections. A comparison of clinical and haematological characteristics of patients having Plasmodium vivax and Plasmodium falciparum mono-infection was observed. The male-female ratio was similar to male predominance in both the groups (2.97:1 in Plasmodium vivax and 2.76:1 in Plasmodium falciparum). Plasmodium vivax patients had a much wider age range compared to Plasmodium falciparum though the median age was similar in both groups. However, the proportion of patients from the younger age group (<5 years) was significantly higher in Plasmodium vivax group. Duration of illness and hospitalization were also similar in both groups with a shorter median duration of symptoms in the Plasmodium falciparum group. Dengue, enteric fever and HIV co-infection were seen in 2, 1 and 1 patients, respectively in Plasmodium vivax group. Among various clinical syndromes, severe anemia, cerebral malaria and hepatitis were most common in both groups. Cerebral malaria, severe anemia and shock were more frequently observed in the Plasmodium falciparum group and this difference was statistically significant (p-value < 0.05). Hepatitis, ARF, acute respiratory distress syndrome (ARDS) and bleeding symptoms were more commonly seen in Plasmodium vivax patients. However, this difference was statistically not significant (p-value >0.05). Among haematological parameters, severe anemia (Hb < 5 g/dl) was significantly more common in the Plasmodium falciparum group and more frequently required packed red cell transfusions (p-value < 0.05). Thrombocytopenia was significantly more common in the Plasmodium vivax group (p-value < 0.05); though no significant difference was observed in the occurrence of severe thrombocytopenia and platelet transfusion requirement in both groups. Leucopenia and leukocytosis were more frequently observed in the Plasmodium falciparum group (p > 0.05). Mortality was highest in mixed infection (11.1%), followed by Plasmodium falciparum (7.6%) and Plasmodium vivax (3%) group. Most of these patients had 2 or more clinical syndromes simultaneously. Cerebral malaria was the most common cause of mortality (4 cases), followed by DIC (2), shock (2), ARF (1), and hepatitis (1) in Plasmodium falciparum patients. In patients with Plasmodium vivax mono-infection, ARDS, ARF and hepatitis (2 each) were the causes of mortality, followed by cerebral malaria and DIC.

They concluded that severe Plasmodium vivax malaria is an emerging recognized entity and all the complications seen with falciparum malaria, are now reported with vivax as well. Thrombocytopenia, respiratory complications, renal dysfunctions and hepatitis are more commonly reported with Plasmodium vivax in the present series. ^[44]

Manoj Kumar Mohapatra and et al. in India in 2013 determined serum procalcitonin semiquantitatively by immunochromatographic test in 41 patients of severe and in 19 cases of uncomplicated falciparum malaria. The diagnosis of malaria was made with the detection of the parasite from the peripheral blood smear. All patients were subjected to detail clinical, biochemical, and haematological workup. The diagnosis of severe malaria was done according to WHO criteria and the severity of organ dysfunction was assessed with Malaria Severity Score (MSS) in all patients by taking different physiological parameters into consideration. The risk stratification of severe malaria was determined with MSS and it is compared with the procalcitonin level. Results: Out of 41 patients of severe falciparum malaria 39 (95.1%) patients had multiple complications and 2 (4.9%) had a single complication. The mean MSS was 8.39 ± 4.35. According to MSS, patients were categorized into low, intermediate, and high-risk groups in 4 (9.7%), 9 (21.9%), and 28 (68.3%) patients respectively. Estimation of PCT showed that 13 (31.7%) patients of severe malaria had procalcitonin value within 2-10 ng/ml (moderately raised) and 28 (68.3%) patients had ≥ 10.0 ng/ml (highly raised). High-risk patients according to MSS were categorized as critical malaria. procalcitonin could able to diagnose such cases with excellent sensitivity and specificity.

Comparison of serum procalcitonin (PCT) in uncomplicated and severe falciparum malaria

Descriptive statistics of the accuracy of serum procalcitonin (PCT) using various cut-off points.

In another study from India by Godse RR in 2013 investigate the effects of severe malaria in infected patients on some biochemical and haematological parameters that could provide credential clues in understanding malaria pathogenesis, diagnosis and management. A total of 54 (28 males and 26 females) blood samples were collected for estimation of haemoglobin in the EDTA coated vial and stored at –20°C while the 54 non-haemolysed blood samples of serum for Liver Function and Kidney Function Test were collected in the plain vial. 1 healthy man and 1 healthy woman blood sample (as a control) were also collected.

The diagnosis of the malaria parasite in the blood samples was confirmed by observing the various stages of the malaria parasite in the stained blood film under a compound microscope. The average haemoglobin level in the male blood samples was found to be 10.91 mg/ml The average serum levels of SGPT and SGOT in plasmodium affected male patients were found to be 38.07 ± 7.44 IU/l and 29.60 ± 10.42 IU/l (Table 1) respectively which were significantly high as compared to the upper limit of the normal range. Out of 28 male patients, 5 patients showed significantly higher values of SGPT and SGOT. While in female patients it is significantly high as compared to the upper limit of the normal range. Out of 26 female patients, 6 patients showed significantly higher values of SGPT and SGOT. The average serum level of ALP in Plasmodium affected male patients was found to be 68.11 ±13.68 IU/l, which is significantly high. Seven patients show significantly higher values of ALP and also in female patients the average serum levels of ALP were found to be 67.20 ± 0.28 IU/l, which is also significantly high and out of 26 female patients, 6 patients showed higher values than the normal range. The average serum levels of Bilirubin in Plasmodium affected male patients were found to be 0.9 ± 0.2 mg/dl which is significantly high. Out of 28 male patients, the 5 patients show significantly higher values of bilirubin and also in female patients the average serum levels of bilirubin were found to be 0.78 ± 0.38 mg/dl. The average serum levels of creatinine and urea in Plasmodium affected male patients were found to be 0.95 ± 0.30 mg/dl and 17.12 ± 6.91 mg/dl respectively which is significantly high. In the male group, 7 patients showed significantly higher values of creatinine whereas in females it was only in 2 patients. In female patients, the average serum levels of creatinine and urea were found to be 1.01 ± 0.22 mg/dl and 22.73 ± 8.94 mg/dl.

The result of this study shows that malaria infection resulted in the alternations of a few parameters. Haemoglobin was significantly reduced in malaria which can be due to the increased breakdown of red blood cells by the parasites. As SGOT, SGPT and ALP are synthesized in the liver hence, it is possible that initial inflammation of the liver may increase its production due to infection 12 of plasmodium to the liver. Also, symptoms of these infections associated with vomiting could have caused increased hemoconcentration and lead to an initial increase in serum SGOT, SGPT and ALP due to the breakdown of liver cells after the infection. This study also observed a significant increase in the level of bilirubin in malaria in Aurangabad region. The concurrent increase in the serum creatinine level is mostly the result of impaired glomerular filtration of urea and creatinine and that is also an indicator for acute malarial severity. Authors have also reported high levels of creatinine in children and adults with malaria infection. This study reports hyperuricemia in malaria patients which can be attributed to the increased catabolic rate which characterizes the disease. There is also increased urea to creatinine ratio in malaria patients also indicate that the causes of uremia in these patients are largely pre-renal and may be due to reduced renal blood flow to the glomeruli due to malaria-associated hypotension and may be responsible for the reduced glomerular filtration rate and 16 hence decreased renal excretion of the analytes

They concluded that malaria has a significant impact on haematological and biochemical profile therefore it must be considered as a leading differential diagnosis in acutely febrile patients with more abnormalities like splenomegaly, fall in hemoglobin level, and raised bilirubin levels and serum creatinine-urea level.^[45]

Debojyoti Sarkar and et al. in India in 2013 conduct a study to find out the clinico‑laboratory profile of severe Plasmodium vivax malaria in a tertiary care center in Kolkata, India. During the study period, a total of 900 cases of Plasmodium vivax malaria were encountered out of which 200 showed features of severe malaria. Severe malaria was diagnosed as per the guidelines of the World Health Organization (WHO). A detailed history and clinical examination were noted. Routine haematological and biochemical investigations were carried out and treated with artemisinin based combination therapy as per hospital policy along with other supportive measures. Patients were followed‑up until discharge or death. Complications such as anaemia, hypoglycemia, convulsion, renal failure, jaundice, and circulatory collapse were treated appropriately. The patients who recovered and those who expired were grouped. They were also grouped under single and multiple complications (MC).

Out of 900 admissions of Plasmodium vivax malaria, 200 patients of severe malaria according to WHO 2000 definition were recruited in the study. The fever before admission was continuous in 54% of the cases, intermittent in 38% cases, and remittent in the remaining 8%. Most of the patients (78%) had presented with the acute illness of 2‑7 days duration of fever, 18% the fever was of 8‑14 days duration and 4% it was >14 days. A large number of patients (132) presented with a history of jaundice (66%). Fifty-six percent of the patients presented with impaired consciousness. In 22%, the presenting feature was delirium and 34% presented with unconsciousness. Generalized convulsions were seen in 28 (14%) patients as the presenting symptom associated with altered sensorium. History of dark coloured urine with oliguria was noted in 30% of cases. Diarrhoea (6‑8 times/day) with diffuse abdominal pain was the presenting symptom in 4% of the patients, although, the bleeding tendency was seen in 4 cases who had purpura. Sixty-six percent of the patients had clinically recognizable icterus. Hepatomegaly was found in 64% of the patients. Splenomegaly was found in 84% of the patients. Four patients had purpuric eruptions. Moderate to severe anaemia was seen in 46% of severe Plasmodium vivax malaria patients among whom 20% required blood transfusion. 20% had leucocytosis and 6% had leucopenia on admission. Thrombocytopenia (platelet count <100,000/cmm) was found in 60 patients (30%) among whom severe thrombocytopenia was noted in 12% [Table 1]. However, bleeding manifestations occurred in only four patients. A total of 132 patients (66%) had a serum total bilirubin level of >3 mg/dl, the highest being 31.2 mg/dl. Serum alanine transaminase (ALT) level was increased >3 fold (120 IU/l) in 48 patients (24%), whereas 76 patients (38%) had aspartate transaminase (AST) level more than 3 times the upper normal limit. A raised serum creatinine (>1.5 mg/dl) was seen in 72 patients (36%). There were 108 (54%) patients with a single complication (SC) and 92 (46%) patients with MC. Patients with SC had jaundice (48.1%) followed by cerebral malaria (25.9%), severe anemia (11.11%), renal failure (7.4%), and acute respiratory distress syndrome (ARDS) (3.7%) [Table 3]. The MC was found in various combinations and the majority (n = 44, 47.8%) had a constellation of two different complications. Renal failure with jaundice (12/44, 27%) was the most common combination. Regardless of the number of complications, jaundice was present in 86.9% (80 of 92) patients with MC. Cerebral malaria was present in 34% (68 of 200) patients alone or combination, whereas jaundice alone or in combination was found in 66% of the patients (132 of 200). The mortality rate of patients with SC and MC.

In their study, complications seen in Plasmodium vivax malaria were cerebral malaria, hyperbilirubinemia, anemia, thrombocytopenia acute renal failure (ARF) and ARDS. The incidence of severe disease among inpatients of Plasmodium vivax malaria in our study (22.2%) was a bit higher as compared to that found by other studies, probably biased by the fact the hospital is a tertiary care center. Out of the 200 patients, 60% had jaundice alone or in combination with other complications. Jaundice may be a combination of prehepatic (due to hemolysis), hepatic and cholestatic components. Hepatic dysfunction due to microvascular sequestration of parasitized red cells causes significant rises in serum bilirubin concentration, mild elevations of AST and ALT and prolongation of prothrombin time. Close to half of patients with cerebral malaria had jaundice in this study. Although consciousness was impaired in almost 56% of cases in this study, only 34% had cerebral malaria as per the WHO definition. History of convulsion was present in 14% of the patients. Seizures are attributed to various factors such as hyperpyrexia, lactic acidosis, hypoglycemia etc. Moderate to severe anemia was noted in 46% of patients, 20% needed a blood transfusion. Severe anemia occurs in Plasmodium vivax malaria due to recurrent bouts of hemolysis of predominantly uninfected erythrocytes with increased fragility. Recurrent infections due to treatment failure and relapse from the liver stages result in up to 80% of patients having recurrent malaria within 4 weeks and provide a plausible explanation for our observations that almost 6% of patients hospitalized with Plasmodium vivax have severe anemia. Sixty percent of the patients were found to have thrombocytopenia. In 12%, thrombocytopenia was severe. Bleeding due to thrombocytopenia was seen in the form of epistaxis, petechiae, ecchymoses, and hematuria. The platelet count increased with the treatment of malaria. Thrombocytopenia was more common in severe Plasmodium vivax malaria as compared to falciparum malaria.

