Malaria-2 Lecture Outline PDF

MALARIA-2 Dr. NANTHA KUMAR JEYAPRAKASAM, PhD Center for Toxicology and Health Risk Studies (CORE) Faculty of Health Science (FSK) Universiti Kebangsaan Malaysia (UKM)  LECTURE OUTLINE Subtopic 7 Subtopic 8...

MALARIA-2 Dr. NANTHA KUMAR JEYAPRAKASAM, PhD Center for Toxicology and Health Risk Studies (CORE) Faculty of Health Science (FSK) Universiti Kebangsaan Malaysia (UKM)  LECTURE OUTLINE Subtopic 7 Subtopic 8 Laboratory diagnosis Treatment for malaria (microscopy & molecular techniques) Subtopic 6 Subtopic 9 Complication of malaria Prevention & control for malaria  COMPLICATION OF MALARIA 1. Cerebral malaria Most severe complications Late stage schizonts of P. falciparum secrete a protein on the surface of RBCs to form knobs. fatal complication, headache, neck stiffness, disorientation, coma, death This knob produces specific adhesive proteins, which promote adhesion of infected RBCs to other non- A multi-factorial process with infected RBCs and adhere to the receptors on capillary sequestration, inflammation and endothelial cells. endothelial dysfunction in the microvascular of the brain These sequestrated RBCs cause obstruction of cerebral leading to coma. microvasculature which results in anoxia, ischaemia and haemorrhage causing cerebral malaria.  COMPLICATIONS (P. falciparum) 2. Hemolytic anemia Causes of anemia in malaria Late stage schizonts of P. falciparum secrete a protein on the surface of RBCs to form knobs. This knob produces specific adhesive proteins, which promote adhesion of infected RBCs to other non- infected RBCs and adhere to the receptors on capillary endothelial cells. These sequestrated RBCs cause obstruction of cerebral microvasculature,which results in anoxia, ischaemia and haemorrhage causing cerebral malaria.  COMPLICATIONS (P. falciparum) 3. Blackwater fever Seen in ▪ in falciparum malaria, ▪ in patients who have experienced repeated infections ▪ inadequate treatment with quinine. Clinical manifestations include: ✓ bilious vomiting and prostration, ✓ with passage of dark red or blackish urine (blackwater). The pathogenesis is due to massive intravascular haemolysis caused by anti-erythrocyte antibodies, giving rise to haemoglobinuria. Complications of blackwater fever include renal failure, acute liver failure, and circulatory collapse. 4. Gastrointestinal syndrome jaundice, hepatomegaly, epigastric pain, nausea, vomitting, diarrhea  COMPLICATIONS (P. falciparum) 5. Algid malaria Adrenal insufficiency like syndrome - leading to shock. Low BP, rapid pulse, difficulty in breathing, hemoconcentration 6. Hypoglycaemia Especially during pregnancy. May cause neurological problems eg. Restlessness, dyspnoea, convulsion, loss of consciousness depletion of liver glycogen from decrease oral intake, glucose consumption by parasite, hypoglycaemic effect of TNF 7. Pulmonary oedema (PO) Increased level of TNF may produce PO or as a result of over infusion 8. Acute renal failure Severe case - Due to tubular necrosis 9. Septicemic malaria It is characterized by high continuous fever Dissemination of the parasite to various organs, leading to multiorgan failure. Death occurs in 80% of the cases. COMPLICATIONS (Other falciparum) P. vivax & P. ovale May develop serious disease but complications are rare P. malariae Mild infection but can usually become very chronic Main complication is nephrotic syndrome Immune complexes may cause structural glomerular damage and the associated nephrotic syndrome. COMPLICATIONS (P. knowlesi) Single or multi system failure : Acute respiratory distress syndrome Acute Renal failure Hepatic dysfunction Metabolic acidosis Hypoglycaemia Hypotension Thrombocytopenia/lymphopenia  RECRUDESCENCE & RELAPSE Differences between recrudescence and relapse  LABORATORY DIAGNOSIS History from the patient Signs and symptoms To confirm diagnosis by laboratory investigations (blood films) Blood smears are stained with Romanowsky staining (Giemsa/Field stain)- The cytoplasm will be stained bluish and the nuclear chromatin reddish 2 types of blood films used; thin and thick blood films Investigating suspected malaria 1. Demonstration of Parasite by Microscopy Two types of smears are prepared from the peripheral blood. One is called thin blood smear and the other is called thick blood smear. Advantages & limitations of microscopy THICK BLOOD FILM Main use is to detect infection because the amount of blood used is more than thin film Cannot identify species because red cells are lysed THIN BLOOD FILM to identify the species RBC not lysed, so that the shape and size of infected cell can be studied  DIAGNOSIS: Identification of malaria parasite Size of infected RBC - only in P. vivax infection RBC becomes enlarged Shape of infected RBC - becomes oval in P. ovale infection Shape of gametocyte - banana or cresent in P. falciparum, the rest round Shape of trophozoite in RBC - band form in P. malariae and P. knowlesi; the rest ring form  DIAGNOSIS: Identification of malaria parasite Stippling of infected RBC - large dots in P. f (Maurer’s cleft) - fine dots in P. v (Schuffner’s dot) - fine dots in P. m (Ziemann’s dot) - fine dots in P. o (James’s dot) Schizont (no. of merozoites) - P. v: 12-24 (RBC enlarged) - P. f: 18-28 Accole form: (red arrow) applique forms. A term applied to the - P. m: 6-12 (usually 8 – rosette) manner in which the ring stage of - P. o: 6-12 plasmodium falciparum parasitises the marginal portion of erythrocytes Multiple infection, Accole form usually seen in P.f Gametocyte – when cytoplasm of trophozoite fills up the cell and the chromatin materials become compact at one corner of the cell.  