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hemoparasiteshemoparasiteshemoparasiteshemoparasites

1. Moderated by Dr.subhranshu mandal md Associate professor Department of laboratory medicine CNCI-Kolkata. Laboratory diagnosis of hemoparasites By dr.b.parthiban PGT MD Laboratory Medicine

2. Blood parasites Protozoan pathogens Malaria ( plasmodium spp.) Babesiosis ( babesia spp.) Trypanosomiasis Chagas disease ( trypanosoma cruzi ) African trypanosomiasis ( trypanosoma brucei ) Leishmaniasis Helminthic parasites Filarial worms Wuchereria bancrofti Mansonella spp. Brugia spp. Loa loa .

3. Laboratory diagnosis of parasites Microscopy remains the gold standard for detection and identification of many parasites. Blood and feces are the most common specimens that are submitted for parasitologic evaluation, but other important specimen types include urine, sputum, duodenal aspirates, tissue biopsies, cyst and abscess material, and skin scrapings. Antigen detection methods , including enzyme immunoassays and lateral flow immunochromatographic assays are widely used for detection of enteric protozoa and Plasmodium species. Antibody detection methods are most useful for detecting evidence of systemic infection in which parasites are not otherwise easily sampled. Nucleic acid amplification methods are becoming increasingly important for detection and identification of parasites, such as the intestinal protozoa, Leishmania species, Toxoplasma gondii, and Trichomonas vaginalis. Culture methods are available for select parasites but are infrequently utilized except in specialized referral centers and research facilities. Formalin and other preservatives lessen, but do not completely eliminate, the infectious disease risk associated with specimens submitted for laboratory analysis.

4. Malaria Malaria is a hematogenous, mosquito-transmitted infection caused by apicomplexan parasites ( Haemosporidia ) in the genus Plasmodium . Four species that cause human malaria are P. Falciparum P. Malariae P. Vivax P. ovale ( P. ovale curtisi and P. ovale wallikeri ) P. knowlesi

5. Distribution Plasmodium vivax - predominant malaria parasite P. falciparum is mostly confined to the tropics and subtropics- fatal form of human malaria - malignant tertian Plasmodium malariae occurs in subtropical and temperate areas-less frequently Plasmodium ovale is confined to West Africa Plasmodium knowlesi - a monkey malaria - Southeast Asia

6. Plasmodium vivax (Benign tertian) Prefer reticulocytes (young RBC)- all erythrocytic stages The infected erythrocytes are enlarged and with good staining will show granules known as schuffner’s dots in the cytoplasm of the RBC The ring is about 1/3 the size of RBC Ring  trophozoite (amoeboid shape) and accumulates malarial pigment There are about 12–24 (usually 18) merozoites per schizont Erythrocytic schizogony takes 48 h Both male and female gametocytes are large, filling almost the enlarged RBC

7. vivax TIETZ TEXTBOOK OF LABORATORY MEDICINE, 7TH EDITION (Tietz Textbook of Clinical Chemistry and Molecular Diagnostics) 

8. Plasmodium falciparum (Malignant tertian) Most pathogenic Both young and old erythrocytes The early ring form is fine, measuring 1/6 the size of red blood cell (RBC) Rings - attached along the margin of the red cell Double chromatins Multiple rings - a single erythrocyte Late trophozoites and schizonts are not usually seen in the peripheral blood. These stages are sequestrated in the internal capillaries The mature schizont has 8–24 (usually 16) merozoites The erythrocytic schizogony takes about 36–48 h The infected erythrocytes are not enlarged Coarse dots - maurer’s clefts Mature gametocytes - crescent or banana shaped

9. flaciparum TIETZ TEXTBOOK OF LABORATORY MEDICINE, 7TH EDITION (Tietz Textbook of Clinical Chemistry and Molecular Diagnostics) 

10. Plasmodium malariae (Quartan malaria) Plasmodium malariae - older erythrocytes The ring forms resemble those of P. Vivax but the cytoplasm is thicker Infected erythrocytes - normal size or slightly smaller Fine stippling - ziemann’s stippling Trophozoites - band form Mature schizont - 8 merozoites - rosette appearance Erythrocytic schizogony takes 72 h Both male and female gametocytes occupy nearly the entire RBC