They concluded that the mortality in Plasmodium vivax malaria increases with increasing age. Thrombocytopenia is very common in severe Plasmodium vivax infection. Renal, hepatic, lung and cerebral involvement are occurring with increasing frequency. ^[47]

In a recent study by Eduardo Rodrigues Alves-Junior et al. in Brazil in 2020, they followed 87 patients with acute Plasmodium vivax mono-infection acquired in an endemic region of the Brazilian Amazon. Forty-two different biochemical and haematological parameters frequently tested in clinical routine were evaluated at the acute phase and the convalescent phase. A total of 42 laboratory tests were performed: biochemical parameters measured were serum lipids levels, aminotransferases, bilirubin, amylase, glucose, urea, creatinine, albumin, globulin, uric acid, C-reactive protein, and alpha-1-acid glycoprotein. Haematological parameters included total and differential white blood cell and platelet counts, haemoglobin concentration, mean platelet volume, platelet width distribution, and plateletcrit.

The patients included in the study were mostly male (82%), with a mean (±SD) age of 40 (±15) years. The majority were occupationally involved in risky activities for malaria transmission, such as mining and truck driving. All cases were from the Brazilian Amazon, a region endemic for malaria, and 23%were prime-infected; the other cases reported at least one previous malaria episode at the time of diagnosis. The median parasite density was 4000/mm³, ranging from 1500/mm3(per-centile 25) to 10,000/mm3 (percentile 75). Most patients had a fever, chills, myalgia, headache, epigastric pain and vomiting, the classic symptoms of malaria. According to WHO criteria there were no cases of severely ill patients. Jaundice and enlarged spleen and liver, classic clinical signs of malaria, were present in some cases.

Out of the 42 laboratory parameters analyzed, 22 were varied significantly from the acute phase to convalescent phase. Odds ratio above 1.0 meant a greater the probability of an abnormal result in the acute phase, whereas an odds ratio below 1.0 meant a greater the probability of an abnormal result in the convalescent phase. The 10 laboratory parameters that were altered in the acute phase and returned to normal in the convalescent phase were C-reactive protein (CRP), plateletcrit, lymphocyte count, platelet count, total (TB), direct(DB) and indirect bilirubin (IB), neutrophil-to-lymphocyte ratio (NLR), α-1-acid glycoprotein (AGP), and eosinophil count. CRP was also increased in acute malaria and this increase was confirmed to be associated with the Plasmodium vivax acute phase in the present study. The median CRP in the acute phase dropped from 91 to 6.6 mg/dL in the convalescent phase (p < 0.001). The proportion of patients with increased CRP levels in the acute phase was 50-fold higher (95% CI: 20.8–136.7; p < 0.001) than in the convalescent phase. Similarly, α-1-acid glycoprotein (AGP) and erythrocyte sedimentation rate (ESR) was significantly higher in the acute phase compared to results in the convalescent phase. These findings suggest that CRP, AGP, and ESR could be used to establish the “base-line” in Plasmodium vivax malaria that could be subsequently used to monitor the therapeutic response of patients. In the present study lactate dehydrogenase (LDH) presented a later clearance and remained altered in the convalescent phase, and creatine phosphokinase (CPK) did not change in any of the phases. The probability of a low platelet count in the acute phase was 25-fold higher (95% CI: 13.3–50.2; p < 0.001) than in the convalescent phase. The probability of patients with low PCT in the acute phase was 39-fold (95% CI: 16.5–98.2; p < 0.001) higher. In addition, a platelet distribution width (PDW) above nor-mal was 2.4-fold (95%CI: 1.2–4.8; p = 0.006) more likely in the acute phase. Compared with the PDW, the mean platelet volume (MPV) was significantly higher in the acute phase (p < 0.001). All these platelet parameters are indicative of the early production of larger and more efficient platelets. Both PCT and platelet count can also help in the clinical evaluation of patients with acute Plasmodium vivax malaria. The probability of low lymphocyte and eosinophil counts were 28-fold (95% CI: 8.8–141.6; p < 0.001) and 7-fold (95% CI: 2.9–21.5; p < 0.001) higher in the acute phase. On the other hand, the probability of an increased NLR was 16-fold (95% CI: 16.6 (7.5–41.1); p < 0.001) in the acute phase. In their study, the median of the NLR changed from 2.5 in the acute phase to 1.4 in the convalescent. Because of a decrease in the number of lymphocytes and an increase in neutrophil count in Plasmodium vivax malaria, the NLR index is considered to be a novel inflammatory biomarker in malaria, indicating poor prognosis; the greater the difference between these parameters, the more severe is the disease. However, in their study, the evaluation of lymphocyte numbers alone was better as an acute phase marker than the NLR index. Basophil count showed a significant decrease in the acute phase. So far, this had not been reported in malaria. Reticulocyte count was in the normal range in the acute phase and increased in the convalescent phase; this shows a late response of this marker. Haemoglobin and haematocrit are also considered by WHO as criteria for the severity of malaria. Although haemoglobin and haematocrit values in the acute phase were decreased, they were not significantly different from those in the convalescence phase. Regarding biochemical serum parameters, other markers were higher in the acute phase of Plasmodium vivax malaria such as indirect bilirubin (OR: 17.3, 95%CI: 7.9–42.9; p < 0.001), direct bilirubin (OR: 10.4, 95%CI:5.0–23.6; p < 0.001), sodium (OR: 1.9, 95%CI: 1.0–3.8; p = 0.047), and potassium (OR: 4.1, 95%CI: 1.1–22.3; p = 0.020). On the other hand, serum total cholesterol (OR: 0.2, 95%CI: 0.1–0.7; p = 0.002), LDL (OR: 0.2, 95%CI: 0.1–0.4; p < 0.001), non-HDL (OR:0.2, 95%CI: 0.1–0.4; p < 0.001), albumin (OR: 0.3, 95%CI: 0.1–0.7;p = 0.003), and amylase (OR: 0.2, 95%CI: 0.1–1.0; p = 0.027) were reduced in the acute phase. Total cholesterol and its fractions, LDL, non-HDL and HDL, decreased in the acute phase, whereas triglyceride values were increased. In the present study, the patients had lower albumin levels (p < 0.001) in the acute phase, but had normal globulin levels. Similarly, the prothrombin time (PT) was higher in the acute phase. Other liver parameters showed no changes in the present study. There was no difference in creatinine (p = 0.541), urea (p = 0.062), and blood glucose (p = 0.080) levels between acute and convalescent phases. In fact, only one patient showed glycemia below 60 mg/dL; this is not frequent among patients with Plasmodium vivax malaria from the Amazon region. Levels of convalescent phase amylase were higher than in the acute phase, demonstrating a late increase of this enzyme or decrease in the acute phase, which has not been reported so far. Sodium and potassium levels were significantly more reduced in the acute phase compared to that in the convalescent phase.

They concluded that the 10 most relevant parameters for evaluating patients in the acute phase of Plasmodium vivax malaria were C-reactive protein, indirect bilirubin, neutrophil-to-lymphocyte ratio, total bilirubin, α-1-acid glycoprotein, and direct bilirubin, which increased expressively in the acute phase. In contrast, plateletcrit, lymphocyte, platelet and eosinophil counts were significantly reduced in the acute phase. All these parameters reverted to normal values during the convalescence period. Considering that these blood parameters are widely used in medical routine, these findings suggest that these parameters could help physicians in the first clinical evaluation and during therapeutic follow-up of uncomplicated Plasmodium vivax malaria infected patient.^[37]

Materials and Methods

1. Study setting:

Department of Pathology and Department of General Medicine,

Ramakrishna Mission Seva Pratishthan,

Vivekananda Institute of Medical Sciences,

99 Sarat Bose Road, Kolkata – 700 026.

• Study design:

It is an institution based observational case-control study.

• Period of study:

January 2019 to June 2020

• Study population:

All OPD, emergency and indoor patients being attended this hospital and who fulfil inclusion criteria.

• Sample size and design:

Participants will be stratified into two groups.

Group I:    Patients of severe malaria will be selected as per WHO guidelines.

Group II:   Patients of malaria based on peripheral blood smear or rapid diagnostic antigen detection test positivity.

Thus 66 patients to each group (cases and controls) will be used to study.

• Inclusion criteria:

Group I:    Cases of severe malaria as per World Health Organization (WHO) guidelines.

Epidemiological and research definition of severe Plasmodium falciparum malaria (For epidemiological and research purposes, severe malaria is defined as one or more of the following, occurring in the absence of an identified alternative cause, and in the presence of Plasmodium falciparum asexual parasitemia)
Impaired consciousness:	A Glasgow Coma Score <11 in adults or a Blantyre coma score <3 in children.
Acidosis:	A base deficit of >8 meq/L or, if unavailable, a plasma bicarbonate of <15 mM or venous plasma lactate >5 mM. Severe acidosis manifests clinically as respiratory distress – rapid, deep and laboured breathing
Hypoglycaemia:	Blood or plasma glucose <2.2 mM (<40 mg/dl)
Severe malarial anaemia:	A haemoglobin concentration <5 g/dL or a haematocrit of <15% in children <12 years of age (<7 g/dL and <20%, respectively, in adults) together with a parasite count >10,000/ μL
Renal impairment (acute kidney injury):	Plasma or serum creatinine >265 μM (3 mg/dL) or blood urea >20 mM
Jaundice:	Plasma or serum bilirubin >50 μM (3 mg/dL) together with a parasite count >1,00,000/μL
Pulmonary oedema:	Radiologically confirmed, or oxygen saturation <92% on room air with a respiratory rate >30/min, often with chest indrawing and crepitations on auscultation
Significant bleeding:	Including recurrent or prolonged bleeding from nose gums or venepuncture sites; haematemesis or melaena
Shock:	Compensated shock is defined as capillary refill ≥3 s or temperature gradient on leg (mid to proximal limb), but no hypotension. Decompensated shock is defined as systolic blood pressure <70 mm Hg in children or <80 mm Hg in adults with evidence of impaired perfusion (cool peripheries or prolonged capillary refill)
Hyperparasitemia:	Plasmodium falciparum parasitaemia >10%

Epidemiological and research definition of severe Plasmodium vivax malaria:

The criteria for severe Plasmodium vivax malaria are the same as for adults and children with severe Plasmodium falciparum malaria but with no parasitaemia density thresholds (and without the criterion of hyperparasitemia).

Group II:   Cases of malaria based on peripheral blood smear or rapid diagnostic antigen detection test positivity for control.

• Exclusion criteria:

Patients with a history of malignancy, alcoholics, congestive cardiac failure and liver disease were excluded from the study. They were also excluded if patients having other co-infections (i.e. enteric fever, dengue fever, sepsis, urinary tract infection, HIV, HBsAg positive patients, meningitis and encephalitis) that interferes with the clinical presentation of malaria.

• Required Parameters:
• Age of patient.
• Sex of patient
• Body temperature per oral by mercury thermometer in adult and children forehead temperature by electronic forehead thermometer.
• Anaemia, jaundice, cyanosis, clubbing, oedema, pulse rate (beats/minute), Glasgow Coma Scale (GCS) score by clinical examination.
• Middle fingertip SpO_2.
• Identification of parasite by light microscopic examination of peripheral blood smear or rapid diagnostic antigen detection test positivity.
• Determination of parasite load and hyperparasitemia.
• Haemoglobin concentration, packed cell volume (PCV) or haematocrit (Hct), total red blood cell count (TRBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) and red cell distribution width as coefficient variation (RDW-CV) by automated haematology analyzer.
• Total leucocyte count (TLC), differential leucocyte count (DLC) and platelet count by automated haematology analyzer and final confirmation by microscopy.
• Estimation of parasite load by microscopic examination of peripheral blood smear.
• Estimation of random blood glucose, serum urea, creatinine, sodium, potassium, bilirubin (total), bilirubin (conjugated), bilirubin (unconjugated), total protein, albumin, globulin, aspartate aminotransferase (AST) or serum glutamic oxalo-acetic transaminase (SGOT), alanine aminotransferase (ALT) or serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase (ALP).
• Estimation of serum procalcitonin
• Calculation of hospital stay in days.

