Morphology, Life Cycle and Lab Diagnosis of Malaria Parasite (P. Vivax and P. Falciparum)

Introduction

The following four species of plasmodium cause malaria: Plasmodium vivax, Plasmodium falciparum, Plasmodium malariae, and Plasmodium ovale. Plasmodium is transmitted through female anopheles mosquito bites. Malaria in humans caused by Plasmodium falciparum is most dangerous and causes acute malaria with symptoms of high fever, postural hypotension, and massive rapture of RBC. That produces redness and swelling in limbs. Malaria in humans caused by Plasmodium vivax is mild and most common malaria. Symptoms of malaria due to Plasmodium vivax are acute fever and chills, heavy sweating, liver and spleen enlargement, anemia, headache lethargy, and abdominal cramps. Malaria by Plasmodium ovale is rarely found. Malaria by Plasmodium malariae is most common in tropical countries.

Morphology of Plasmodium vivax and Plasmodium falciparum

• Malaria Parasites exists in various form in their hosts depending upon their stage (phases) of development inside the host. Their primary host is a female anopheles mosquito and their secondary host is human. The following are the stages of their development in host cells.

  • · Inside female anopheles mosquito (Primary host): Sporozoites

  • In the Human host (Secondary host)

    • · Inside the human liver (Tissue phase): Pre-erythrocytic Schizonts.

    • · Inside human RBCs (Erythrocytic phase): Erythrocytic Schizonts. This is Gametocytes.

• Morphology of Malaria Sporozoite: It is visible under a light microscope. It is a thread-like structure with a pointed anterior end and a round posterior end. Its length varies depending on the types of malaria parasites. The pointed anterior end has secretion to help in penetration into the liver cells of humans. An elongated nucleus is present in the center of the sporozoite. It has a small amount of cytoplasm surrounded by the plasma membrane. It does not contain any pigment.

• Morphology of Malaria Pre-erythrocytic Schizont: It is typically a small and round structure. The diameter varies up to 10 micrometers. It has a cytoplasm surrounded by a plasma membrane with one nucleus in the center. Inside the nucleus several nuclei are present.

• Morphology of Malaria erythrocytic schizont: The size of erythrocytic schizont is larger than pre-erythrocytic schizont. It contains multiple nuclei and organelles in the granular cytoplasm. The cytoplasm contains dark brown or black pigment Mature nuclei in schizonts form merozoites. The rapture of schizonts releases merozoites. Merozoites invade other RBC.

• Morphology of Malaria gametocytes: Malaria gametocytes are the sexual form of malaria parasites. Their morphology depends upon their development stage. There is also variation depending upon the types of malaria parasites.

• · Stage I: Gametocytes appear round or oval at the early stage, diameter of 5 to 8 micrometers. They have pale cytoplasm and one nucleus. Gametocytes of Plasmodium vivax are smaller than Plasmodium falciparum.

• · Stage II: It is the intermediate stage. It has a larger size than stage-I gametocyte. Its center has a pigment called “chromatin dot”. Cytoplasm is darker and more granular. Gametocytes of Plasmodium vivax are smaller than Plasmodium falciparum.

• · Stage III: It is the late stage. It is larger and elongated than stage II gametocyte. More prominent and elongated chromatin dot. It has a banana shape. Gametocytes of Plasmodium vivax gametocytes are less banana-shaped than Plasmodium falciparum.

• · Stage IV: It is a mature gametocyte. It is further elongated and possesses a banana shape. More condensed cytoplasm and more prominent chromatin dot. Gametocytes of Plasmodium vivax gametocytes are less banana shape than Plasmodium falciparum.

The Life Cycle of Malarial Parasites

Malarial parasites (Plasmodium) have two hosts i.e. mosquito and human. The life cycle of malarial parasites can be divided into two phases: The Asexual phase in humans and the sexual phase in female anopheles mosquitoes.

• A. Asexual Phase (In Human): Human is a secondary host of plasmodium. It has the following four stages: Pre-erythrocytic schizogony, Exo-erythrocytic schizogony, Erythrocytic schizogony, Gametogony, and latent stage.