11. malariae TIETZ TEXTBOOK OF LABORATORY MEDICINE, 7TH EDITION (Tietz Textbook of Clinical Chemistry and Molecular Diagnostics) 

12. Plasmodium ovale (Tertian malaria) Rarest Trophozoites resemble - P. Vivax Schuffner’s dots Infected erythrocytes - slightly enlarged In thin films- appear oval shape with fimbriated margins Schizonts resemble - P. Malariae The erythrocytic schizogony takes 48 h Both male and female gametocytes occupy nearly the entire RBC

13. ovale TIETZ TEXTBOOK OF LABORATORY MEDICINE, 7TH EDITION (Tietz Textbook of Clinical Chemistry and Molecular Diagnostics) 

14. Plasmodium knowlesi (Quotidian malaria) Ring stage resembles P. Falciparum Accole form, double chromatins and multiple infections are common in an infected RBC Infected RBC - not enlarged Trophozoite stage has a band form resembling P. Malariae Mature schizont - 10 merozoites, with a maximum of 16 Pigment collects into 1 or more yellowish-black masses and eventually into a single mass in the mature schizont The erythrocytic schizogony takes 24 h Both male and female gametocytes occupy nearly the entire RBC

15. Life Cycle Malaria parasite completes its life cycle in 2 hosts Definitive host - female anopheles mosquito. Intermediate host - humans

16. Modes of transmission are via bite of infected Anopheles mosquito, blood transfusion, congenital transmission and shared syringes. Asexual phase (in intermediate host) – schizogony Red blood cells (erythrocytic schizogony) Liver cells (exoerythrocytic or pre-erythrocytic schizogony) Sexual phase (in definitive host) - sporogony Human cycle (schizogony) sporozoites  infective forms of the parasite  salivary gland

17. Exoerythrocytic cycle Within 30 min, the sporozoites reach the liver and enter the hepatocytes to initiate the stage of pre-erythrocytic schizogony In P. vivax and P. ovale , they form schizonts which persist and remain dormant (hypnozoite Erythrocytic cycle The merozoites released by pre-erythrocytic schizonts in the liver invade the RBCs and form rings or young trophozoites The parasite feeds on the haemoglobin  leaves behind haemozoin or malaria pigment

18. Sporogonic cycle Ingests parasitized erythrocytes  asexual forms of malaria parasite are digested Gametocytes  undergo further development in the midgut M ale gametocytes divide to produce 8 microgametes ( exflagellation ) F emale gametocyte (macrogamete) is fertilized by the microgamete to form zygote The zygote develops into a motile form called ookinete - forms an oocyst  within which numerous sporozoites are formed - mature oocyst ruptures releasing sporozoites - salivary glands The time taken for completion of sporogony in the mosquito is about 1–4 weeks, depending on the species and environmental temperature

19. Review The Laboratory Diagnosis of Malaria: A Focus on the Diagnostic Assays in Non-Endemic Areas Adriana Calderaro , Giovanna Piccolo and Carlo Chezzi Int. J. Mol. Sci. 2024 , 25 , 695. https:// doi.org /10.3390/ijms25020695

20. Babesia Intraerythrocytic sporozoan parasites that morphologically resemble P. Falciparum Tick-borne parasite - Ixodid ticks Morphology Trophozoites - 2–5 μ m in diameter pyriform, amoeboid, or spindle-like, usually in pairs and are often mistaken as ring form of Plasmodium

21. Life cycle Sporozoites - salivary glands of tick - human or other mammals Sporozoites - transform to trophozoites - invade the rbcs and multiply asexually by binary fission Differentiated from malarial parasites by the absence of pigments in the infected RBC s In humans, liver is not involved in the life cycle of babesia Gametocyte and schizont stages are not found in humans Humans usually are dead-end hosts Human-to-human transmission is well recognized to occur via contaminated blood transfusions

22. Babesia TIETZ TEXTBOOK OF LABORATORY MEDICINE, 7TH EDITION (Tietz Textbook of Clinical Chemistry and Molecular Diagnostics) 

23. Diagnosis Microscopic examination Absence of schizonts and gametocytes and presence of tetrads ( maltese cross ) in peripheral blood smear Molecular diagnosis - PCR on blood Animal inoculation - Blood from suspected cases are inoculated into hamsters to be examined later for infected RBCs