1. Instruments used:
2. Hicks Mercury thermometer to record the oral temperature of adults and Omron MC-720 forehead thermometer to record forehead temperature of children:

Hicks Mercury thermometer	Omron MC-720 forehead thermometer

Procedure to record oral temperature:

The patients are asked to open his or her mouth, and gently insert the thermometer under the tongue, next to the frenulum. This is adjacent to a large artery (sublingual artery), so the temperature will be close to the core temperature. The patient is advised to close their lips, but not their teeth, around the thermometer, to prevent cool air circulating in the mouth. Leave in position for 3 minutes. Remove the thermometer, taking care not to touch the part that has been in the patient’s mouth and recording of temperature by noting the length of the mercury column corresponding to marking at eye level.

• BPL SmartOxy Finger-tip pulse oximeter to record SpO₂:

BPL SmartOxy Finger-tip pulse oximeter

Procedure to record SpO₂:

Turn the pulse oximeter on. It will go through internal calibration and checks. Make sure the fingertip is clean. Remove any nail varnish if present. Position the pulse oximeter carefully; make sure it fits easily without being too loose or too tight. Allow several seconds for the pulse oximeter to detect the pulse and calculate the oxygen saturation. Look for the displayed pulse indicator that shows that the machine has detected a pulse.

• Malaria parasite detection by light microscopy:

Procedure to collect blood and preparation of blood smear:

At first patient’s details are recorded on the form and the glass slides are marked. Wearing protective latex gloves, hold the patient’s left hand, palm facing upwards, and select the third finger from the thumb, called the ‘ring finger’. For infants, the big toe can be used, not the heel.

Clean the finger with cotton wool dampened with alcohol. Then dry the finger with clean cotton, using firm strokes to stimulate blood circulation. After that using a sterile lancet and a quick rolling action, puncture the ball of the finger or toe. Apply gentle pressure to the finger or toe and express the first drop of blood; wipe it away with dry cotton wool. Apply gentle pressure to the finger and collect a single small drop of blood in the middle of the slide. This is for the thin film.

Apply further gentle pressure to express more blood, and collect two or three larger drops on another slide. Wipe the remaining blood off the finger with cotton wool. This is for the thick film.

Alternatively, EDTA mixed blood obtained by peripheral venepuncture is used to prepare blood film when the preparation of direct blood film by finger puncture is not possible.

Using another clean slide as a spreader and with the slide with the blood resting on a flat, firm surface, touch the small drop of blood with the edge of the spreader, allowing the blood to run right along the edge. Firmly push the spreader along with the slide, keeping it at an angle of 45^o. The edge of the spreader must remain in even contact with the surface of the other slide while the blood is being spread.

Handling the slides 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. The circular thick film is about 1 cm in diameter. Then the films are air-dried, stained with Leishman’s stain and examined under a light microscope.

Examination of blood smear:

• Place the slide to be examined on the stage, and position the film in line with the objective lens.
  • Place a drop of immersion oil on the film, and allow it to spread.
  • Using paired x10 oculars and a x40 objective, scan the film for microfilariae, other large blood parasites and obvious debris. Select the part of the film that is well stained, free of debris and has evenly distributed white blood cells.
  • Raise the revolving nosepiece away from the stage, and swivel the x100 oil immersion objective over the selected portion of the film.
  • Raise the mechanical stage until the objective lens gently touches the immersion oil.
  • Using the fine adjustment, focus on the cell elements and confirm that the portion of the film is acceptable for routine examination: 15–20 white blood cells per thick film field will give a satisfactory film thickness. Films with fewer white blood cells per field will require more extensive examination.
  • Starting at the X mark shown in the diagram below, examine the film carefully, field by field, moving to each contiguous field as in the pattern.

Method of examination of blood film

• Routine examination of a thick film is based on an examination of 100 good fields; i.e. a slide can be pronounced negative only when a minimum of 100 fields have been carefully examined for the presence of parasites. If parasites are found but the diagnosis of species is uncertain, then further 100 fields are examined to identify a potential mixed infection.
• Finish the examination by recording the findings on the form.

• Malaria parasite detection by STANDARD Q Malaria P.f/P.v Ag Rapid Kit:

STANDARD Q Malaria P.f/P.v Ag Rapid Kit

•   Sample used – EDTA mixed blood obtained by peripheral venipuncture or capillary whole blood collected aseptically by puncturing fingertip.

•   Specimen volume required – 3μL

•   Unacceptable specimens – Clotted samples or those containing clots or fibrin strands.

•   Principle of the test:             STANDARD Q Malaria P.f/P.v Ag Test contains two pre-coated lines, “P.f” (Plasmodium falciparum), “P.v” (Plasmodium vivax) as test lines and “C” as control line on the surface of the nitrocellulose membrane. The test lines and control line in the result window of the test device is not visible before applying any specimens. Monoclonal anti-Plasmodium falciparum HRP-2 is coated on the P.f test line region, monoclonal anti-Malaria Plasmodium vivax LDH is coated on the P.v test line region and monoclonal anti-chicken IgY is coated on the control line region. During the test, the Plasmodium falciparum

specific HRP-2 and/or Plasmodium vivax specific pLDH in the specimen reacts to the gold-conjugated monoclonal anti-Malaria HRP-2 and/or gold-conjugated monoclonal anti-Malaria Plasmodium vivax LDH and then bind to them respectively. Any Plasmodium falciparum specific HRP-2 antigen-antibody gold particle complex and/or Plasmodium vivax specific pLDH antigen-antibody gold particle complex also migrates with the buffer and are immobilized by monoclonal anti-Plasmodium falciparum HRP-2 and/or monoclonal anti-Malaria Plasmodium vivax LDH at the two individual test lines to the formation of violet test coloured band(s) which confirms a positive result. The absence of this violet coloured bands indicates a negative result. The control line is used for procedural control, and should always appear if the test procedure is performed properly and the test reagents of the control line are working.

• Test procedure:
• Using a specimen transfer device takes 3μL of whole venous blood.
• Add the collected whole blood to the specimen well of the cassette.
• Add 3 drops (60μL) of buffer into buffer well.
• Read the test result in 15 minutes.
• Interpretation of result:

A coloured band will appear in the top section of the result window to show that the test is working properly. This band is the control line (C).

Colored bands will appear in the middle and lower section of the result window. These bands are the Plasmodium vivax line (P.v) and the Plasmodium falciparum line (P.f) respectively.

If there is no coloured band in the control line (C), then the test is invalid.

Interpretation of result:  STANDARD Q Malaria P.f/P.v Ag Rapid Kit

1. Estimation of parasite load:

The glass slide is placed on the microscope stage with the label to the left. This allows a standardized approach for the start point for counting and also to record parasite locations using the marked divisions on the slide holder. In the top section of the thin film, a field with about 250 red cells is identified. The total number of red cells in that field and the number of parasitized red cells are counted. A typical field (at 100x magnification) should contain approximately 250 red cells. Asexual forms (in either single or mixed species infections) are counted without sexual (gametocyte) forms, which are not counted but just reported. In mixed infections, all asexual parasites are counted together. Using a multiple type tally counter, parasitized and other red cells are counted by clicking the assigned keys for parasitized and nonparasitized red cells. After counting all the parasites and red blood cells in one field, move to the next field, following the zigzag pattern. Counting procedure to be continued in each field with attention not to overlap fields. All parasitized and other red cells in each field are counted even if the total red cell count per field exceeds 250. Stop counting when about 20 fields with about 250 red cells (about 5000 red cells) have been counted. Record the actual numbers of parasitized and other red cells counted on an appropriate worksheet. When counting is completed, parasite density can be calculated from the patient’s actual red cell count using the following formula. The final result is rounded to the nearest whole number

Number of parasitized red cells X Total RBC Count Parasites / μL =           20 fields X 250 RBCs

Number of parasitized red cells X 100 Parasite % =    20 fields X 250 RBCs

• Sysmex XN-550 – Auto analyzer:

Sysmex XN-550 – Auto analyser

An auto-analyzer is used for complete blood count (CBC).

•   Sample used – 2 mL blood sample from each patient was collected into a vial containing 2.0 mg/ml K2EDTA and preserved at 37^oC for complete blood count by a qualified phlebotomist using 24G needle.

•   Specimen volume required – Optimal draw is a tube drawn to capacity. The collection tube must be filled to a minimum of one-half full for acceptable results. A minimum of 1 mL of whole blood is required for sample analysis. To maintain the proper anticoagulant ratio tube has been filled to the 250 microliter line at the time of collection.

•   Unacceptable specimens –

1. Clotted samples or those containing clots or fibrin strands.
2. Samples were drawn above an IV (intravenous) cannula or line.

•   Principle – It is a multi-parameter quantitative automated haematology analyzer for in vitro diagnostic use in determining whole blood diagnostic parameters. The device performs hematology analyses based on hydrodynamically focused impedance measurement, the flow cytometry method (using a semiconductor laser) and the SLS-hemoglobin method. The device counts and sizes red blood cells (RBC) and platelets (PLT) using hydrodynamic impedance counting. At the same time, the hematocrit (HCT) is measured as a ratio of the total RBC volume to whole blood via the RBC pulse height detection method. Cytometry is used to analyze the physiological and chemical characteristics of cells and other biological particles. Flow cytometry is a method used to analyze those cells and particles as they pass through extremely small flow cells.

• Ortho Clinical Diagnostics VITROS 4600 Chemistry System:

Ortho Clinical Diagnostics VITROS 4600 Chemistry System

VITROS 4600 Chemistry System is used for random blood glucose, serum urea, creatinine, sodium, potassium, bilirubin (total), bilirubin (conjugated), bilirubin (unconjugated), total protein, albumin, globulin, aspartate aminotransferase (AST) or serum glutamic oxalo-acetic transaminase (SGOT), alanine aminotransferase (ALT) or serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase (ALP).

• Sample used – Serum.
• Specimen volume required – 2 mL.
• Unacceptable specimens – Hemolysed sample.
• Principle – VITROS 4600 Chemistry System MicroSlide is an entire integrated test environment on a thin piece of film. Spreading, masking, scavenger, and reagent layers are discretely combined on one postage-stamp-sized slide. When serum comes into contact with these dry chemical layers, a spectral reaction occurs which can be measured by the analyzer. Layering enables separate reaction domains so that each step can be optimized.

• bioMérieux MINI VIDAS immunoanalyzer:

bioMérieux MINI VIDAS immunoanalyzer

MINI VIDAS® is a compact automated immunoassay system based on the Enzyme Linked Fluorescent Assay (ELFA) principles.

•   Sample used – Serum

•   Specimen volume required – Sample volumes between 50 μL and 200 μL

• Unacceptable specimens – Hemolysed sample.

•   Principle – The assay principle combines a one-step immunoassay sandwich method with a final fluorescent detection (ELFA). The Solid Phase Receptacle (SPR®), serves as the solid phase as well as the pipetting device. Reagents for the assay are ready-to-use and pre-dispensed in the sealed reagent strips. All of the assay steps are performed automatically by the instrument. The sample is transferred into the wells containing anti-procalcitonin antibodies labelled with alkaline phosphatase (conjugate). The sample/conjugate mixture is cycled in and out of the SPR® several times. This operation enables the antigen to bind with the immunoglobulins fixed to the interior wall of the SPR® and the conjugate to form a sandwich. Unbound compounds are eliminated during washing steps. Two detection steps are performed successively. During each step, the substrate (4-Methyl-umbelliferyl phosphate) is cycled in and out of the SPR®. The conjugate enzyme catalyzes the hydrolysis of this substrate into a fluorescent product (4-Methyl-umbelliferone) the fluorescence of which is measured at 450 nm. The intensity of the fluorescence is proportional to the concentration of antigen present in the sample. At the end of the assay, results are automatically calculated by the instrument in relation to two calibration curves corresponding to the two detection steps. A fluorescence threshold value determines the calibration curve to be used for each sample. The results are then printed out.