Overall morphological characteristics of malaria parasites in blood P. falciparum P. falciparum P. vivax P. vivax P. malariae  P. malariae (Ring-form early trophozoite) https://www.cdc.gov/dpdx/malaria/index.html Ring-form trophozoite of P. malariae in a thin blood smear. Ring-form trophozoites one (rarely two) chromatin dots and a cytoplasm ring that tends to be thicker than P. falciparum. ‘Bird’s-eye’ forms may appear. Ring-form (lower right) and developing (upper left) No enlargement of infected RBCs. trophozoites of P. malariae in a thick blood smear.  P. ovale (Ring-form trophozoite) Ring-form trophozoite of P. ovale in a thin blood smear. Ring-form trophozoites Usually contain a single chromatin dot, but may contain double-chromatin dots. The single rings may be difficult to differentiate from P. vivax, as the cytoplasm is usually thick with a large chromatin dot. As the trophozoites mature, they are less amoeboid than P. vivax and may exhibit fimbriation and Schüffner’s dots. Infected RBCs are not usually enlarged as in P. vivax infections. P. ovale (Ring-form trophozoite) Plasmodium ovale rings Sturdy cytoplasm and large chromatin dots. RBCs are normal to slightly enlarged (1 1/4×), may be round to oval, and are sometimes fimbriated. Schüffner's dots are visible under optimal conditions.  P. knowlesi (Early ring-form trophozoite) Ring-form trophozoite of P. knowlesi in a thin blood smear. Early ring-form trophozoites Similar to P. falciparum, as rings may show double chromatin dots. Appliqué forms may appear, as well as rectangular rings harboring one or more accessory chromatin dots. Red blood cells may also be multiply-infected. https://www.cdc.gov/dpdx/malaria/index.html  P. knowlesi (Older, Developing trophozoite) Ring-form trophozoites of P. knowlesi in a Band-form (upper) and ring-form (lower) Giemsa-stained thin blood smear trophozoites of P. knowlesi Older, developing ring-form trophozoites P. knowlesi Band forms may appear that are similar in appearance to P. malariae. https://www.cdc.gov/dpdx/malaria/index.html Don’t get confused with white blood cells! Types of WBC. (a) Neutrophils, (b) Basophils, (c) Eosinophils, (d) Monocytes, and (e) Lymphocytes. Species Differentiation on Thin Blood Films Feature Pf Pv Po Pm Pk Enlarged - + + - - infected RBC Infected RBC Round Round, distorted Oval, Round Round shape fimbriated Stippling Mauer clefts Schuffner spots Schuffner spots None Sinton and infected RBC Mulligan’s Trophozoites Small ring, Large ring, Large ring, Small ring, Small ring, shape accolé amoeboid compact compact, band Appliqué forms /appliqué, form may appear, single or two band form in chromatins older & developing trophozoites Chromatin Often double single large single Single or double  Species Differentiation on Thin Blood Films Feature Pf Pv Po Pm Pk Mature schizont Rare, 8-24 12-24 4-12 6-12 8-16 merozoites merozoites merozoites, merozoites, merozoites, (average 10), may almost fill with large with large fills the host RBC nuclei; clustered nuclei, clustered RBC, merozoites around mass of around mass of may appear dark-brown coarse, dark- ‘segmented’ and pigment brown pigment; the pigment has occasional collected into a rosettes single mass Gametocyte Banana/Cresce Large, round Large, round Compact, round Round, and fill nt shape the RBC DIAGNOSIS 2. Serodiagnosis In cases where we clinically suspect malaria, but films are negative May be parasitaemia is low therefore, can miss the diagnosis using blood films Useful epidemiological study to know the population immunity towards malaria To identify the infected donors in transfusion malaria. Not useful in acute cases since Ab might not be present yet The tests used are indirect hemagglutination (IHA), indirect fluorescent antibody (IFA) test and enzyme-linked immunosorbent assay (ELISA).  