24. Falciparum vs babesia TIETZ TEXTBOOK OF LABORATORY MEDICINE, 7TH EDITION (Tietz Textbook of Clinical Chemistry and Molecular Diagnostics) 

25. Hemoflagellates TIETZ TEXTBOOK OF LABORATORY MEDICINE, 7TH EDITION (Tietz Textbook of Clinical Chemistry and Molecular Diagnostics) 

26. Trypanosoma Endemic in scattered foci in west and central africa . The principal vectors are glossina palpalis and glossina tachynoides (riverine tsetse flies )

27. The diagnosis of both types of human African trypanosomiasis (HAT) is similar Microscopic examination Wet mount preparation of lymph node aspirates, CSF, and chancre fluid are used for demonstration of trypomastigotes Animal inoculation - Inoculation of specimens from suspected cases to rats/mice is a highly sensitive procedure Serodiagnosis - Specific antibodies or antigens can be detected in serum and CSF Molecular diagnosis - PCR on clinical specimens

28. cruci bruci TIETZ TEXTBOOK OF LABORATORY MEDICINE, 7TH EDITION (Tietz Textbook of Clinical Chemistry and Molecular Diagnostics) 

29. Leishmania donovani Morphology The parasite exists in 2 forms. Amastigote form is found in humans and other mammals. The amastigote form of the parasite seen in human samples is called Leishman Donovan (LD) body and it is intracellular. LD body is an ovoid or rounded cell, about 2–4 μ m in size Intracellular, found inside macrophages in the reticuloendothelial system Promastigote form is found in the sandfly and in culture. Promastigote is a flagellate and is present in sandfly and in culture. It is long, spindle shaped, 15–25 μ m in length and 1.5–3.5 μ m in breadth.

30. Diagnosis Microscopic examination Demonstration of amastigotes in blood smears and tissue aspirates (bone marrow, spleen, lymph nodes) is the gold standard for diagnosis.                                                                   Culture - Tissue specimens or blood are cultured in NNN medium Molecular diagnosis - PCR on clinical specimens Skin test - Leishmanin skin test ( Montenegro test )

31. Leishmania TIETZ TEXTBOOK OF LABORATORY MEDICINE, 7TH EDITION (Tietz Textbook of Clinical Chemistry and Molecular Diagnostics) 

32. Filarial Worms The filarial worms reside in the subcutaneous tissues, lymphatic system, or serous cavities of humans The female worms - ovoviviparous - release larvae known as microfilariae Detected in the peripheral blood or cutaneous tissues Life cycle - completed in 2 hosts: definitive host (human) and intermediate host (blood-sucking arthropods) The microfilariae complete their development in the arthropod host to produce the infective larval stage Adult worms have a lifespan of many years in the human body whereas microfilariae survive for a few months

33. helminths TIETZ TEXTBOOK OF LABORATORY MEDICINE, 7TH EDITION (Tietz Textbook of Clinical Chemistry and Molecular Diagnostics) 

34. Key diagnostic morphologic features Wuchereria bancrofti Usually colorless sheath (Giemsa), anucleate tail , short headspace, relatively loose nuclear column Burgia malayi Usually hot-pink sheath (Giemsa), terminal and subterminal tail nuclei separated by large gaps, long headspace Burgia timori Usually colorless sheath (Giemsa), terminal nuclei and subterminal tail long separated by large gaps, long headspace Loa loa Usually colorless sheath (Giemsa), tail nuclei randomly distributed to the tip of the tail, short headspace, relatively dense nuclear column Mathison BA, Couturier MR, Pritt BS. 2019. Diagnostic identification and differentiation of microfilariae . J Clin Microbiol 57:e00706-19. https:// doi.org /10.1128/JCM .00706-19 .

35. Mathison BA, Couturier MR, Pritt BS. 2019. Diagnostic identification and differentiation of microfilariae . J Clin Microbiol 57:e00706-19. https:// doi.org /10.1128/JCM .00706-19 .

36. Microscopy remains the cornerstone of the laboratory diagnosis of infections due to blood and tissue parasites Examination of thick and thin peripheral blood smears stained with Giemsa or other appropriate stains is used for detection and identification of species of Plasmodium, Babesia, Trypanosoma, Brugia, Mansonella, and Wuchereria Thank you