1.      Method:

Results and Analysis

In our study, a total of 133 patients of any age and sex were taken. 66 patients were diagnosed with severe malaria as per WHO guidelines and another 67 patients were diagnosed with uncomplicated malaria. Then routine clinical examinations, relevant haematological, biochemical and microscopic examinations were done to compare these two diseases.

Statistical Methods:

Categorical variables are expressed as the number of patients and percentage of patients and compared across the groups using Pearson’s Chi-Square test for Independence of Attributes/ Fisher’s Exact Test as appropriate.

Continuous variables are expressed as Mean and Standard Deviation and compared across the groups using unpaired t-test.

Associations between continuous variables are captured using Pearson’s Correlation Coefficient.

The statistical software SPSS version 20 has been used for the analysis.

An alpha level of 5% has been taken, i.e. if any p-value is less than 0.05 it has been considered as significant.

Table 1:     Comparison of mean values of age between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
Age (Years)	37.47	20.07	33.78	15.16	0.233	Not Significant

Figure 1:      Comparison of mean age between severe malaria and uncomplicated malaria

The mean value of age in cases of severe malaria is 37.47 with a standard deviation of 20.07 and in cases of uncomplicated malaria is 33.78 with a standard deviation of 15.16. As per this test, the p-value is 0.233 which is not significant.

Table 2:     Distribution of gender of the patients

Parameter		GROUP		Total	p-Value	Significance
		Severe Malaria	Uncomplicated Malaria
Sex	Female	23(34.85)	17(25.37)	40(30.08)	0.233	Not Significant
	Male	43(65.15)	50(74.63)	93(69.92)
Total		66(100)	67(100)	133(100)

Figure 2:    Distribution of gender of the patients

Out of 66 severe malaria patients, 23 are female and 43 are male. Out of 67 uncomplicated malaria patients, 17 are female and 50 are male. As per this test, the p-value is 0.233 which is not significant.

Table 3:     Comparison of mean values of temperature between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
Temperature (Fahrenheit)	101.03	1.75	100.15	1.23	0.001	Significant

Figure 3:    Comparison of mean values of temperature between severe malaria and uncomplicated malaria

The mean value of temperature in cases of severe malaria is 101.03 with a standard deviation of 1.75 and in cases of uncomplicated malaria is 100.15 with a standard deviation of 1.23. As per this test, the p-value is 0.001 which is significant.

Table 4:     Comparison of mean values of systolic BP between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
Systolic BP (mm Hg)	95.67	20.75	113.04	8.78	<0.001	Significant

Figure 4:    Comparison of mean values of systolic BP between severe malaria and uncomplicated malaria

The mean systolic BP of severe malaria is 95.67 with a standard deviation of 20.75 and in cases of uncomplicated malaria is 113.04 with a standard deviation of 8.78. As per this test, the p-value is <0.001 which is significant.

Table 5:     Comparison of mean values of diastolic BP between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
Diastolic BP (mm Hg)	65.52	10.16	72.87	7.11	<0.001	Significant

Figure 5:    Comparison of mean values of diastolic BP between severe malaria and uncomplicated malaria

The mean diastolic BP of severe malaria is 65.52 mm Hg with a standard deviation of 10.16 and in cases of uncomplicated malaria is 72.87 mm Hg with a standard deviation of 7.11. As per this test, the p-value is <0.001 which is significant.

Table 6:     Comparison of mean values of pulse rate between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
Pulse rate (Number of beats/Min)	92.64	12.31	86.52	7.60	0.001	Significant

Figure 6:    Comparison of mean values of pulse rate between severe malaria and uncomplicated malaria

The mean pulse rate of severe malaria is 92.64 with a standard deviation of 12.31 and in cases of uncomplicated malaria is 86.52 with a standard deviation of 7.60. As per this test, the p-value is 0.001 which is significant.

Table 7:     Comparison of mean values of GCS between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
GCS	14.85	1.11	15	0.00	0.267	Not Significant

Figure 7:    Comparison of mean values of GCS between severe malaria and uncomplicated malaria

The mean value of GCS of severe malaria is 14.85 with a standard deviation of 1.11 and in cases of uncomplicated malaria is 15 with a standard deviation of 0.00. As per this test, the p-value is 0.267 which is significant.

Table 8:     Comparison of mean values of SpO₂ between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
SpO2	93.18	8.69	96.61	1.54	0.002	Significant

Figure 8:    Comparison of mean values of SpO2 between severe malaria and uncomplicated malaria

The mean value of SpO₂ in severe malaria is 93.18 with a standard deviation of 8.69 and in cases of uncomplicated malaria is 96.61 with a standard deviation of 1.54. As per this test, the p-value is 0.002 which is significant.

Table 9:     Comparison of mean values of haemoglobin concentration between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
Haemoglobin concentration (gm/dL)	9.82	2.27	12.14	1.68	<0.001	Significant

Figure 9:    Comparison of mean values of haemoglobin concentration between severe malaria and uncomplicated malaria

The mean Haemoglobin Concentration in severe malaria is 9.82 with a standard deviation of 2.27 and in cases of uncomplicated malaria is 12.14 with a standard deviation of 1.68. As per this test, the p-value is <0.001 which is significant.

Table 10:   Comparison of mean values of PCV between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
PCV (%)	30.28	6.26	36.81	4.76	<0.001	Significant

Figure 10:  Comparison of mean values of PCV between severe malaria and uncomplicated malaria

The mean value of PCV in severe malaria is 30.28 with a standard deviation of 6.26 and in cases of uncomplicated malaria is 36.81 with a standard deviation of 4.76.

Table 11:   Comparison of mean values of total red blood cell count between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
Total red blood cell count (Number of cells in million/μL)	3.48	0.72	4.29	0.63	<0.001	Significant

Figure 11:  Comparison of mean values of total red blood cell count between severe malaria and uncomplicated malaria

The mean value of total red blood cell count in severe malaria is 3.48 with a standard deviation of 0.72 and in cases of uncomplicated malaria is 4.29 with a standard deviation of 0.63.

Table 12:   Comparison of mean values of MCV between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
MCV (fL)	87.19	6.17	86.13	4.92	0.273	Not Significant

Figure 12:  Comparison of mean values of MCV between severe malaria and uncomplicated malaria

The mean value of MCV in severe malaria is 87.19 with a standard deviation of 6.17 and in cases of uncomplicated malaria is 86.13 with a standard deviation of 4.92.

Table 13:   Comparison of mean values of MCH between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
MCH (pg)	28.19	3.14	28.42	2.34	0.640	Not Significant

Figure 13: Comparison of mean values of MCH between severe malaria and uncomplicated malaria

The mean value of MCH in severe malaria is 28.19 with a standard deviation of 3.14 and in cases of uncomplicated malaria is 28.42 with a standard deviation of 2.34.

Table 14:   Comparison of mean values of MCHC between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
MCHC (gm/dL)	32.31	2.46	33.00	2.06	0.082	Not Significant

Figure 14:  Comparison of mean values of MCHC between severe malaria and uncomplicated malaria

The mean MCHC of severe malaria is 32.31 with a standard deviation of 2.46 and in cases of uncomplicated malaria is 33.00 with a standard deviation of 2.06.

Table 15:   Comparison of mean values of RDW-CV between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
RDW-CV	16.20	1.41	14.34	1.33	<0.001	Significant

Figure 15:  Comparison of mean values of RDW-CV between severe malaria and uncomplicated malaria

The mean RDW-CV of severe malaria is 16.20 with a standard deviation of 1.41 and in cases of uncomplicated malaria is 14.34 with a standard deviation of 1.33. As per this test, the p-value is <0.001 which is significant.

Table 16:   Comparison of mean values of platelet count between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
Platelet count (Number of cells/μL)	93893.94	37187.73	131029.85	51614.24	<0.001	Significant

Figure 16:  Comparison of mean values of platelet count between severe malaria and uncomplicated malaria

The mean platelet count of severe malaria is 93893.94 with a standard deviation of 37187.73 and in cases of uncomplicated malaria is 131029.85 with a standard deviation of 51614.24. As per this test, the p-value is <0.001 which is significant.

Table 17:   Comparison of mean values of total leucocyte count between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
Total leucocyte count (Number of cells/μL)	4551.52	1337.76	6152.24	1828.76	<0.001	Significant

Figure 17:  Comparison of mean values of total leucocyte count between severe malaria and uncomplicated malaria

The mean leukocyte count of severe malaria is 4551.52 with a standard deviation of 1337.76 and in cases of uncomplicated malaria is 6152.24 with a standard deviation of 1828.76. As per this test, p-value is <0.001 which is significant.

Table 18:   Comparison of mean values of neutrophil count between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
Neutrophil count (%)	59.85	10.48	67.43	9.58	<0.001	Significant

Figure 18:  Comparison of mean values of neutrophil count between severe malaria and uncomplicated malaria

The mean neutrophil count of severe malaria is 59.85 with a standard deviation of 10.48 and in cases of uncomplicated malaria is 67.43 with a standard deviation of 9.58. As per this test, the p-value is <0.001 which is significant.

Table 19:   Comparison of mean values of lymphocyte count between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
Lymphocyte count (%)	30.32	9.71	24.63	8.14	<0.001	Significant

Figure 19:  Comparison of mean values of lymphocyte count between severe malaria and uncomplicated malaria

The mean lymphocyte count of severe malaria is 30.32 with a standard deviation of 9.71 and in cases of uncomplicated malaria is 24.63 with a standard deviation of 8.14. As per this test, the p-value is <0.001 which is significant.

Table 20:   Comparison of mean values of monocyte count between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
Monocyte count (%)	7.15	2.43	5.48	2.26	<0.001	Significant

Figure 20:  Comparison of mean values of monocyte count between severe malaria and uncomplicated malaria

The mean monocyte count of severe malaria is 7.15 with a standard deviation of 2.43 and in cases of uncomplicated malaria is 5.48 with a standard deviation of 2.26. As per this test, the p-value is <0.001 which is significant.

Table 21:   Comparison of mean values of eosinophil count between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
Eosinophil count (%)	2.68	1.36	2.46	1.12	0.312	Not Significant

Figure 21:  Comparison of mean values of eosinophil count between severe malaria and uncomplicated malaria

The mean eosinophil count of severe malaria is 2.68 with a standard deviation of 1.36 and in cases of uncomplicated malaria is 2.46 with a standard deviation of 1.12.

Table 22:   Comparison of mean values of parasite load % (hyperparasitemia) between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
Parasite load % (hyperparasitemia)	8.53	3.80	2.79	1.04	<0.001	Significant

Figure 22:  Comparison of mean values of parasite load % (hyperparasitemia) between severe malaria and uncomplicated malaria

The mean parasite load % (hyperparasitemia) of severe malaria is 8.53 with a standard deviation of 3.80 and in cases of uncomplicated malaria is 2.79 with a standard deviation of 1.04. As per this test, the p-value is <0.001 which is significant.

Table 23:   Comparison of mean values of parasite load per microliter between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
Parasite load (number of parasites/μL)	5986.15	2952.07	2408.30	980.16	<0.001	Significant

Figure 23:  Comparison of mean values of parasite load per microliter between severe malaria and uncomplicated malaria

The mean parasite load per microliter of severe malaria is 5986.15 with a standard deviation of 2952.07 and in cases of uncomplicated malaria is 2408.30 with a standard deviation of 980.16. As per this test p-value is <0.001 which is significant.

Table 24:   Comparison of mean values of random blood glucose between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
Random blood glucose (mg/dL)	79.98	12.88	89.28	21.71	0.004	Significant

Figure 24:  Comparison of mean values of random blood glucose between severe malaria and uncomplicated malaria

The mean random blood glucose of severe malaria is 79.98 with a standard deviation of 12.88 and in cases of uncomplicated malaria is 89.28 with a standard deviation of 21.71. As per this test, the p-value is 0.004 which is significant.

