Laboratory Diagnosis Malaria Parasit.pptx

Laboratory Diagnosis Malaria Parasit.pptx

• 1.
  Laboratory Diagnosis Malaria Parasite -ARKADAS -DMLT 2ND YEAR -The Calcutta Heart Clinic & Hospital
• 2.
  Laboratory Diagnosis MalariaParasite Microscopic Tests: 1. Peripheral Blood Smear - a)Thick smear—more sensitive b)Thin smear—speciation can be done 2.Fluorescence Microscopy 3.Quantitative Buffy Coat Examination Non-Microscopic tests: 1.Antigen Detection Tests (RDTs) 2.Antibody Detection - ELISA 3.Culture - RPMI 640 Medium 4.Molecular Diagnosis - PCR
• 3.
  Microscopic Tests 1. PeripheralBlood Smear Peripheral smear study gold standard confirmatory test for detection of malarial parasites. • Specimen : Peripheral blood is collected from ear lobe or by finger prick in older children & adults. In case of EDTA anticoagulated blood, smears should be made within an hour of collection of blood. • Time for taking blood: Blood should be collected few hours after the heigh fever and because parasite density is maximum during this period. • Types of peripheral blood smear : It is of two types-thin and thick smears. Thick Smear - A big drop of blood is spread over half inch square area on a clean glass slide. It can detect as low as 5–10 parasites per μL of blood. Thin Smear - A small drop of blood is taken on a corner of a slide. It is spread by another slide at an angle of 45. Th in smear is useful in speciation of malaria parasite.
• 4.
  Microscopic Tests
• 5.
  Microscopic Tests 2. FluorescenceMicroscopy It is a fluorescent staining method for demonstrating malaria parasites. Blood smears are prepared on a slide and re stained with acridine orange and examined under a fluorescence microscope. Nuclear DNA is stained green. 3. Quantitative Buffy Coat Examination Th e quantitative buff y coat (QBC) malaria test is an advanced microscopic technique for malaria diagnosis. It consists of three basic steps— i. concentration of blood by centrifugation ii. staining with acridine orange stain and iii. (examination under ultraviolet (UV) light source QBC is faster more sensitive.
• 6.
  Non-Microscopic Tests 1. AntigenDetection by Rapid Diagnostic Tests : Several malarial antigens can be detected by rapid test kit. Parasite lactate dehydrogenase : It is produced by trophozoites and gametocytes of all Plasmodium species. Parasite Aldolase: Produced by all Plasmodium species Plasmodium falciparum specific histidine rich protein-2 : It is produced by trophozoites and young (but not mature) gametocytes of P. falciparum. Most of the kits are designed to detect a combination of two antigens, one is P. falciparum specific antigen other is a pan malarial antigen.
• 7.
  Non-Microscopic Tests 1. AntigenDetection by Rapid Diagnostic Tests :
• 8.
  Non-Microscopic Tests 2. AntibodyDetection: ELISA (enzyme-linked immunosorbent assay),IFA (indirect fluorescent antibody test) and IHA (indirect hemagglutination test) formats are available using soluble malarial antigens. Detection of antibody in serum indicates past malaria infection 3. Culture: RPMI 1640 medium is the most commonly used and found superior to other media for cultivation of P. falciparum 4. Molecular Diagnosis: I. DNA probe: Highly sensitive, detects even if the parasite count is low less than 10/μL. II. PCR: It can detect a single P. falciparum in 20 μL of blood. It is 100 times more sensitive than that of thick blood smear.
• 9.
  Laboratory Diagnosis Kala-Azar -AMRITA NATH -DMLT2ND YEAR -The Calcutta Heart Clinic & Hospital
• 10.
  Laboratory Diagnosis Kala-Azar DirectExamination: 1. Microscopy (Detects LD Bodies) 2. Culture (Detects Promastigotes) Indirect Examination : 1.Antibody Detection In Serum 2.Non Specific Tests To Detect Hypergammaglobulinemia Napier’s Aldehyde Test Chopra Antimony Test 3.Molecular Method 4.Leishmanin Test
• 11.
  Direct Examination 1. Microscopy Demonstrationof Leishman Donovan bodies or LD bodies is the gold standard method for the diagnosis of VL. • Splenic aspiration: The sensitivity of splenic smear examination is excellent (>95%) but splenic puncture is associated with risk of hemorrhage. • Bone marrow aspiration: It is the most commonly preferred sample though the sensitivity is around 60–85%. • Lymph node aspiration: It is useful only in African cases of kala-azar. • Liver biopsy: Less sensitive and carries the risk of hemorrhage. • Peripheral blood smear: Thick blood film is examined for demonstration of amastigote form. • Biopsy specimens of various organs: Like oropharynx, stomach, or intestine.
• 12.
  Direct Examination 1. Microscopy AmastigoteForm Smear Shows Promastigote Form Giemsa Stain From Culture
• 13.
  Direct Examination 2. Culture Asa sample Aspirations from spleen, bone marrow or other tissue and also buffy coat collected.  Samples are culture in NNN medium.  Inoculated specimens are incubated at specific temperature (22–26°C) up to 4 weeks  Amastigotes transform into promastigotes in the culture fluid which are detected by staining with Giemsa stain.  Culture is found to be positive in 75% of cases. NNN Medium
• 14.
  Indirect Examination 1. AntibodyDetection In Serum  Enzyme-linked immunosorbent assay (ELISA) and indirect fluorescent antibody(IFA) test, (Fig. 5.7A) are the newer tests found to be more sensitive 2. Nonspecific Tests To Detect Hypergammaglobulinemia: Napier’s Aldehyde test: Patient’s serum is added with a drop of 40% formalin in a test tube. Positive test is indicated by jellification of the serum forming milk white opacity like that of white of a boiled egg within 20 minutes. Disadvantage - It is negative in the first three months Chopra’s antimony test: Positive test is indicated by formation of flocculation when patient’s serum is mixed with 4% urea stibamine solution.
• 15.
  Indirect Examination 3. Molecularmethods Detection of Leishmania specific mitochondrial DNA by polymerase chain reaction (PCR) 4. Leishmanin test (Montenegro test)  It is a delayed hypersensitivity skin test to a suspension of killed L. donovani promastigote injected intradermally.  A positive test is indicated by induration of more than or equal to 5 mm in 72 hours.  Positive test indicates prior exposure to Leishmania antigens. 5. Nonspecific Tests a. Complete blood count—to detect pancytopenia b. Elevated liver enzymes c. Reversal of albumin globulin.
• 16.
  THANK YOU