  • a. Pre-erythrocytic schizogony or Primary Exo-erythrocytic schizogony: Sporozoites enter into human blood during an infected anopheles mosquito bite. These sporozoites enter into human liver cells. Here it grows to form schizont. Schizont divides to form cryptozoites (merozoites). This division is called schizogony. Infected liver cells rapture to release cryptozoites (merozoites) into space inside the liver having blood. Pre-erythrocytic schizogony lasts for 8 days for Plasmodium vivax and 6 days for Plasmodium falciparum.

  • b. Exo-erythrocytic schizogony: Cryptozoites (merozoites) from pre-erythrocytic phase enter into other liver cells. Here they multiply to form metacryptozoites. Cells rapture to release metacryptozoites. Smaller ones (micrometacryptozoites) enter into the blood while larger ones (macrometacryptozoites) re-enter other cells of the liver.

  • These two stages do not show any clinical symptoms. Inoculation of this blood does not show any infection.

  • c. Erythrocytic schizogony: Micrometacryptocytes (micromerozoites) enter into RBC (erythrocyte) from blood plasma. Parasites inside RBC undergo the following changes

    • i. Young trophozoites and Signet ring stage: Inside RBC, micrometacryptozoites become round to form trophozoites. A large central vacuole appears inside the trophozoite as it grows. This vacuole pushes the cytoplasm and nucleus of trophozoite to form a signet ring-like structure in trophozoites.

    • ii. Amoeboid stage: Trophozoites become amoeboidal in shape and vacuoles disappear. They also secrete digestive enzymes that digest erythrocytes cytoplasm. They live on hemoglobin of RBC. These trophozoites are also called erythrocytic schizonts.

    • iii. Erythrocytic Micrometacryptocytes (Merozoites): RBC rapture to release these erythrocytic merozoites. These released erythrocytic merozoites attack other RBCs. During this release fever appears in infected humans.

  • The blood smear shows the presence of parasites after 3 to 4 days from the date of completion of pre-erythrocytic schizogony. Entry of sporozoites into human blood and appearance of erythrocytic schizonts in blood takes 14 days in Plasmodium vivax infection and 12 days in Plasmodium falciparum infection. This period is called the incubation period for malaria parasites.

  • d. Gametogony: The formation of gametocytes from erythrocytic merozoites is called gametogony. During erythrocytic schizogony, some merozoites enter into fresh RBC while some others form gametocytes. Gametocytes are a sexual form of plasmodium parasite. Female gametocytes are larger in shape called macrogametocytes while male gametocytes are smaller in shape called microgametocytes. It takes four days to form gametocytes from erythrocytic schizonts.

• B. Sexual phase (In Female Anopheles Mosquito): Female anopheles mosquitoes are the Primary host for the malarial parasite. Female anopheles mosquitoes live on human blood. The malarial parasite does not harm mosquitoes.

• The sexual phase of plasmodium parasites (Malaria parasites) starts inside the human body with the formation of male gametocytes (microgametocytes) and female gametocytes (macrogametocytes). During blood-sucking from infected humans by female anopheles mosquitoes, gametocytes (a sexual form of parasites) of parasites enter into the mosquito's midgut. Here development in gametocytes occurs.

• One male gametocyte (microgametocyte) develops 4 to 8 thread-like filamentous structures inside the mosquito gut. This filamentous microgametocyte is called microgamete. Development in macrogametocyte occurs to form macrogamete. One macro gametocyte forms only one macrogamete. One male gamete (microgamete) fuses with the periphery of one female gamete (macrogamete) to form a zygote.

• A mature zygote is called an Ookinete. Ookinete develop to form oocysts. An oocyst is a spherical mass covered by capsules. Oocyte size increases to form a mature oocyte that undergoes cell division to form a large sporozoite. Sporozoites enter into several tissues of mosquitoes. A maximum number of sporozoites is found in the salivary gland and salivary duct of female anopheles mosquitoes. At this stage, the mosquito is capable of transmitting infection to healthy the  human through mosquito bite.