Table 25:   Comparison of mean values of urea between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
Urea (mg/dL)	40.81	28.91	29.18	12.81	0.004	Significant

Figure 25:  Comparison of mean values of urea between severe malaria and uncomplicated malaria

The mean urea of severe malaria is 40.81 with a standard deviation of 28.91 and in cases of uncomplicated malaria is 29.18 with a standard deviation of 12.81. As per this test, the p-value is 0.004 which is significant.

Table 26:   Comparison of mean values of creatinine between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
Creatinine (mg/dL)	1.16	0.74	0.91	0.27	0.008	Significant

Figure 26:  Comparison of mean values of creatinine between severe malaria and uncomplicated malaria

The mean value of creatinine in severe malaria is 1.16 with a standard deviation of 0.74 and in cases of uncomplicated malaria is 0.91 with a standard deviation of 0.27. As per this test, the p-value is 0.008 which is significant.

Table 27:   Comparison of mean values of sodium between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
Sodium (mEq/L)	133.89	5.79	138.04	3.74	<0.001	Significant

Figure 27:  Comparison of mean values of sodium between severe malaria and uncomplicated malaria

The mean value of sodium in severe malaria is 133.89 with a standard deviation of 5.79 and in cases of uncomplicated malaria is 138.04 with a standard deviation of 3.74. As per this test, the p-value is <0.001 which is significant.

Table 28:   Comparison of mean values of potassium between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
Potassium (mEq/L)	3.86	0.60	4.01	0.43	0.083	Not Significant

Figure 28:  Comparison of mean values of potassium between severe malaria and uncomplicated malaria

The mean value of potassium in cases of severe malaria is 3.86 with a standard deviation of 0.60 and in cases of uncomplicated malaria is 4.01 with a standard deviation of 0.43.

Table 29:   Comparison of mean values of total bilirubin between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
Total bilirubin (mg/dL)	1.77	0.97	1.39	0.55	0.006	Significant

Figure 29:  Comparison of mean values of total bilirubin between severe malaria and uncomplicated malaria

The mean value of total bilirubin in cases of severe malaria is 1.77 with a standard deviation of 0.97 and in cases of uncomplicated malaria is 1.39 with a standard deviation of 0.55. As per this test, the p-value is 0.006 which is significant.

Table 30:   Comparison of mean values of AST / SGOT between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
AST / SGOT (U/L)	70.14	45.59	43.61	31.51	<0.001	Significant

Figure 30:  Comparison of mean values of AST / SGOT between severe malaria and uncomplicated malaria

The mean value of AST / SGOT in cases of severe malaria is 81.89 with a standard deviation of 38.93 and in cases of uncomplicated malaria is 44.75 with a standard deviation of 21.42. As per this test, the p-value is <0.001 which is significant.

Table 31:   Comparison of mean values of ALT / SGPT between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
ALT / SGPT (U/L)	70.14	45.59	43.61	31.51	<0.001	Significant

Figure 31:  Comparison of mean values of ALT / SGPT between severe malaria and uncomplicated malaria

The mean value of ALT / SGPT in cases of severe malaria is 70.14 with a standard deviation of 45.59 and in cases of uncomplicated malaria is 43.61 with a standard deviation of 31.51. As per this test, the p-value is <0.001 which is significant.

Table 32:   Comparison of mean values of ALP between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
ALP (U/L)	134.79	81.73	82.61	29.12	<0.001	Significant

Figure 32:  Comparison of mean values of ALP between severe malaria and uncomplicated malaria

The mean value of ALP in cases of severe malaria is 134.79 with a standard deviation of 81.73 and in cases of uncomplicated malaria is 82.61 with a standard deviation of 29.12. As per this test, the p-value is <0.001 which is significant.

Table 33:   Comparison of mean values of duration of hospital stay between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
Duration of hospital stay (Days)	5.53	0.83	4.36	0.90	<0.001	Significant

Figure 33:  Comparison of mean values of duration of hospital stay between severe malaria and uncomplicated malaria

The mean value of duration of hospital stay in cases of severe malaria is 5.53 with a standard deviation of 0.83 and in cases of uncomplicated malaria is 4.36 with a standard deviation of 0.90. As per this test, the p-value is <0.001 which is significant.

Table 34:   Distribution status of the parasite species in cases of severe malaria and uncomplicated malaria

		GROUP		Total	p-Value	Significance
		Severe Malaria	Uncomplicated Malaria
Parasite species	P. falciparum	30(45.45)	14(20.9)	44(33.08)	0.004	Significant
	P. vivax	33(50)	52(77.61)	85(63.91)
	P. vivax & falciparum	3(4.55)	1(1.49)	4(3.01)
Total		66(100)	67(100)	133(100)

Figure 34:  Distribution status of the parasite species in cases of severe malaria and uncomplicated malaria

Out of 66 severe malaria cases, there are 30 (45.45%) cases of Plasmodium falciparum, 33 (50%) cases of Plasmodium vivax and 3 (4.55%) cases of both Plasmodium falciparum and Plasmodium vivax. Out of 67 uncomplicated malaria cases, there are 14 (20.9%) cases of Plasmodium falciparum, 52 (77.61%) cases of Plasmodium vivax and 1 (1.49%) cases of both Plasmodium falciparum and Plasmodium vivax.

Table 35:   Comparison of mean values of procalcitonin between severe malaria and uncomplicated malaria

Parameter	GROUP				p-Value	Significance
	Severe Malaria		Uncomplicated Malaria
	Mean	Standard Deviation	Mean	Standard Deviation
Procalcitonin (ng/mL)	6.33	2.41	2.02	0.89	<0.001	Significant

Figure 35:  Comparison of mean values of procalcitonin between severe malaria and uncomplicated malaria

The mean value of procalcitonin in cases of severe malaria is 6.33 with a standard deviation of 2.41 and in cases of uncomplicated malaria is 2.02 with a standard deviation of 0.89. As per this test, the p-value is <0.001 which is significant.

Table 36:   Comparison of different clinical parameters between severe malaria and uncomplicated malaria

Parameters	Range	Severe Malaria No of Patient (%)	Uncomplicated Malaria No of Patient (%)	Total No of Patient (%)	p Value	Significance
Temperature (Fahrenheit)	Below 98.4	5 (7.6%)	6 (9.0%)	11 (8.3%)	0.002	Significant
	98.5 – 100	17 (25.8%)	32 (47.8%)	49 (36.8%)
	100 – 102	25 (37.9%)	25 (37.3%)	50 (37.6%)
	Above 102	19 (28.8%)	4 (6.0%)	23 (17.3%)
Systolic BP (mm Hg)	Below 80	29 (43.9%)	0 (0.0%)	29 (21.8%)	<0.001	Significant
	80 – 100	11 (16.7%)	8 (11.9%)	19 (14.3%)
	100 – 120	19 (28.8%)	52 (77.6%)	71 (53.4%)
	Above 120	7 (10.6%)	7 (10.4%)	14 (10.5%)
Diastolic BP ( mm Hg)	Below 60	16 (24.2%)	0 (0.0%)	16 (12.0%)	<0.001	Significant
	60 – 80	47 (71.2%)	58 (86.6%)	105 (78.9%)
	Above 80	3 (4.5%)	9 (13.4%)	12 (9.0%)
SpO2  (%)	Below 92	17 (25.8%)	0 (0.0%)	17 (12.8%)	<0.001	Significant
	92 – 95	18 (27.3%)	15 (22.4%)	33 (24.8%)
	Above 95	31 (47.0%)	52 (77.6%)	83 (62.4%)
	5 – 10	46 (69.7%)	4 (6.0%)	50 (37.6%)
	Above 10	17 (25.8%)	0 (0.0%)	17 (12.8%)
	40 – 80	30 (45.5%)	19 (28.4%)	49 (36.8%)
	Above 80	34 (51.5%)	45 (67.2%)	79 (59.4%)

It is evident that a total of 19 (28.8%) patients have a temperature above 102^o F in the severe malaria group. Whereas only 4 (6.0%) patients have a temperature above 102^o F in the uncomplicated malaria group. As per this test, the p-value is 0.002 which is significant.  29 (43.9%) cases have systolic blood pressure below 80 mm Hg in patients with severe malaria and no case has systolic blood pressure below 80 mm Hg in patients with uncomplicated malaria. Similarly, no case has diastolic blood pressure below 60 mm Hg in patients with uncomplicated malaria. Whereas 16 (24.2%) cases have diastolic blood pressure below 60 mm Hg in patients with severe malaria. A total of 17 (25.8%) cases have SpO₂ below 92% in patients with severe malaria and there is no case of SpO₂ below 92% in patients with uncomplicated malaria.

Table 37:   Comparison of different haematological parameters between severe malaria and uncomplicated malaria

Parameters	Range	Severe Malaria No of Patient (%)	Uncomplicated Malaria No of Patient (%)	Total No of Patient (%)	p Value	Significance
Haemoglobin Concentration (gm/dL)	Below 5.0	1 (1.5%)	0 (0.0%)	1 (0.8%)	<0.001	Significant
	5.0 – 7.0	14 (21.2%)	0 (0.0%)	14 (10.5%)
	7.0 – 11.0	28 (42.4%)	18 (26.9%)	46 (34.6%)
	Above 11.0	23 (34.8%)	49 (73.1%)	72 (54.1%)
PCV (%)	Below 20	4 (6.1%)	0 (0.0%)	4 (3.0%)	0.003	Significant
	20 – 40	61 (92.4%)	55 (82.1%)	116 (87.2%)
	40 – 45	1 (1.5%)	9 (13.4%)	10 (7.5%)
	Above 45	0 (0.0%)	3 (4.5%)	3 (2.3%)
Red Blood Cell Count (Number of cells in million/μL)	Below 2.5	9 (13.6%)	0 (0.0%)	9 (6.8%)	<0.001	Significant
	2.5 – 3.8	32 (48.5%)	15 (22.4%)	47 (35.3%)
	3.8 – 5.5	25 (37.9%)	49 (73.1%)	74 (55.6%)
	Above 5.5	0 (0.0%)	3 (4.5%)	3 (2.3%)
RDW-CV (%)	12 – 14	4 (6.1%)	30 (44.8%)	34 (25.6%)	<0.001	Significant
	Above 14	62 (93.9%)	37 (55.2%)	99 (74.4%)
Platelet Count (Number of cells/μL)	Below 50000	7 (10.6%)	0 (0.0%)	7 (5.3%)	0.003	Significant
	50000 – 100000	30 (45.5%)	22 (32.8%)	52 (39.1%)
	100000 – 150000	24 (36.4%)	30 (44.8%)	54 (40.6%)
	Above 150000	5 (7.6%)	15 (22.4%)	20 (15%)
Total Leucocyte Count (Number of cells/μL)	Below 4000	24 (36.4%)	5 (7.5%)	29 (21.8%)	<0.001	Significant
	4000 – 11000	42 (63.6%)	61 (91.0%)	103 (77.4%)
	Above 11000	0 (0.0%)	1 (1.5%)	1 (0.8%)
Neutrophil (%)	Below 40	3 (4.5%)	0 (0.0%)	3 (2.3%)	0.158	Not Significant
	40 – 80	61 (92.4%)	63 (94.0%)	124 (93.2%)
	Above 80	2 (3.0%)	4 (6.0%)	6 (4.5%)
Lymphocyte (%)	Below 20	6 (9.1%)	13 (19.4%)	19 (14.3%)	0.011	Significant
	20 – 40	51 (77.3%)	53 (79.1%)	104 (78.2%)
	Above 40	9 (13.6%)	1 (1.5%)	10 (7.5%)
Monocyte (%)	2 – 10	62 (93.9%)	65 (97.0%)	127 (95.5%)	0.393	Not Significant
	Above 10	4 (6.1%)	2 (3.0%)	6 (4.5%)

Haemoglobin concentration below 5.0 mg/dL and 5.0 – 7.0 mg/dL is seen in 1 (1.5%) and 14 (21.2%) cases respectively in patients with severe malaria. Whereas there is no case of haemoglobin concentration below 5.0 mg/dL and 18 (26.9%) cases with haemoglobin concentration 5.0 – 7.0 mg/dL in patients with uncomplicated malaria. As per this test, the p-value is <0.001 for all of these parameters which is significant. There is also a statistically significant fall in packed cell volume (PCV) and total red blood cell count in patients with severe malaria in comparison to uncomplicated malaria. A total of 62 (93.9%) patients with severe malaria show RDW-CV value above 14 and in the uncomplicated malaria group, there are only 37 (55.2%) cases. Thrombocytopenia is more evident in cases of severe malaria group in comparison to the uncomplicated malaria group. As per this test, the p-value is 0.003 which is significant. Low total leukocyte count is observed in patients with severe malaria. Statistically significant lymphocytosis is seen in cases of severe malaria.

