By Shervin Ghaffari Hoseini MD. PhD Malaria is the world's most important parasitic disease, and kills more people than any other communicable disease

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Slide 2 By Shervin Ghaffari Hoseini MD. PhD Slide 3 Malaria is the world's most important parasitic disease, and kills more people than any other communicable disease except tuberculosis. Each year 350-500 million cases of malaria occur worldwide, and over one million people die Slide 4 Slide 5 Laboratory diagnosis of malaria Microscopic Diagnosis Blood smear Fluorescent Microscopy Quantitative Buffy Coat (QBC ) Antigen Detection Immunochromatographic Dipstick: RDT Serology IFA ELISA Molecular Diagnosis PCR Real time PCR Slide 6 Malaria Blood Smear Remains the gold standard for diagnosis Blood sample from vein or Puncture from finger pulp New and clean slide Both thin and thick films for all patients Slide 7 Malaria Blood Smear Thick films: Dry Do not fix but dehemoglobinate Stain Thin films: Dry Fix Stain Staining methods: Giemsa stain Leishman's stain Fields stain Slide 8 Malaria Blood Smear Thick films: first step: Examination of a thick blood film 20 fold more concentrated species identification difficult minimum of 200 oil immersion fields about 15 minutes for an experienced observer Slide 9 Malaria Blood Smear Thin films: determine the species entire thin film should be examined about 20-40 minutes for an experienced observer Slide 10 Interpreting Thick and Thin Films THICK FILM lysed RBCs larger volume 0.25 l blood/100 fields more difficult to diagnose species good screening test THIN FILM fixed RBCs, single layer smaller volume 0.005 l blood/100 fields good species differentiation requires more time to read low density infections can be missed Slide 11 Calculating Parasite Density - 1 Count the number of parasitized and nonparasitized RBCs in the same fields on thin smear Count 500-2000 RBCs % parasitemia = # parasitized RBCs total # of RBCs X 100 Slide 12 Calculating Parasite Density -2 Count 200 WBCs on thick film Assume WBC is 8000/ l (or count it) parasites/ l = parasites counted WBC counted X WBC count/ l Slide 13 Estimating Parasite Density Alternate Method Count the number of asexual parasites per high-power field (HPF) on a thick blood film 1-10 parasites per 100 HPF+ 11-100 parasites per 100 HPF++ 1-10 parasites per each HPF+++ > 10 parasites per each HPF++++ Slide 14 Malaria Blood Smear Prepare smears as soon as possible Don not fix thick smear time-honoured peripheral smear study Identify P. falciparum in a dual infection with P. vivax Mixed infections are not uncommon. Slide 15 Malaria Blood Smear negative test DOES NOT rule out malaria Repeat tests partial antimalarial treatment sequestration of parasitised cells in deep vascular beds malarial pigment in circulating neutrophils and monocytes is useful Slide 16 Malaria Blood Smear Advantages Distinguishes between species and life cycle stages Parasitemia is quantifiable Threshold of detection thin film: 100 parasites/ 1 lit thick film: 5 -20 parasites/ 1 lit Simple and inexpensive Slide 17 Malaria Blood Smear Disadvantages labor-intensive equipment, training and supervision needed Slide 18 Malaria Parasite Erythrocytic Stages Ring form Trophozoite Schizont Gametocytes Slide 19 Slide 20 Fluorescent Microscopy Fluorescent dyes detect RNA and DNA that is contained in parasites Nucleic material not normally in mature RBCs Stain thin film with acridine orange (AO) Requires special equipment fluorescent microscope Staining itself is cheap Sensitivities around 90% Slide 21 Quantitative Buffy Coat (QBC ) Slide 22 Advantages Useful for screening large numbers of samples Quick, saves time Due to larger volume of blood observed, method is more sensitive diagnosis of other diseases such as Babesiosis, Trypanosomiasis and Filariasis is possible Slide 23 Quantitative Buffy Coat (QBC ) Disadvantages Species identification and quantification difficult: thick/thin films on QBC-positive samples is required High cost of capillaries and equipment Cant store capillaries for later reference Slide 24 An adapted light microscope for the viewing of QBC tubes. Slide 25 Trophozoites of P. falciparum. Slide 26 Malaria Antigen Detection Immunologic assays to detect specific antigens Commercial kits now available as immunochromatographic rapid diagnostic tests (RDTs), used with blood P. falciparum histidine-rich protein 2 (PfHRP-2) parasite LDH (pLDH) Slide 27 Mode of action of common malaria RDT format Slide 28 Slide 29 Malaria Antigen Detection - RDTs FeaturePfHRP-2 testspLDH tests Sensitivity/ Specificity* Sensitivity 92-100% Specificity 85- 100% Sensitivity P.f. 88-98% P.v. 89-94% Specificity P.f. 93-99% P.v. 99-100% Commercial cost/test** Approximately US$ 0.60 1.00Approximately US$ 2.50 Commercial products 1)PATH falciparum Malaria IC Strip test Program for Appropriate Technology in Health 2)MAKROmed 3)Orchid 1)OptiMAL - Flow, Inc. 2)Binax NOW ICT