Advanced Haematology MEDI4004 General Haematology Lecture Notes PDF

Curtin University is a trademark of Curtin University of Technology. CRICOS Provider Code 00301J (WA)  Advanced Haematology MEDI4004 General Haematology Lecture 1. Microorganisms that may be encountered in haematological assessment: Malariae Presenter: Professor Phillip...

Curtin University is a trademark of Curtin University of Technology. CRICOS Provider Code 00301J (WA)  Advanced Haematology MEDI4004 General Haematology Lecture 1. Microorganisms that may be encountered in haematological assessment: Malariae Presenter: Professor Phillip Clark Tel: x7378 Email: [email protected]  Learning outcomes At the end of this lecture you should be able to: Understand the morphological characteristics of Plasmodium species, that may infect people, when assessed by light microscopy Understand methods for the detection of malaria(e) Understand the limitations of methods used for the detection of malaria(e)  References http://imagebank.hematology.org Bain et al. (2012) Dacie & Lewis, Practical Haematology 11th edn. Charles et al. (2005)Notifications of imported malaria in Western Australia, 1990–2001: incidence, associated factors and chemoprophylaxis MJA 182: 164–167 Douglas et al. (2012) Malaria Journal 11:135 Kantele A & Jokiranta TS (2011) Review of Cases With the Emerging Fifth Human Malaria Parasite, Plasmodium knowlesi Clin Inf Dis 52(11):1356–1362 Houzé, Sandrine, et al. "Performance of rapid diagnostic tests for imported malaria in clinical practice: results of a national multicenter study." PloS one 8.9 (2013): e75486. Laishram et al. (2012) Malaria Journal 11:29 Lamikanra et al. (2012) Malaria Journal 11:201 Pereira et al. (2011) Atlas of Peripheral blood: The primary diagnostic tool. www.rph.wa.gov.au/malaria.html Prescott et al. (2012) Malaria Journal. 11:155 Robinson P et al. (2001)Imported Malaria Treated in Melbourne, Australia: Epidemiology and Clinical Features in 246 Patients J Travel Med 8:76–81. Speers D et al. (2003) Diagnosis of malaria aided by polymerase chain reaction in two cases with low-level parasitaemia Int Med J 33: 613–615 Ta, T. H., Hisam, S., Lanza, M., Jiram, A. I., Ismail, N., & Rubio, J. M. (2014). First case of a naturally acquired human infection with Plasmodium cynomolgi. Malar J, 13(1), 68. White (2011) Malaria Journal 10:278 http://www.cdc.gov/dpdx/malaria/dx.html ***  The Worldwide Impact of Malaria WHO world malaria report – https://www.who.int/teams/global-malaria-programme/reports/world- malaria-report-2023 Malaria cases Estimated cases malaria that occurred worldwide: – 249 million in 2022 – The WHO African Region, with an estimated 233 million cases in 2022, accounted for about 94% of cases globally. – Twenty-nine countries accounted for 95% of malaria cases globally. – Four countries – Nigeria (27%), the Democratic Republic of the Congo (12%), Uganda (5%) & Mozambique (4%) – accounted ~50% of all cases Malaria deaths Estimated deaths from malaria worldwide: – 608,000 in 20233 Children aged under 5 years are the most vulnerable group affected by malaria. – Since 2015, they accounted for 76% of all malaria deaths worldwide. http://www.cdc.gov/malaria/about/biology/  Life cycle stages Stages within humans – Shizont – Trophozoite – Gametocyte Stages within mosquitoes – Gametocytes Anopheles mosquito (Female) – Ookinete – Oocyst – Sporzoites  Malaria: clinical Clinical spectrum – Mild-asymptomatic – Acute self-limiting febrile, myalgia, nausea, vomiting, diarrhoea – Severe, life-threatening Poor prognosis – Severe anaemia, respiratory distress, cerebral malaria, jaundice, renal failure, shock, acidosis, haemostatic abnormalities  Malaria: Haematological changes Anaemia due to complex, multifactorial mechanism – Sequestration of parasitized RBC within bone marrow – Maturation defects (persist >3 weeks post- parasitaemia) – Dyserythropoiesis (P. vivax) – Destruction of parasitized RBC at merogony – Enhanced splenic clearance of infected RBC (extravascular haemolytic anaemia) – Blood loss  Malaria: Haematological changes Thrombocytopenia – Common P. vivax, P. falciparum – Decreased platelet survival (2-4 days) – Enhanced splenic clearance, destruction/sequestration – DIC in severe cases Leukopenia – Mild in uncomplicated cases of malaria Leukocytosis – Associated with severe P. falciparum malaria – TNFα mediated? – Concurrent bacteraemia? Overview of Mechanisms of Anaemia General mechanisms – Intravascular haemolysis – Extravascular haemolysis – Dyserythropoiesis – Bone marrow insufficiency Comparison of Plasmodium vivax (red font) with Plasmodium falciparum (black font) Douglas et al. (2012)  