Values of other haematological parameters like MCV, MCH, MCHC, differential count of neutrophil and monocyte between the two groups do not show any statistically significant findings.

Table 38:   Comparison of hyperparasitemia (%) and parasite load between severe malaria and uncomplicated malaria

Parameters	Range	Severe Malaria No of Patient (%)	Uncomplicated Malaria No of Patient (%)	Total No of Patient (%)	p Value	Significance
Hyperparasitemia (%)	Below 5	3 (4.5%)	63 (94.0%)	66 (49.6%)	<0.001	Significant
	5 – 10	46 (69.7%)	4 (6.0%)	50 (37.6%)
	Above 10	17 (25.8%)	0 (0.0%)	17 (12.8%)
Parasite Load (Number of parasites/μL)	Below 2500	6 (9.1%)	41 (61.2%)	47 (35.3%)	<0.001	Significant
	2500 – 5000	23 (34.8%)	25 (37.3%)	48 (36.1%)
	5000 – 10000	31 (47.0%)	1 (1.5%)	32 (24.1%)
	Above 10000	6 (9.1%)	0 (0.0%)	6 (4.5%)

17 (25.8%) patients show a parasite density above 10% in the severe malaria group. Whereas there is no patient with parasite density above 10% in the uncomplicated malaria group.

Table 39:   Comparison of biochemical parameters between severe malaria and uncomplicated malaria

Parameters	Range	Severe Malaria No of Patient (%)	Uncomplicated Malaria No of Patient (%)	Total No of Patient (%)	p Value	Significance
Blood Glucose (mg/dL)	Below 40	2 (3.0%)	0 (0.0%)	2 (1.5%)	0.029	Significant
	40 – 80	30 (45.5%)	19 (28.4%)	49 (36.8%)
	Above 80	34 (51.5%)	45 (67.2%)	79 (59.4%)
	Above 120	0 (0.0%)	3 (4.5%)	3 (2.3%)
Urea (mg/dL)	≤ 20	13 (19.7%)	20 (29.9%)	33 (24.8%)	0.175	Not Significant
	Above 20	53 (80.3%)	47 (70.1%)	100 (75.2%)
Creatinine (mg/dL)	≤ 1.1	46 (69.7%)	59 (88.1%)	105 (78.9%)	0.009	Significant
	Above 1.1	20 (30.3%)	8 (11.9%)	28 (21.1%)
Sodium (mEq/L)	Below 136	37 (56.1%)	11 (16.4%)	48 (36.1%)	<0.001	Significant
	≥ 136	29 (43.9%)	56 (83.6%)	85 (63.9%)
Potassium (mEq/L)	Below 3.5	18 (27.3%)	8 (11.9%)	26 (19.5%)	0.026	Significant
	≥ 3.5	48 (72.7%)	59 (88.1%)	107 (80.5%)
Bilirubin (Total) (mg/dL)	Below 1.3	23 (34.8%)	32 (47.8%)	55 (41.4%)	0.009	Significant
	1.3 – 3.0	35 (53.0%)	35 (52.2%)	70 (52.6%)
	Above 3.0	8 (12.1%)	0 (0.0%)	8 (6.0%)
AST / SGOT (U/L)	Below 40	1 (1.5%)	30 (44.8%)	31 (23.3%)	<0.001	Significant
	40 – 80	36 (54.5%)	32 (47.8%)	68 (51.1%)
	80 – 120	25 (37.9%)	3 (4.5%)	28 (21.1%)
	Above 120	4 (6.1%)	2 (3.0%)	6 (4.5%)
ALT / SGPT (U/L)	Below 40	12 (18.2%)	38 (56.7%)	50 (37.6%)	<0.001	Significant
	40 – 80	37 (56.1%)	23 (34.3%)	60 (45.1%)
	80 – 120	11 (16.7%)	2 (3.0%)	13 (9.8%)
	Above 120	6 (9.1%)	4 (6.0%)	10 (7.5%)
ALP (U/L)	Below 40	1 (1.5%)	1 (1.5%)	2 (1.5%)	<0.001	Significant
	40 – 95	24 (36.4%)	50 (74.6%)	74 (55.6%)
	95 – 300	38 (57.6%)	16 (23.9%)	54 (40.6%)
	Above 300	3 (4.5%)	0 (0.0%)	3 (2.3%)
Procalcitonin (ng/mL)	Below 0.5	0 (0.0%)	1 (1.5%)	1 (0.8%)	<0.001	Significant
	0.5 – 2.0	0 (0.0%)	36 (53.7%)	36 (21.7%)
	2.0 – 10	64 (97.0%)	30 (44.8%)	94 (70.7%)
	Above 10	2 (3.0%)	0 (0.0%)	2 (1.5%)

Clinically significant hypoglycaemia is seen in cases of severe malaria in comparison to uncomplicated malaria. A low level of serum sodium and potassium is seen in severe malaria patients compared to uncomplicated malaria patients. High level of AST/SGOT, ALT/SGPT, alkaline phosphatase (ALP), and serum procalcitonin level is found in severe malaria patients.

Table 40:   Comparison of duration of hospital stay and outcome between severe malaria and uncomplicated malaria

Parameters	Range	Severe Malaria No of Patient (%)	Uncomplicated Malaria No of Patient (%)	Total No of Patient (%)	p Value	Significance
Duration of Hospital Stay (Days)	Less than 4	0 (0.0%)	11 (16.4%)	11 (8.3%)	<0.001	Significant
	4 – 6	59 (89.4%)	56 (83.6%)	115 (86.5%)
	More than 6	7 (10.6%)	0 (0.0%)	7 (5.3%)
Outcome	Discharged	65 (98.5%)	67 (100%)	132 (99.2%)	0.314	Not Significant
	Expired	1 (1.5%)	0 (0.0%)	1 (0.8%)

11 (16.4%) patients with uncomplicated malaria get discharge from the hospital in less than 4 days. Whereas, no patient with severe malaria gets discharge from the hospital in less than 4 days. 7 (10.6%) patients with severe malaria stay in the hospital for more than 6 days. None of the patients with uncomplicated malaria has to stay in the hospital for more than 6 days.

Only one patient with severe malaria is expired whereas all other patients of both study groups are discharged from the hospital after cure.

Discussion

Malaria is a major health issue for people residing in tropical and sub-tropical areas. Many factors influence the disease manifestations of the infection and the likelihood of progression. Mortality and morbidity are mainly due to the delayed diagnosis and treatment of the potentially treatable disease.

In this study out of 133 patients, 66 patients were with severe malaria another 67 patients were with uncomplicated malaria. In our sample, out of 133 patients, 40 were female and 93 were male. Out of 40 female patients, 57.5% (n=23) were diagnosed with severe malaria & 42.5% (n=17) were diagnosed with uncomplicated malaria. Out of 93 male patients, 44% (n=43) were diagnosed with severe malaria & 56% (n=50) were diagnosed with uncomplicated malaria (Table 2; Figure 2).  Male predominance was seen in both groups, which is possibly due to higher health-seeking behaviour for males.^[43] In this population, the mean age (mean ± SD) of severe malaria patients was 37.47 ± 20.07 years and uncomplicated malaria patients were 33.78 ± 4.16 years (Table 1; Figure 1).

Our study is focused on whether haematological (Hb%, TRBC, MCV, MCH, MCHC, RDW-CV, platelet count, TLC, DLC, parasite load), biochemical (random blood glucose, urea, creatinine, sodium, potassium, bilirubin, liver enzymes) and procalcitonin can differentiate between severe and uncomplicated malaria. We did the clinical examinations, haematological and biochemical tests. Both the outcomes were compared and analyzed.

The diagnosis of malaria was done based on peripheral blood smear or rapid diagnostic antigen detection test. At diagnosis out of the total 133 cases, 33.08% (n=44) were Plasmodium falciparum positive, 63.91% (n=85) were Plasmodium vivax positive and 3.01% (n=4) were both Plasmodium falciparum and Plasmodium vivax (Table 34; Figure 34).

In this study, we found the mean value of temperature in cases of severe malaria is 101.03 ± 1.75 (range 98^o F – 105.6^o F) and in cases of uncomplicated malaria is 100.15 ± 1.23 (range 98^o F– 102.6^o F). As per the test, the p-value is 0.001 which is significant (Table 3; Figure 3).

While comparing with other studies, we found that the outcomes of haemoglobin concentration (gm/dL) are almost similar to the study done by Mohapatra MK et al. in India.^[44] They showed that the mean value of haemoglobin is 9.61 ± 2.44 in severe malaria and 11.31 ± 0.93 in uncomplicated malaria. In our studies, the mean value of haemoglobin concentration (gm/dL) is 9.82 ± 2.27 in severe malaria and 12.14 ± 1.68 in uncomplicated malaria. The overall outcomes are also matches with other studies by different authors.

Malaria is associated with a reduction of PCV and total RBC count in this study. Reduction of PCV and total RBC count are more evident in severe malaria compared to uncomplicated malaria with p-value <0.001 which is significant (Table 10 and 11; Figures 10 and 11).  This reduced PCV observed was associated with reduced total RBC count. It is evident that in malaria RBC count reduction leads to PCV reduction which subsequently causes a reduction in haemoglobin concentration. These findings are concordant with the study by Motchan PA et al.^[49]

Malaria infection is also associated with elevated RDW-CV which is more in severe malaria with p-value <0.001 which is significant in this study. (Table 15; Figure 15) Elevated RDW-CV is also been noted in other studies by Motchan PA et al.^[49] and Jairajpuri ZS et al.^[50] Elevated RDW-CV may arise from the enlargement of RBCs due to parasites.

MCV, MCH & MCHC are not found to be much different between the two groups and statistically not significant in the present study (Table 12, 13 and 14; Figure 12, 13 and 14), as well as other studies by Jairajpuri ZS et al.^[50] and Koltas IS et al.^[51]

As expected, thrombocytopenia is associated with malaria. Further, thrombocytopenia is more evident in severe malaria in comparison with uncomplicated malaria and statistically significant. (Table 16; Figure 16)   These findings are concordant with a study by Motchan PA et al.^[49], Shaikh QH et al. ^[52], Beale PJet et al.^[53], Kochar DK et al.^[54] and Coelho HC et al.^[55]

Our analysis of WBC counts showed decreased count in severe malaria in comparison with uncomplicated malaria which is statistically significant (p-value 0.001) (Table 17; Figure 17). The same finding was observed by McKenzie FE et al.^[56] In this study, we found a statistically significant decrease in differential neutrophil count, increased differential lymphocyte and monocyte in the severe malaria group in comparison to uncomplicated malaria (Table 18, 19 and 20; Figure 18, 19 and 20). In a study, Ladhani S et al.^[43] found the neutrophil counts were decreased in hyperparasitemia and in contrast, monocytosis was associated with hyperparasitemia. We also observed hyperparasitemia is associated with severe malaria in comparison to uncomplicated malaria (Table 22; Figure 22).

Lymphocytes, particularly T cells, play a major role in immunity to falciparum malaria by releasing proinflammatory cytokines, such as tumour necrosis factor-alpha (TNF-α), interferon-γ and other cytokines, and activating other inflammatory cells. Ladhani S et al.^[43] showed that higher lymphocyte count with features of severe malaria. This may represent overstimulation of the proinflammatory pathway, with deleterious consequences. Our study has a similar outcome.

We noticed a statistically significant (p-value 0.004) decreased in blood glucose level in severe malaria (mean value 79.98 ± 12.88) in comparison to uncomplicated malaria (mean value 89.98 ± 12.88). (Table 24; Figure 24). A similar type of finding was observed by Mohapatra MK et al.^[44] although their finding was statistically not significant.