DIAGNOSIS 3. Rapid diagnostic tests (RDT) Based on the detection of circulating parasite antigens in the human blood. RDT tests (immunochromatographic tests) cannot replace microscopy but can be supplementary when malaria diagnosis is being performed by relatively inexperienced staff (e.g., in low prevalence areas and outside normal working hours). Result of RDTs should be always interpreted together with the results of microscopy, read by an experienced laboratory technician  DIAGNOSIS 4. Molecular diagnosis Whole blood in EDTA or dried blood spot on filter paper are the preferable specimens for PCR analysis PCR for confirmation and species determination is indicated in certain situation such as: ▪ With clinical symptoms of malaria but no malaria parasite seen in blood film ▪ Mortality cases ▪ Cases having microscopic appearance of P. malariae Morphological similarities between human & simian Plasmodium Simian Human Plasmodium sp. Plasmodium sp. P. malariae & P. P. knowlesi falciparum P. cynomolgi P. vivax P. inui P. malariae P. coatneyi P. falciparum P. fieldi P. ovale (Coatney et al., 1971)  Molecular detection methods for simian Plasmodium Molecular diagnosis Nested PCR Real-time PCR Single step PCR LAMP Consist of two PCR reactions Real time PCR or also known as Consist of a single PCR reaction Loop-mediated isothermal where the initial reaction is quantitative PCR (qPCR), is a where the amplification amplification (LAMP) amplifies carried out using primers set PCR-based method that process is performed in a single DNA rapidly with high that cover the target sequence enables amplification of a tube premixed with DNA specificity and efficiency under as well as some extra sequence target DNA sequence while polymerase and other PCR isothermal condition. This flanking both ends. On the monitoring the DNA reagents. Due to single method uses a set of four to six other hand, second PCR amplification process in real reaction process, it increases primers that recognizes six to reaction uses the primers that time by inspecting the intensity the speed of Plasmodium eight regions of specific target bind to the target sequence of the fluorescence signal. parasites detection. DNA sequences. within the amplified sequence of the first PCR reaction.  TREATMENT FOR MALARIA Specific treatment Use of antimalarial drugs aimed at terminating parasitaemia, prevent relapse, recrudescence, prevent transmission and prophylaxis Drugs of choice depends on the policy of the country, types of malaria infection and whether there is any drug resistance. Drug resistance is a major problem this days TREATMENTS Supportive treatment Aimed at relieving patients' symptoms such as to bring down high temperature, rehydration, blood replacement in cases of severe anemia, control of convulsion with anticonvulsive drugs, treatment of DIC with fresh frozen plasma or plasma rich platelet Disseminated Intravascular Coagulation (DIC) TREATMENTS Anti-malarial drugs Species, strain and parasite stage may have some influence on the effectiveness of the drugs Drugs that act on tissue schizont are called tissue schizonticide (eg Primaquine). It acts on tissue schizont therefore preventing symptomatic malaria and can be used for prophylaxis Drugs that act on blood schizont are called blood schizonticide and can also be used for prophylaxis (eg. Chloroquine, Quinine) Drugs that act on gametocytes are called gametocidal agents Chloroquine is a potent schizonticide and commonly used drugs, but there are problems with resistance TREATMENT Management Guidelines of Malaria in Malaysia Artemisinin-based combination therapy (ACT) is recommended for the treatment of P. falciparum malaria. Fast acting artemisinin-based compounds are combined with a drug from a different class. Artemisinin derivatives include dihydroartemisinin, artesunate and artemether. Benefits of ACTs: high efficacy, fast action and the reduced likelihood of resistance developing. Chloroquine is still the first line treatment for P. vivax and P. ovale, while primaquine can be used to treat liver stage parasites of P. vivax TREATMENTS Quinine is a derivative from the Cinchona plant - potent schizonticide used in cases where there is resistance to Chloroquine and for severe P. f infection Mefloquine is related to Quinine and good or treating multi resistant strain of P. f Halofantrine also good for treating multi resistant strain of P. f Qinghaosu is a derivative from the plant Artemesia annua good for treating multi-resistant strain of P. f and severe P. f infection TREATMENT  TREATMENT Management Guidelines of Malaria in Malaysia  PREVENTION & CONTROL FOR MALARIA There are four principles—adapted from the WHO’s ABCD of malaria protection— of which all travelers to malarious areas should be informed:  PREVENTION & CONTROL Treat cases to prevent transmission Prophylaxis for people entering endemic areas (Cq, Mefloquine, Doxycycline, Proguanil) Vector control, elimination of standing water, and mosquito breeding sites, use of insecticide spray Personal protection by using bed nets, insect repellant, wearing long sleeve clothing Use of vaccine once ready PREVENTION & CONTROL  Questions List the species of malaria parasites Describe the life cycle of a malaria parasite Explain the possible reasons for increasing cases of P. k in Malaysia Describe the various mode of malaria transmission Discuss the clinical differences between the 5 malaria spp. Discuss the general clinical manifestations of malaria Discuss the complications of Pf and Pk malaria Discuss on the diagnosis of malaria Discuss the prevention and control of malaria  Dr. NANTHA KUMAR JEYAPRAKASAM, PhD Center for Toxicology and Health Risk Studies (CORE) Faculty of Health Science (FSK) Universiti Kebangsaan Malaysia (UKM)