Laboratory Diagnosis Demonstration of parasites in blood film: - Thin film - Thick film Additional methods – Rapid diagnostic tests – Molecular biology methods  Blood Film Examination Blood collected when the patient's temperature ↑ Prepare – Thick films x3; screening – Thin films x2; confirmation  Thick Film Preparation Place a drop of blood onto microscope slide & spread the drop until it is about 1-2 cm2 Adjust thickness=> just possible to see through it Allow the films to dry DO NOT fix thick films Stain: – Prepare a 10% solution of Giemsa stain in phosphate buffer pH 7.2 – Filter into a coplin jar – Stain slides for 20 min – Allow to air dry If available use a positive control  Thin Film Preparation Thin films are made in the standard manner – Air dry – fix – Stain with May-Grünwald & Giemsa (or equivalent Romanowsky stain) pH of the stain – Slightly alkaline stain is recommended (pH 7.2) – Staining in an acid (pH < 6.8) may fail to show parasites  Effects of staining pH on parasite staining characteristics (P. vivax). At pH 7.2 (A) the parasite is readily distinguished from the adjacent blue/grey erythrocytes, chromatin staining is prominent and Schüffner's dots are readily distinguished. At pH 6.9 (B) the parasite remains visible, but there is less contrast from surrounding erythrocyte cytoplasm, relatively weak chromatin staining, and poor resolution of Schüffner's dots (which may not be visible at this pH). pH 7.2 pH 6.9 From Bain et al 2012  Blood Film Examination Examine the thick films using an oil immersion or high dry lens to determine if parasites are present Confirm presence of parasite using thin film Use thin film to identify species Examine each: Thick film - 3 minutes Thin film - 5 minutes Thick Film Concentrating technique => more likely to detect Plasmodium organisms Organisms distorted so more difficult to determine species Thin Film Allows better assessment of morphology of parasites Allows speciation Low density infections may be difficult to detect organisms  Parasitaemia No. parasites 100-106 per uL of blood Varies with species Combined circulating RBC & sequestered RBC => circulating alone underestimates parasitaemia Parasitaemia determined as no. parasitised RBC per 1000 RBC Can be used to follow course of disease/response to treatment Malaria 4 common species affecting people – Plasmodium falciparum – Plasmodium vivax – Plasmodium ovale – Plasmodium malariae In Australia; – P. vivax > P. falciparum >> P. ovale, P. malariae – NB these infections are in travellers/immigrants, not endemic  Species of Plasmodium in Australia Robinson et al. (2001), Melbourne – P. vivax 69.8% – P. falciparum 26.9% – P. ovale 1.9% – P. malariae 0.4% Charles et al. (2005), Western Australia – P. vivax 58% – P. falciparum 28% – P. ovale 1% – P. malariae 2% – Mixed 5% – Unspecified 7% [sic] Malaria Species - Characteristics Feature P. falciparum P. vivax P. malariae P. ovale RBCs Enlargement None Yes None Yes Trophozoite Size 1/3 of RBC >1/3 of RBC >1/3 of RBC Multiple common rare rare rare Parasites Shape delicate; often double rough; single compact; inverted rough; single chromatin dot chromatin dot chromatin dot chromatin dot Schizont Frequency Very rare Common Common Common Configuration Random Random Daisy-head Daisy-head # Merozoites 8 - 24 12 - 24 8 – 12 8-12 Gamete Crescent forms Large and round Small and round Small and round Central chromatin Fills RBC Fills 1/3-1/2 of RBC Fills 1/3–1/2 of RBC  Schuffner’s dots Light microscopic morphologic alterations in infected RBC visible in Romanowsky-stained blood smears as multiple brick-red dots. P. vivax P. ovale P. malariae P. falciparum Presence of Schuffner's Yes, fine Yes, large No No dots Trophozoite of P. ovale in a thin blood smear. Note Schüffner's dots. http://www.cdc.gov/dpdx/malaria  Plasmodium vivax Most commonly seen Plasmodium species in Australia Usually not life threatening Invades reticulocytes ~48 hour cycle Diagnostic points – Infected red cells are typically enlarged – Ring forms usually large ~ 1/3 size of red cell – May see range of morphological forms Usually responds well to treatment Plasmodium vivax http://www.cdc.gov/dpdx/malaria Plasmodium vivax Plasmodium vivax ring form trophozoites (2). http://www.cdc.gov/dpdx/malaria Plasmodium vivax Trophozoite of P. vivax in a thin blood smear. Note the amoeboid appearance, Schüffner's dots and enlargement of infected RBC http://www.cdc.gov/dpdx/malaria Plasmodium vivax Plasmodium vivax gametocytes (2). http://www.cdc.gov/dpdx/malaria Plasmodium vivax Plasmodium vivax mature schizont. NB ‘random’ arrangement. http://www.cdc.gov/dpdx/malaria  Plasmodium