In this study, we found serum urea and creatinine values were increased in severe malaria in respect to uncomplicated malaria (Table 25, 26; Figure 25, 26). A similar type of findings was observed by Mohapatra MK et al.^[46], Godse RR et al.^[47], Mangal, Praveen et al.^[57]

Jaundice in malaria may be a combination of prehepatic (due to hemolysis), hepatic and cholestatic components. Hepatic dysfunction due to microvascular sequestration of parasitized red cells causes significant rises in serum bilirubin concentration, elevations of liver enzymes. In this study we found the mean value of AST (SGOT), ALT (SGPT) and ALP are 81.89 ± 38.93, 70.14 ± 45.59 and 134.79 ± 81.73 respectively in the severe malaria group. In the uncomplicated malaria group, the mean value of AST (SGOT), ALT (SGPT) and ALP are 44.75 ± 21.42, 43.61 ± 31.51 and 82.61 ± 29.12 respectively. (Table 30, 31 and 32; Figure 30, 31 and 32). As per the test, the p-value of all three enzymes is <0.001 which is significant. Other researchers like U Hollenstein et al.^[7] and Sarkar D et al.^[48] found a similar type of result. Although Mohapatra MK et al.^[46] noticed elevated mean values of AST (SGOT), ALT (SGPT) and ALP in severe malaria in respect to uncomplicated malaria, their findings were not significant.

Procalcitonin (PCT) is a pro-hormone of calcitonin containing 116 amino acids with a molecular weight of 13 kDa. Under physiological conditions, calcitonin is produced and secreted from C-cells of the thyroid gland after intracellular proteolysis to circulation with a plasma half-life of a few minutes. Under normal condition procalcitonin level is low (<0.5 ng/ml)^[58]. Procalcitonin is found elevated in bacterial infection in response to inflammation and in addition to procalcitonin, other inflammatory mediators like IL-6, TNF, and acute phase reactants like ESR, CRP are also raised. Out of all, procalcitonin is found to be elevated in bacterial infection and correlate well with severe sepsis and septic shock i.e. when organ dysfunction is present^[59].The origin of procalcitonin in infection is thought to be extrathyroidal and the predominance of procalcitonin without an increase in calcitonin indicates the presence of a constitutive pathway within the cell that bypasses the enzymatic conversion of procalcitonin to calcitonin ^[58]. In falciparum malaria IL-6, TNF was found to increase and impairment of microcirculatory blood flow due to blockade by the parasitized RBC has a pathogenetic role in severe malaria.^[7] All these factors may induce the production of procalcitonin in falciparum malaria.

In our study we divided procalcitonin value into four groups:

Group	Value
Normal	<0.5 ng/ml
Low	– 2 ng/ml
Moderate	>2 – 10 ng/ml
High	≥ 10 ng/ml

Table 41:   Comparison of mean values of serum procalcitonin between severe malaria and uncomplicated malaria

		GROUP		Total	p-Value	Significance
		Severe Malaria	Uncomplicated Malaria
Procalcitonin (ng/mL)	Normal ( <0.5)	0(0)	1(1.49)	1(0.75)	<0.001	Significant
	Low (0.5-2)	0(0)	36(53.73)	36(27.07)
	Moderate (>2-10)	64(96.97)	30(44.78)	94(70.68)
	High (≥10)	2(3.03)	0(0)	2(1.5)
Total		66(100)	67(100)	133(100)

We found no case within the normal procalcitonin value range in the severe malaria group and only 1 case (1.49%) in the uncomplicated malaria group. There was again no case within the low procalcitonin value range from the severe malaria group and 36 cases (53.73%) were from the uncomplicated malaria group. 64 cases (96.97%) from the severe malaria group and 30 cases (44.78%) uncomplicated malaria group respectively were within the moderate procalcitonin value range. There were 2 cases (3.03%) from the severe malaria group and no cases from the uncomplicated malaria group within the high procalcitonin value range. As per the test, the p-value is <0.001 and significant.

Table 42:   Correlations between Parasite load % (hyperparasitemia) and Parasite Load per microliter with serum procalcitonin value

		Procalcitonin Value
Parasite load % (Hyperparasitemia)	Pearson Correlation	0.620
	p-Value	<0.001
Parasite load per microliter	Pearson Correlation	0.532
	p-Value	<0.001

Pearson’s Correlation Coefficient shows the strength of association between the parasite load % (hyperparasitemia) and procalcitonin value is high (r = 0.620), and that the correlation coefficient is significant (p-value < 0.001). Similarly, the association between the parasite load per microliter and procalcitonin value is also high (r = 0.532), and that the correlation coefficient is highly significant (p-value < 0.001).

In our study, one patient who expired had the highest procalcitonin level of 17.60 ng/dL

Table 43:   Comparisons between parasite load % (hyperparasitemia) and laboratory parameters of all studied patients.

Parameters	Parasite load %			p-Value	Significance
	< 5%	5% – 10%	> 10%
	Mean ± Std. Deviation	Mean ± Std. Deviation	Mean ± Std. Deviation
Systolic BP	111.61 ± 11.09	99.96 ± 20.87	88.35 ± 18.09	<0.001	Significant
Diastolic BP	72.36 ± 8.28	66.12 ± 9.47	65.18 ± 10.77	<0.001	Significant
Hb%	11.93 ± 1.93	10.09 ± 2.36	10.02 ± 2.05	<0.001	Significant
PCV	36.33 ± 5.34	31.01 ± 6.65	30.38 ± 4.97	<0.001	Significant
TRBC	4.22 ± 0.7	3.6 ± 0.76	3.44 ± 0.59	<0.001	Significant
RDW-CV	14.47 ± 1.4	16.1 ± 1.46	15.99 ± 1.73	<0.001	Significant
PLT COUNT	126439.39 ± 51577.61	99352.94 ± 42655.28	95411.76 ± 38767.03	0.003	Significant
TLC	6154.55 ± 1789.3	4527.45 ± 1436.95	4817.65 ± 1368.5	<0.001	Significant
Neutrophil	67.44 ± 10.08	60.41 ± 10.3	58.76 ± 9.07	<0.001	Significant
Lymphocyte	24.59 ± 8.56	29.69 ± 9.45	31.82 ± 8.76	0.001	Significant
Monocyte	5.53 ± 2.33	7.27 ± 2.43	6.53 ± 2.21	0.001	Significant
Blood Glucose	89.35 ± 22	79.88 ± 13.55	80 ± 9.21	0.011	Significant
Sodium	137.94 ± 3.76	134.37 ± 5.09	132.88 ± 7.69	<0.001	Significant
Potassium	4.06 ± 0.48	3.8 ± 0.49	3.85 ± 0.67	0.020	Significant
Bilirubin (Total)	1.38 ± 0.51	2.03 ± 1.13	1.25 ± 0.64	<0.001	Significant
AST / SGOT	46.32 ± 22.24	75.39 ± 44.45	91.88 ± 14.56	<0.001	Significant
ALT / SGPT	45.44 ± 31.73	64.84 ± 49.64	79.06 ± 32.49	0.002	Significant
ALP	82.33 ± 27.74	130.94 ± 82.66	151.18 ± 81.94	<0.001	Significant
Procalcitonin	2.11 ± 1.02	6.02 ± 2.69	6.65 ± 2.05	<0.001	Significant
Duration of Hospital Stay	4.41 ± 0.88	5.59 ± 1	5.12 ± 0.49	<0.001	Significant

We have divided all the patients into three groups depending on the parasite load % (Table 43). The first group is with parasite load % less than 5, the second one is with parasite load % between 5 and 10 and the third one is with parasite load % more than 10. We noticed a fall in both systolic and diastolic blood pressure with an increase in parasite density which is significant (p-value <0.001). Haemoglobin concentration (Hb%), packed cell volume (PCV), Total red blood cell count (TRBC), total leucocyte count (TLC), platelet count, blood glucose, sodium and potassium level decrease with an increased value of parasite load % and the observation is significant (p-value <0.001). Differential count of lymphocytes and monocyte, total bilirubin, AST / SGOT, ALT / SGPT, ALP, Procalcitonin and duration of hospital stay increased as parasite density increased from <5% to >10%.

We noticed there is nothing significant changes in the parameters like  MCV, MCH, MCHC, eosinophil count, serum urea, creatinine, total protein, albumin, globin with an increase in parasite density.

We noticed that anaemia, leucopenia with lymphocytosis and thrombocytopenia along with low haemoglobin concentration, PCV, TRBC (Table: 37) are more evident in severe malaria in comparison to uncomplicated malaria. Increased Parasite load and hyperparasitemia are seen in severe malaria in comparison to uncomplicated malaria (Table: 38).

Among the biochemical parameters, low values of random blood glucose, sodium and potassium are observed in patients with severe malaria. Serum creatinine, total bilirubin, liver enzymes (AST, ALT and ALP) including procalcitonin values rise in severe malaria patients and this is statistically significant.

The duration of hospital stay is much more in severe malaria patients in comparison to uncomplicated malaria cases.

In our study, one patient with severe malaria is expired and other patients from both groups are discharged from the hospital after treatment.

Summary and Conclusion

Malaria continues to be a major health problem in many areas of the globe including India. It is one of the most important causes of febrile illness in our part of the globe. Clinical diagnosis of malaria is challenging because of the non-specific nature of signs and symptoms, which overlap considerably with other febrile illnesses. Haematological abnormalities are a hallmark of malaria. Many of these abnormalities may lead to clinical suspicion for malaria, thus initiating prompt therapy even in absence of a positive smear or rapid diagnostic report for malaria.

The objective of our study is to find out the prognostic significance of relevant haematological, biochemical parameters including procalcitonin value and their co-relation in severe malaria and to assess co-relation between procalcitonin value and disease outcome (morbidity & mortality).

Comparing with other studies, our study outcome for haematological and biochemical parameters are relatable. Though some statistical variations are present in our study.

To summarize, the values of haemoglobin concentration, total red blood cell count, packed cell volume, RDW-CV, platelet count, parasite load, blood glucose, serum urea, creatinine, bilirubin including liver enzymes are a good tool for assessing morbidity and mortality in malaria.

We conclude that haematological and biochemical parameters are a fair and valid tool to assess disease outcomes (morbidity & mortality).

Procalcitonin has a strong correlation with parasite load and disease outcome. But a large number of sample size is needed to validate these tools.

References

1. J. Larry Jameson, Dennis L. Kasper, Dan L. Longo, Anthony S. Fauci, Stephen L. Hauser, Joseph Loscalzo. Harrison’s Principles of Internal Medicine: 20^th edition. pp.1575–1590.

• India, M., 2020. MAGNITUDE OF THE PROBLEM: National Vector Borne Disease Control Programme (NVBDCP). [online] Nvbdcp.gov.in. Available at: <https://nvbdcp.gov.in/index4.php?lang=1&level=0&linkid=420&lid=3699>.

• Park, K., 2019. Park’s Textbook of Preventive and Social Medicine, 25th ed. pp 280-294.

• Directorate General of Health Services, Government of India. The National Strategic Plan (NSP) for Malaria Elimination: (2017–2022); pp.7-9

• Wilmer E. Villamil-Gómez, Melisa Eyes-Escalante and Carlos Franco- Paredes. Severe and Complicated Malaria due to Plasmodium vivax, Current Topics in Malaria, Available at <https://www.intechopen.com/books/current-topics-in-malaria/severe-and-complicated-malaria-due-to-plasmodium-vivax>.

• Bassat Q, Alonso PL. Defying malaria: Fathoming severe Plasmodium vivax disease. Nat Med [Internet]. 2011;17(1):48–9.

• U Hollenstein, S Looareesuwan, A Aichelburg, F Thalhammer, B Stoiser, S Amradee, S Chullawichit, I El Menyawi, H Burgmann. Serum procalcitonin levels in severe Plasmodium falciparum malaria, The American Society of Tropical Medicine and Hygiene, The American Journal of Tropical Medicine and Hygiene, Volume 59, Issue 6, Dec 1998, p. 860 – 863.
• Assicot M, Gendrel D, Garsin H, Raymond J, Guilbaud J, Bohuon C, 1993. High serum procalcitonin concentrations in patients with sepsis and infection. Lancet 341: 515–518.

