بسم الله الرحمن الرحیم

By Shervin Ghaffari Hoseini MD. PhD Diagnosis of Malaria By Shervin Ghaffari Hoseini MD. PhD

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

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

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

Malaria Blood Smear Thin films: Dry Fix Stain Dry Thick films: Dry Do not fix but dehemoglobinate Stain Staining methods: Giemsa stain Leishman's stain Field’s stain

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

Malaria Blood Smear Thin films: determine the species entire thin film should be examined about 20-40 minutes for an experienced observer

Interpreting Thick and Thin Films fixed RBCs, single layer smaller volume 0.005 μl blood/100 fields good species differentiation requires more time to read low density infections can be missed THICK FILM lysed RBCs larger volume 0.25 μl blood/100 fields more difficult to diagnose species good screening test

Calculating Parasite Density - 1 Count the number of parasitized and nonparasitized RBCs in the same fields on thin smear Count 500-2000 RBCs # parasitized RBCs total # of RBCs % parasitemia = X 100

Calculating Parasite Density -2 Count ≥ 200 WBCs on thick film Assume WBC is 8000/l (or count it) parasites counted WBC counted X WBC count/l parasites/l =

Estimating Parasite Density Alternate Method Count the number of asexual parasites per high-power field (HPF) on a thick blood film Parasites observed Percentage of red cells parasitized 10-20 per field 1 1-2 per field 0.1 1-2 per 10 fields 0.01 1-2 per 100 fields 0.001 1-2 per 1000 fields 0.0001 1-10 parasites per 100 HPF + 11-100 parasites per 100 HPF ++ 1-10 parasites per each HPF +++ > 10 parasites per each HPF ++++

Mixed infections are not uncommon. 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.

malarial pigment in circulating neutrophils and monocytes is useful 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

Malaria Blood Smear Advantages Distinguishes between species and life cycle stages Parasitemia is quantifiable Threshold of detection thin film: 100 parasites/ 1 μ lit thick film: 5 -20 parasites/ 1 μ lit Simple and inexpensive

Malaria Blood Smear Disadvantages labor-intensive equipment, training and supervision needed

Malaria Parasite Erythrocytic Stages Ring form Trophozoite Schizont Gametocytes

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%

Quantitative Buffy Coat (QBC ®)

Quantitative Buffy Coat (QBC ®) 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

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 Can’t store capillaries for later reference

An adapted light microscope for the viewing of QBC tubes.

Trophozoites of P. falciparum.

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)

Mode of action of common malaria RDT format

Malaria Antigen Detection - RDTs Feature PfHRP-2 tests pLDH 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% Commercialcost/test** Approximately US$ 0.60 –1.00 Approximately US$ 2.50 Commercial products PATH falciparum Malaria IC Strip test – Program for Appropriate Technology in Health MAKROmed™ Orchid ® OptiMAL® - Flow, Inc. Binax NOW ®ICT Malaria - Binax, Inc. * Compared to microscopy, results from multiple studies ** Varies by size of order and vendor

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)

Antigen Detection Malaria Immunochromatographic Dipstick OptiMAL Assay P. falciparum specific monoclonal antibody Control Plasmodium pan specific monoclonal antibody

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

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

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

Para Sight F test

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

Malaria Serology Valuable epidemiologic tool in some settings Useful for Identifying infective donor in transfusion-transmitted malaria Investigating congenital malaria, esp. if mom’s smear is negative Retrospective confirmation of empirically-treated non-immunes

Indirect fluorescent antibody (IFA) test 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).

Polymerase Chain Reaction (PCR) Molecular technique to identify parasite genetic material Uses whole blood collected in anticoagulated tube (200 µl) or directly onto filter paper (5 µl)

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/µl if whole blood in tube 2 parasites/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

Polymerase Chain Reaction (PCR) Disadvantages Parasitemia 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

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

Real-Time PCR

Quantitative Real-Time PCR

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 Schüffner’s dots are stained parasitised cells are decolourised. Blood films for this purpose can be sealed in plastic slide boxes and frozen

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

BCSH Guidelines for Quality Control continue Websites can be used for on-going training. www.dpd.cdc.gov/dpdx/HTML/Image_Library.htm (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)

Plasmodium falciparum Infected erythrocytes: normal size M I Gametocytes: mature (M)and immature (I) forms (I is rarely seen in peripheral blood) Rings: double chromatin dots; appliqué forms; multiple infections in same red cell Schizonts: 8-24 merozoites (rarely seen in peripheral blood) Trophozoites: compact (rarely seen in peripheral blood)

Plasmodium vivax Infected erythrocytes: enlarged up to 2X; deformed; (Schüffner’s dots) Rings Trophozoites: ameboid; deforms the erythrocyte Schizonts: 12-24 merozoites Gametocytes: round-oval

“malariae - like parasite in vivax - like erythrocyte” Plasmodium ovale Infected erythrocytes: moderately enlarged (11/4 X); fimbriated; oval; (Schüffner’s dots) “malariae - like parasite in vivax - like erythrocyte” Trophozoites: compact Rings Gametocytes: round-oval Schizonts: 6-14 merozoites; dark pigment; (“rosettes”)

Plasmodium malariae Infected erythrocytes: size normal to decreased (3/4X) Trophozoite: typical band form Trophozoite: compact Schizont: 6-12 merozoites; coarse, dark pigment Gametocyte: round; coarse, dark pigment