Laboratory diagnosis of Malarial parasite infection

Malaria parasite infection is diagnosed by the microscopic examination of blood smear, rapid diagnostic test, and molecular method. Do not collect blood samples after administration of antimalarial drugs. Collect the blood sample after no sign of fever.

Microscopic examination:

Principle: Plasmodium in blood smear is detected by specific stains such as Geimsa stain, Leishman’s stain, or Wright’s stain. Laboratory technicians identify plasmodium species on the basis of their shape, size, and presence of pigment inside the Plasmodium and THE arrangement of infected RBCs. Field’s stain is used to identify parasites by chromatin dot. It is a time-consuming procedure and requires skill to identify plasmodium. Do not use methanol to fix the smear.

Procedure: The following steps are followed to detect and identify malaria infection

• · Sample collection: Collect blood samples through finger prick or venipuncture.

• · Slide preparation: Place a drop of blood sample on a clean microscopic slide. Spread the blood on the slide to make a film smear. Cover a small area of the slide.

• · Air drying: Properly dry the slide in the air. Do not heat it or blow hot air to dry the slide.

• · Fixation: Fix the smear by using a suitable fixative for 1 to 2 minutes. Avoid methanol as a fixative.

• · Staining: Stain the blood smear using the suitable staining solution. Geimsa stain is most suitable.

• · Washing: Rinse the slide with distilled water or buffer solution.

• · Microscopic examination: Examine the slide under a light microscope. Use 10X or 20X magnification to locate infected RBCs. Examine it using 100X magnification.

• · Identification: Identify malaria parasites on the basis of their morphology.

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Rapid Diagnostic Test (RDTs):

Principle: It is an immune-chromatographic process to detect malaria-specific antigens in a patient blood. Specific antigen present in the reagent binds with malaria antigen present in the blood sample. If malaria antigen is present in the sample, it will develop a visible colored line. However, it may have low sensitivity to detect malaria parasites.

Procedure: The following steps are followed to identify RDTs.

• · Gather necessary materials such as RDT kit, lancet or needle, timer, globes, alcohol swab, etc.

• · Prepare the patient and collect blood simply.

• · Apply blood sample on sample pad or well of RDT device. Do not allow it to overflow or contaminate.

• · Add buffer solution: RDT kit has buffer solution. Add it into the buffer well of the RDT device. Buffer solution helps the blood sample to move through the device and interact.

• · Set a timer as mentioned in the RDT kit. Typically it is 15 minutes.

• · Results: Interpret the result after A specific time. RDT device has normally two lines a control line (C) and a test line (T). If both lines appear, it indicates the presence of malaria antigen in the blood sample. The appearance of only the control line indicates the absence of malaria antigen in the blood sample.

· Molecular method:

• Principle: Polymerase chain reaction (PCR) is a highly sensitive method to detect malaria. It can detect low levels of malaria parasites in the blood sample. However, it may fail to distinguish plasmodium species.

• Procedure: There are two techniques to diagnose malaria. These are Polymerase chain reaction (PCR) and Loop-mediated isothermal amplification (LAMP).

Polymerase Chain Reaction (PCR):

• o Collect the sample and isolate malaria parasite DNA by using a DNA extraction kit.

• o Prepare PCR reaction mixture containing isolated DNA, primers, DNA polymerase, nucleotide, and buffer.

• o Amplify targeted DNA sequence in a thermal cycler.

• o Separate DNA fragments gel electrophoresis. Visualize them under UV light.

• o The presence of a specific-sized band on the gel indicates the presence of malaria.

Loop-Mediated Isothermal Amplification (LAMP)

• o Collect the sample and extract the DNA by using a DNA extraction kit.

• o Prepare a LAMP reaction mixture containing isolated DNA, LAMP primers, DNA polymerase, and reaction buffer.

• o Incubate THE reaction mixture at 60 degrees C for 60 minutes.

• o Add DNA interacting dye to the LAMP reaction mixture. Colour change indicates a positive reaction

Dr Pramila Singh