Malaria - Binax, Inc. * Compared to microscopy, results from multiple studies ** Varies by size of order and vendor Slide 30 Detection of Plasmodium antigens A: HRP-2 (histidine-rich protein 2) (ICT) B: pLDH (parasite lactate dehydrogenase)(Flow) C: HRP-2 (histidine-rich protein 2) (PATH) Slide 31 Antigen Detection Malaria Immunochromatographic Dipstick OptiMAL Assay Control Plasmodium pan specific monoclonal antibody P. falciparum specific monoclonal antibody Slide 32 Slide 33 Slide 34 Malaria Antigen Detection - RDTs Disadvantages The use of the RDT does not eliminate the need for malaria microscopy Cannot detect mixed infections may not be able to detect infections with lower parasitemia Cannot detect P. ovale and P. malariae microscopy is needed to quantify parasitemia Slide 35 Positive control: A panel of wells containing different antigens Using quality control parasite dilutions to test the sensitivity of malaria RDTs: Producing and storing quality control dilutions of parasites obtained from field samples Slide 36 Application of RDTs Potential uses Epidemics and emergencies Inadequate or absent lab services, unskilled staff Mobile clinics self-diagnosis by travelers entering endemic areas outbreak investigation and surveys of parasite prevalence Slide 37 Para Sight F test Slide 38 Malaria Serology antibody detection Methods IFA ELISA Not practical for routine diagnosis of acute malaria because: Delaied development of antibody persistence of antibodies Serology does not detect current infection but rather measures past experience Slide 39 Malaria Serology Valuable epidemiologic tool in some settings Useful for Identifying infective donor in transfusion-transmitted malaria Investigating congenital malaria, esp. if moms smear is negative Retrospective confirmation of empirically-treated non-immunes Slide 40 Indirect fluorescent antibody (IFA) test. The fluorescence indicates that the patient serum being tested contains antibodies that are reacting with the antigen preparation (here, Plasmodium falciparum parasites). Slide 41 Polymerase Chain Reaction (PCR) Molecular technique to identify parasite genetic material Uses whole blood collected in anticoagulated tube (200 l) or directly onto filter paper (5 l) Slide 42 Polymerase Chain Reaction (PCR) Advantages PCR is a reference method. It is at least 10-fold more sensitive than microscopy. Threshold of detection 0.1 parasite/l if whole blood in tube 2 parasites/l 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 Slide 43 Polymerase Chain Reaction (PCR) Disadvantages Parasitemia not quantifiable Requires specialized equipment, reagents, and training Slide 44 analysis of a PCR diagnostic test for species- specific detection of Plasmodium DNA. PCR was performed using nested primers Slide 45 Real-Time PCR potential to quantify parasitemia, may detect multiple wavelengths in same tube identifying multiple species in one run Needs further research and validation for malaria Slide 46 Real-Time PCR Slide 47 Quantitative Real-Time PCR Slide 48 BCSH Guidelines for Quality Control All malaria films should be examined by two observers All new batches of Giemsa stain should be tested with a known P. vivax infection ensure that Schffners dots are stained parasitised cells are decolourised. Blood films for this purpose can be sealed in plastic slide boxes and frozen Slide 49 BCSH Guidelines for Quality Control continue All laboratories must ensure that new staff are adequately trained and maintain their skills: Sets of mixed positive and negative thick and thin films should be available for examination reference laboratories can often provide spare films for training purposes. High quality photographs of malaria parasites should be available for reference Slide 50 BCSH Guidelines for Quality Control continue Websites can be used for on-going training. www.dpd.cdc.gov/dpdx/HTML/Image_Library.ht m (Centres for Disease Control and Prevention, USA) www.rph.wa.gov.au (Royal Perth Hospital, West Australia, click on malaria information for learn and test yourself site) Slide 51 Slide 52 Plasmodium falciparum Rings: double chromatin dots; appliqu forms; multiple infections in same red cell Gametocytes: mature (M)and immature (I) forms (I is rarely seen in peripheral blood) Trophozoites: compact (rarely seen in peripheral blood) Schizonts: 8-24 merozoites (rarely seen in peripheral blood) Infected erythrocytes: normal size MI Slide 53 Plasmodium vivax Trophozoites: ameboid; deforms the erythrocyte Gametocytes: round-oval Schizonts: 12-24 merozoites Rings Infected erythrocytes: enlarged up to 2X; deformed; (Schffners dots) Slide 54 Plasmodium ovale Infected erythrocytes: moderately enlarged (1 1/4 X); fimbriated; oval; (Schffners dots) malariae - like parasite in vivax - like erythrocyte Rings Trophozoites: compact Schizonts: 6-14 merozoites; dark pigment; (rosettes) Gametocytes: round-oval Slide 55 Infected erythrocytes: size normal to decreased (3/4X) Plasmodium ma