falciparum Second most common malarial infection in Australia Invades reticulocytes & mature RBCs ~48 hour cycle Typically greater parasitaemia than other Plasmodium species Most pathogenic – Cerebral sequestration – & sequestration other organs – Life threatening => multiple organ failure, death – Children most vulnerable Diagnostic points – RBC typically not enlarged – Small, fine ring forms – Occasional gametocytes recognised Treatment often difficult due to drug resistance Plasmodium falciparum Plasmodium falciparum Plasmodium falciparum ring form trophozoite. http://www.cdc.gov/dpdx/malaria Plasmodium falciparum Plasmodium falciparum gametocyte (extracellular) Also present is a trophozoite http://www.cdc.gov/dpdx/malaria Plasmodium falciparum Plasmodium falciparum schizont – note disorganised(‘random’) arrangement Also present are trophozoites http://www.cdc.gov/dpdx/malaria  Plasmodium ovale Uncommonly encountered in Australia RBC enlarged ~48 hour cycle Pathogenicity: typically not life-threatening Diagnostic Points – Red cells enlarged. – Comet forms common (top right). – Rings large and coarse. – Schuffner's dots, when present, may be prominent. – Mature schizonts similar to those of P. malariae but larger and more coarse Plasmodium ovale http://www.cdc.gov/dpdx/malaria Plasmodium ovale Plasmodium ovale trophozoite. http://www.cdc.gov/dpdx/malaria Plasmodium ovale Plasmodium ovale gametocyte. http://www.cdc.gov/dpdx/malaria Plasmodium ovale Plasmodium ovale schizont. Note the more organised ‘daisy- head’ appearance. http://www.cdc.gov/dpdx/malaria  Plasmodium malariae Uncommonly encountered in Australia RBC not enlarged ~72 hour cycle Pathogenicity: typically not life-threatening Diagnostic Points – Ring forms may have a squarish appearance. – Band forms are a characteristic of this species. – Mature schizonts may have a typical daisy head appearance with up to ten merozoites. – Red cells are not enlarged. – Chromatin dot may be on the inner surface of the ring Plasmodium malariae http://www.cdc.gov/dpdx/malaria Plasmodium malariae Plasmodium malariae trophozoite. http://www.cdc.gov/dpdx/malaria Plasmodium malariae Plasmodium malariae gametocyte. http://www.cdc.gov/dpdx/malaria Plasmodium malariae Plasmodium malariae schizont. Note the more organised ‘daisy- head’ appearance. http://www.cdc.gov/dpdx/malaria http://www.cdc.gov/dpdx/malaria  A 17-year-old teenager presented with fever and jaundice for 1 week that was preceded by 3 weeks of abdominal pain and malaise. Lori D. Racsa, and Marcos Coutinho Schechter Blood 2015;126:1042 ©2015 by American Society of Hematology  A 17-year-old teenager presented with fever and jaundice for 1 week that was preceded by 3 weeks of abdominal pain and malaise. The patient was born in The Gambia but had lived in the United States for most of his life except for the last 2 years, when he had returned to The Gambia. He was in the United States for 10 days at time of admission. Physical examination revealed depressed sensorium with a Glasgow Coma Scale of 15. He was afebrile and hemodynamically stable on admission but developed hypotension refractory to intravenous fluids, leading to transfer to the intensive care unit within 12 hours of presentation. A complete blood count showed a haemoglobin level of 61 g/L with a nadir of 58 g/L. The peripheral blood smear was diagnostic for Plasmodium falciparum, showing numerous intracellular parasites, with a calculated 28% parasitaemia. There were crescent-shaped gametocytes (panel A, arrowhead), ring forms with split chromatin giving the typical “headphone” appearance (panel B, thick arrow), erythrocytes infected with multiple parasites, and appliqué forms (rings appearing on the periphery of the erythrocyte) (panel B, thin arrow). He recovered after a transfusion of 4 units of packed red blood cells and treatment with quinidine and doxycycline, both administered intravenously.  Plasmodium; mixed infections Infection with > 1 Plasmodium species Underestimated – 30% E.g. – Patients with acute P. falciparum, >33% also harbour P. vivax – Patients with P. vivax, 8% also harbour P. falciparum Effect of method – E.g. 0/23 microscopy; 4/23 PCR  Additional species of malaria Plasmodium knowlesi Plasmodium cynomolgi ‘Plasmodium brasilianum’ ‘Plasmodium simium’ Plasmodium inui Plasmodium fieldi  5th Plasmodium species causing malaria of humans Plasmodium Definitive host: macaque monkeys knowlesi => Zoonotic Present throughout SE Asia Causes severe disease in humans Lifecycle of 24h Infects young & mature RBC Often misdiagnosed as P. falciparum, P. malariae Confirmed by PCR & DNA sequencing (NB 26 species of Plasmodium described for non-human primates) Plasmodium knowlesi Ring-form trophozoites of P. knowlesi in a Giemsa-stained thin blood smear from a human patient that travelled to the Philippines. Note a multiply-infected RBC in this image. http://www.cdc.gov/dpdx/mal aria/gallery.html#pknowtrophs Plasmodium cynomolgi First diagnosed naturally acquired case in a human reported in 2014 Normal host: Macaca fascicularis (Crab- eating macaque, Cynomolgus monkey)? Lifecycle of 48h Plasmodium cynomolgi is morphologically indistinguishable from P. vivax Furthermore one of the most used PCR methods for malaria infection detection may identify a P. cynomolgi infection as P. vivax.  ‘Plasmodium brasilianum’ Plasmodium brasilianum causes a clinical syndrome in New World monkeys very similar to Plasmodium malariae in humans P. brasilianum & P. malariae have a very similar morphology “Although the quartan parasites were The genetics of the two parasites genetically distinct from the other are ‘nearly identical’ and it has Plasmodium species, there was no genetic long been speculated that the differentiation between the P. malariae and P. brasilianum isolates (distance, d = two are the same species 0.005). The average genetic distance First infection with parasites between all quartan isolates from human termed as P. brasilianum and monkey is comparable to intra- reported in humans in 2015(?) species genetic distance in other Plasmodium spp.” doi:10.1016/j.ebiom.2015.07.033  ‘Plasmodium simium’ Cases of human malaria in Brazil have recently been attributed to Plasmodium simium Malarial parasite that commonly infects non-human primates Close similarity at both the morphological and molecular level to Plasmodium vivax => the diagnosis of P. simium is problematic ‘Only two unique single nucleotide polymorphisms (3535 T>C and 3869 A>G) in the whole mitochondrial genome sequence differentiate P. simium from P. vivax’ DOI:10.1038/s41598-017-18216-x  Plasmodium simium Following introduction of P. vivax to the Rougeron, V., Daron, Americas with successive waves of J., Fontaine, M.C. et al. human emigration… Evolutionary history of Plasmodium vivax and It is now believed that the existence of Plasmodium simium in Plasmodium simium is explained by a P. the Americas. Malar J vivax transfer from humans to monkeys 21, 141 (2022). in the Americas… https://doi.org/10.118 Giving rise to a new species that is 6/s12936-022-04132-7 genetically very close to P. vivax, and was named Plasmodium simium. Anthropozoonosis (syn zoonosis) = transfer of disease from Animals to humans Zooanthroponosis = transfer of disease from humans to animals  Plasmodium inui, Plasmodium fieldi Zoonotic species of Plasmodium Putaporntip C et al, Reservoir in macaque species Cryptic Plasmodium Study in Thailand: 5271 blood samples inui and from acute febrile patients from 5 malaria Plasmodium fieldi endemic provinces Infections Among Symptomatic Examined for Plasmodium species by Malaria Patients in microscopy and species-specific Thailand, Clinical polymerase chain reaction Infectious Diseases, ‘Human’ malaria species also present Volume 75, Issue 5, Most P. inui & all P. fieldi infected patients 1 September 2022, Pages 805–812, had simultaneous infections with other https://doi.org/10.1 Plasmodium species 093/cid/ciab1060 P. knowlesi P. cynomolgi P. inui P. fieldi 15 21 19 3 0.29% 0.40% 0.36% 0.06% Risk factors for misidentification using morphology Poorly prepared/stained preparations Deteriorated organisms Low number of parasites Mixed infections Novel organisms (Inexperience)  Additional testing methods Rapid diagnostic tests PCR & DNA Sequencing Automated analysers  Rapid Diagnostic Tests Several commercially available RDTs use small blood samples obtained by finger prick or by venepuncture In general, a blood specimen to be tested (2–50μl) is lysed in buffer solution containing one or more malaria- specific ‘detection antibodies’. Important to follow up with thick and thin films on negatives especially with suggestive history/clinical signs/FBC results Published studies on effectiveness available – E.g. Houzé et al. 2013 Rapid Diagnostic Tests BinaxNOW Malaria test Immunochromatographic assay for the qualitative detection of Plasmodium antigens Targets the histidine-rich protein II (HRPII) antigen specific to Plasmodium falciparum (P.f.) and a pan-malarial antigen common to all four malaria species capable of infecting humans - P. falciparum, P. vivax (P.v.), P. ovale (P.o.), and P. malariae (P.m.). https://www.globalpointofcare.abbott/us/en/ product-details/binaxnow-malaria.html PCR Useful to detect low concentration of parasites Possible to detect