MALARIA, ROLE OF SEROLOGY: ELISA AND PCR Groups 7: Lailiya Vina Rochmatika (115090100111005) Dita Fitriana Kusuma D. (115090101111003) Dian Cahyaningtyas P. (115090107111007)

MALARIA Malaria is a disease caused by parasites of the genus Plasmodium including protozoa through the mediation group puncture (bite) mosquito Anopheles spp. Malaria is one of the world's problems are the main public health. Malaria spreads in many countries, especially in Asia, Africa, and America Latin.

4 TYPES OF MALARIA PARASITE Plasmodium falciparum P. vivax P. malariae P. ovale

Plasmodium falciparum Falciparum malaria malignant (malaria tropica) causes fever patients every day. The incubation period of malaria or the time between the mosquito bite and the appearance of clinical symptoms about 7-14 days. Plasmodium vivax Cause disease vivax malaria (benign tertian malaria). The incubation period of malaria or the time between the mosquito bite and the appearance of clinical symptoms about 8-14 days.

Plasmodium malariae Cause disease malaria quartana. The incubation period of malaria or the time between the mosquito bite and the appearance of clinical symptoms about 7-30 days. Plasmodium ovale This type is generally a lot in Africa and the Western Pacific, causing ovale malaria.

Symptoms of Malaria

Transmission of Malaria Natural Infection This transmission occurs through the bite of an infective female Anopheles mosquito . Mosquitoes bite people sick with malaria parasite will then participate sucked the blood of patients with malaria . Parasite in the mosquito body will grow and multiply , then a mosquito bites a healthy person, the parasite transmitted through the bite to other people.

Unnatural Infection a. Congenital Malaria Occurs in newborns because her mother suffered from malaria . Due to abnormalities in the placental barrier so that there is no barrier of infection from mother to baby. b. Accidental Transmission occurs through blood transfusion or through a syringe . Transmission through needle prevalent in drug addicts who use needles that are not sterile.

Treatment of Malaria Benign malaria can be given orally. Whereas malignant malaria have clinical symptoms of bleeding should be observed in the hospital with intravenous medication.

Laboratory Diagnosis of Malaria 1. Microscopic Diagnosis a. Blood smear b. Fluorescent Microscopy c. Quantitive Buffycoat (QBC) 2. Antigen Detection a. Immunochromatographic Dipstick : RDT 3. Serology a. IFA b. ELISA 4. Molecular Diagnosis a. PCR

Serology Serology is the scientific study of blood serum. In practice, the term usually refers to the diagnostic identification of antibodies in the serum.

Serologycal Test Serologycal test is a test using serum to diagnose some disorders such as malaria. Using this methods it will be possible to do research to the reaction of antigen-antibody (Ag-Ab) in vitro. This method is based on the process of precipitation, agglutination, or complemen activation.

ELISA (Enzyme-linked Immunosorbent Assay) is a widely used biochemical technique for the detection of an antigen in a sample. ELISA assays are frequently used in viral diagnostics , for example in detecting cases of HIV infection. And than used diagnostic of malaria.

A Typical Indirect-Detection ELISA

A Typical Sandwich ELISA

ELISA have three detection for malaria: 1. Antibody detection (Seroepidemiology) 2. Antigen detection (Diagnosis) 3. Antigen detection (epidemiology)

ELISA for Diagnosis of Malaria ELISA used to identify the epidemiology of malaria-infected mosquitoes. The use of monoclonal antibodies specific for protein of Circum Sporozoite Plasmodium knowlesi. This monoclonal antibody used the solid phase and conjugated with an enzyme, as signifying the presence of protein in the sporozoite Circum mosquito homogenates that incubated in microplate wells.

ELISA for Diagnosis of Malaria Circum sporozoite protein (CSP) is an important antigen found on the surface of sporozoites, causing a role in antibody-mediated protection against parasites. Monoclonal antibodies produced to the antigen specificity was determined. By antigen capture ELISA is a useful method for rapidly detecting specific protein antigen as well as in homogenates of mosquitoes (mosquitoes scours pul).

Diagnosis of Humoral Respons with ELISA Ratio Result Analysis IgM < 0,9 Negative Nothing infection of malaria IgM 0,9 – 1,1 ekuivocal Repeat test IgM > 1,1 Positive Supposition new infection of malaria IgG < 1,8 Nothing secondary infection IgG 1,8 – 2,2 Ekuivocal IgG > 2,2 positive

PCR (Polymerase Chain Reaction) PCR (Polymerase Chain Reaction) is the process of enzymatic synthesis to amplify nucleotides in vitro

The Primary Basic Cycle of PCR The primary basic cycle of PCR happens 30-35 cycle, including: Denaturation (950C), for 30 seconds. In this step, the double helix of DNA separates into two units of single DNA. Annealing (550C-600C), for 30 seconds. In this step happens to describe the binding of single DNA probes, the temperature of this step is defined by the primary. Extension (720C), in this step, polymerase happens to form a new single DNA. The duration depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified.

PCR (Polymerase Chain Reaction) PCR can be used to identify: Genetic disorders Infection by viruses Early diagnose disorders such as AIDS The genetic profile of forensic The application of biodiversity Biologycal evolution Direct genetic mutation Measure the quantification of mRNA expression in cells or tissue

PCR for Diagnosis Malaria Molecular teqnique to identify parasite genetic material of malaria in Plasmodium DNA. Uses whole blood collected in anticoagulated tube (200 μl) or directly onto filter paper (5 μl)

Advantages PCR is a reference method. It is at least 10- fold more sensitive than microscopy. - Threshold detection * 0,1 parasite of malaria / μl if whole blood in tube * 2 parasite of malaria / μl if using filter paper More reliable for determining species in a mixed infection. Can identify mutations- try to correlate to drug resistance May have use in epidemiologic studies

Disadvantages Parasitemia malaria not quantifiable Requires specialized equipment, reagents, and training.

Analysis of a PCR diagnostic test for species specific detection of Plasmodium DNA. PCR was performed using nested primers.

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