• Dandona P, Nix D, Wilson MF, Aljada A, Love J, Assicot M, Bohuon C, 1994. Procalcitonin increase after endotoxin injection in normal subjects. J Clin Endocrinol Metab 79:1605–1608.

1. World Health Organization. Severe malaria. Trop Med Int Health 2014; 19(Supp 1): S7‐ S131.

1. Gupta NK, Bansal SB, Jain UC, Sahare K. Study of thrombocytopenia in patients of malaria. Trop Parasitol 2013; 3:58-61.

1. Fábio A Leal-Santos, Soraya BR Silva, Natasha P Crepaldi, Andréia F Nery, Thamires OG Martin, Eduardo R Alves-Junior, Cor JF Fontes. Altered platelet indices as potential markers of severe and complicated malaria caused by Plasmodium vivax: a cross-sectional descriptive study. Malaria Journal, 2013, Volume 12, Number 1, Page 1.

1. Robert N Maina, Douglas Walsh, Charla Gaddy, Gordon Hongo, John Waitumbi, Lucas Otieno, David Jones, Bernhards R Ogutu. Impact of Plasmodium falciparum infection on haematological parameters in children living in Western Kenya. Malaria Journal, 2010, Volume 9, Number 3, Page 1.

1. Hesselink DA1, Burgerhart JS, Bosmans-Timmerarends H, Petit P, van Genderen PJ. Procalcitonin as a biomarker for severe Plasmodium falciparum disease: a critical appraisal of a semi-quantitative point-of-care test in a cohort of travellers with imported malaria.Malar J. 2009 Sep 1;8:206. doi: 10.1186/1475-2875-8-206.

1. World Health Organization. World Malaria Report 2019; pp.1-12

1. Chatterjee, K., 2019. Parasitology (Protozoology And Helminthology). 13th ed. pp.91-107.

1. Cdc.gov. 2020. CDC – Malaria – About Malaria – Biology. [online] Available at: <https://www.cdc.gov/malaria/about/biology/index.html>.

1. Bastidas AR, Quiroga LJ, Aponte JE, Aztorquiza MH, Perez CE. Multiorgan Failure caused by a splenic hematoma secondary to Plasmodium vivax infection. 2014;18(3): 116–9.

1. Getachew Geleta and Tsige Ketema. Severe Malaria Associated with Plasmodium falciparum and P. vivax among Children in Pawe Hospital, Northwest Ethiopia, Malaria Research and Treatment, Volume 2016, Article ID 1240962, 7 pages.

• Price, Ric Na; Douglas, Nicholas Ma; Anstey, Nicholas Ma. New developments in Plasmodium vivax malaria: severe disease and the rise of chloroquine resistance, Current Opinion in Infectious Diseases: October 2009 – Volume 22 – Issue 5 – p 430–435.

• Emiliana Tjitra, Nicholas M Anstey, Paulus Sugiarto, Noah Warikar, Enny Kenangalem, Muhammad Karyana, Daniel A Lampah, Ric N Price. Multidrug-Resistant Plasmodium vivax Associated with Severe and Fatal Malaria: A Prospective Study in Papua, Indonesia, June 17, 2008.

• Bernard Uzzan, Arezki Izri, RémyDurand, MichèleDeniau, Olivier Bouchaud, Gérard-Yves Perreta. Serum procalcitonin in uncomplicated falciparum malaria: A preliminary study, Travel Medicine and Infectious Disease, Volume 4, Issue 2, March 2006, Pages 77-80.

• Braun Nele, Marfo Yeboah, von Gartner Christiane, Burchard Gerd D., Zipfel P. F., Browne N. Edmund N., Fleischer Bernhard, Broker Barbara M. (2003) CTLA-4 positive T cells in contrast to procalcitonin plasma levels discriminate between severe and uncomplicated Plasmodium falciparum malaria in Ghanaian children. Tropical Medicine and International Health, 8 (11), 1018.

• Pierre Hausfater, Gaëlle Juillien, Beatrice Madonna-Py, Julien Haroche, Maguy Bernard and Bruno Riou. Serum procalcitonin measurement as diagnostic and prognostic marker in febrile adult patients presenting to the emergency department.

• Choudhary H, Choudhary PR. Study of clinical presentation of falciparum malaria and correlation with laboratory indices of poor prognosis. Int J Adv Med 2017; 4:1591-8.

• Te Witt, R., van Wolfswinkel, M. E., Petit, P. L., van Hellemond, J. J., Koelewijn, R., van Belkum, A., & van Genderen, P. J. (2010). Neopterin and procalcitonin are suitable biomarkers for exclusion of severe Plasmodium falciparum disease at the initial clinical assessment of travellers with imported malaria. Malaria journal, 9, 255. doi:10.1186/1475-2875-9-255

• Tran TH, Day NP, Ly VC, Nguyen TH, Pham PL, Nguyen HP, Bethell DB, Dihn XS, Tran TH, White NJ. Blackwater fever in southern Vietnam: a prospective descriptive study of 50 cases. Clin Infect Dis 1996; 23: 1274-81.

• Day N, Phu NP, Mai NTH, et al. Prognostic significance of acidosis in severe malaria. Crit Care Med 2000; 28: 1833-40.

• Dondorp AM, Thi Hong Chau T, Hoan Phu N, Thi Hoang Mai N, Phu Loc P, Van Chuong L, Xuan Sinh D, Taylor A, Tinh Hien T, White NJ, Day NPJ. Unidentified acids of strong prognostic significance in severe malaria. Crit Care Med 2004; 32: 1683-8.

• Molyneux ME, Taylor TE, Wirima JJ, Borgstein A. Clinical features and prognostic indicators in paediatric cerebral malaria: a study of 131 comatose Malawian children. Q J Med 1989; 71: 441-59.

• The South East Asian Quinine Artesunate Malaria Trial (Seaquamat) Group. Artesunate versus quinine for treatment of severe falciparum malaria: a randomised trial. Lancet 2005; 366: 717-25.

• Taylor TE, Fu WJ, Carr RA, Whitten RO, Mueller JS, Fosiko NG, Lewallen S, Liomba NG, Molyneux ME. Differentiating the pathologies of cerebral malaria by postmortem parasite counts. Nat Med 2004; 10: 143-5.

• Brewster DR, Kwiatkowski D, White NJ. Neurological sequelae of cerebral malaria in children. Lancet 1990; 336: 1039-43.

• Al-Nawas B, Shah P (1997) Procalcitonin in acute malaria. Eur J Med Res 2:206-208.

• Muñoz P, Simarro N, Rivera M, Alonso R, Alcalá L, Bouza E: Evaluation of procalcitonin as a marker of infection in a nonselected sample of febrile hospitalized patients. Diagn Microbiol Infect Dis 2004, 49:237-241.

• Chiwakata CB, Manegold C, Bönicke L, Waase I, Jülch C, et al. (2001) Procalcitonin as a parameter of disease severity and risk of mortality in patients with Plasmodium falciparum malaria. J Infect Dis 183: 1161-1164.

• ALVES-JUNIOR, Eduardo Rodrigues et al. New laboratory perspectives for evaluation of vivax malaria infected patients: a useful tool for infection monitoring. Braz J Infect Dis [online]. 2020, vol.24, n.2 [cited 2020-10-15], pp.120-129.

• Kochar DK, Saxena V, Singh N, Kochar SK, Kumar SV, Das A. Plasmodium vivax malaria. Emerg Infect Dis 2005; 11: 132-4.

• Silamut K, White NJ. Relation of the stage of parasite development in the peripheral blood to prognosis in severe falciparum malaria. Trans R Soc Trop Med Hyg 1993; 87: 436–43.

• Newton CR, Peshu N, Kendall B, Kirkham FJ, Sowunmi A, Waruiru C, Mwangi I, Murphy SA, Marsh K. Brain swelling and ischaemia in Kenyans with cerebral malaria. Arch Dis Child 1994; 70: 281-7.

• Dondorp, A.M. Pathophysiology, clinical presentation and treatment of cerebral malaria. Neurol. Asia. 2005, 10, 67-77.

• Duffy F, Bernabeu M, Babar PH, Kessler A, Wang CW, Vaz M, Chery L, Mandala WL, Rogerson SJ, Taylor TE, Seydel KB, Lavstsen T, Gomes E, Kim K, Lusingu J, Rathod PK, Aitchison JD, Smith JD. 2019. Meta-analysis of Plasmodium falciparum var signatures contributing to severe malaria in African children and Indian adults. mBio 10:e00217-19.

• Ladhani S, Lowe B, Cole A, Kowuondo K, Newton C. Changes in white blood cells and platelets in children with falciparum malaria: relationship to disease outcome. British Journal of Haematology. 2002;119(3):839-847.

• Hesselink, D.A., Burgerhart, J., Bosmans-Timmerarends, H. et al. Procalcitonin as a biomarker for severe Plasmodium falciparum disease: a critical appraisal of a semi-quantitative point-of-care test in a cohort of travellers with imported malaria. Malar J 8, 206 (2009).

• Yadav, D., Chandra, J., Aneja, S. et al. Changing Profile of Severe Malaria in North Indian Children. Indian J Pediatr 79, 483–487 (2012).

• Mohapatra MK. Serum Procalcitonin: as a Triage Tool for Severe Plasmodium falciparum Malaria. Journal of Tropical Diseases. 2013;01(04).

• Godse RR. Hematological and biochemical evaluation in malaria patients with clinical correlation. Indian Journal of Research and Reports in Medical Sciences. 2013;3(4):28-31.

• Sarkar D, Ray S, Saha M, Chakraborty A, Talukdar A. Clinico-laboratory profile of severe Plasmodium vivax malaria in a tertiary care centre in Kolkata. Trop Parasitol 2013;3:53-7.

• Motchan PA, Subashchandrabose P, Basavegowda M, Suryanarayan A. Hematological features in malarial infection and their variations with parasite density: A retrospective analysis of 6‑year data in an Indian city. Int J Health Allied Sci 2019;8:53-60.

• Jairajpuri, Z., Rana, S., Hassan, M., Nabi, F. and Jetley, S., 2014. An Analysis of Hematological Parameters as a Diagnostic test for Malaria in Patients with Acute Febrile Illness: An Institutional Experience. Oman Medical Journal, 29(1), pp.12-17.

• Koltas IS, Demirhindi H, Hazar S, Ozcan K. Supportive presumptive diagnosis of Plasmodium vivax malaria. Thrombocytopenia and red cell distribution width. Saudi Med J 2007 Apr;28(4):535-539.

• Shaikh QH, Ahmad SM, Abbasi A, et al. Thrombocytopenia in malaria. Journal of the College of Physicians and Surgeons–pakistan : JCPSP. 2009 Nov;19(11):708-710.

• Beale PJ, Cormack JD, Oldrey TB. Thrombocytopenia in malaria with immunoglobulin (IgM) changes. Br Med J 1972;1:345‑9.

• 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;21:623‑7.

• Coelho HC, Lopes SC, Pimentel JP, Nogueira PA, Costa FT, Siqueira AM, et al. Thrombocytopenia in Plasmodium vivax malaria is related to platelets phagocytosis. PLoS One 2013;8:e63410.

• McKenzie FE, Prudhomme WA, Magill AJ, Forney JR, Permpanich B, Lucas C, et al. White blood cell counts and malaria. J Infect Dis 2005;192:323‑30.

• Mangal, Praveen et al. “Analysis of the Clinical Profile in Patients with Plasmodium falciparum Malaria and Its Association with Parasite Density.” Journal of Global Infectious Diseases 9 (2017): 60 – 65.

• Müller B, White JC, Nylén ES, Snider RH, Becker KL, et al. (2001) Ubiquitous expression of the calcitonin-i gene in multiple tissues in response to sepsis. J Clin Endocrinol Metab 86: 396-404.

Aikawa N, Fujishima S, Endo S, Sekine I, Kogawa K, Yamamoto Y, et al. (2005) Multicenter prospective study of procalcitonin as an indicator of sepsis. J Infect Chemother 11:152-5.