New Hemoparasites malaria filaria lymph nodes

Hemaparasite

1. HEMOPARASITES AND RECENT DIAGNOSTIC MODALITIES Dr. R aabiah begum M Pathology PG

2. Overview Introduction Malaria including recent diagnostic modalities Microfilaria Trypanosoma Babesiosis Ehrilichiosis Summary References

3. Introduction Hemoparasites are those parasites that lives within host blood stream Malaria Filaria Leishmania Babesia Trypanosoma Ehrlichiosis/anaplasmosis

4. Malaria Plasmodium Species P . v ivax P. falciparum P. ovale P. malariae 5 th species - P. knowlesi (monkey malaria) Macaque monkeys

5. CLINICAL FEATURES 3 paroxysms:- I – Cold stage – 20 mins to 1 hr. II – Hot stage – 1 to 4 hr. III – Sweating stage – 2 to 3 hr. Black water fever. Cerebral malaria.

6. DIAGNOSTICS MODALITIES - MALARIA Microscopy Parasite density Quantification of parasite Rapid antigen detection tests Serodiagnosis Molecular diagnosis Fluorescent microscopy Flow cytometry

7. Diagnostic Modalities Microscopy : Thick smear :- Detection of parasite Thin smear :- Detection of species Examine 100 oil immersion fields → Thick smears 200 oil immersion fields → Thin smears Stains used : Giemsa Field’s satin Leishman stain Jaswath singh Battacharya stain Thick smear sensitive test detects 10 parasites /µL

8. What to be looked for in PS? Appearance of RBCs Appearance of parasite. Stage of parasite.

9. Plasmodium vivax RBC – Enlarged. All stages are seen. Single parasite in one RBC Schffner’s dots Medium parasite density.

10. Ring-stage Trophozoite Blue cytoplasmic ring Plasmodium vivax – Stages In Human Blood Mature Trophozoite One side of ring (ameboid); large granular yellow-brown. Thicker; red chromatin dot pigment in center.

11. Plasmodium vivax – Stages In Human Blood Schizont 12-24 merozoites arranged rosette-like, granular yellow brown pigment in centre Gametocyte Large and spherical

12. Plasmodium falciparum RBC – Normal size. Stage - Only ring forms and gametocytes seen. Multiple rings in single cell. High parasite density .

13. Plasmodium falciparum – Stages In Human Blood Ring-stage Trophozoite Delicate, small uniformly fine cytoplasmic 1-2 red chromatin dots, ring may be attached to the red cell margin ( accole form). Mature Trophozoites and Schizonts disappear from peripheral blood and are not seen Gametocyte Cresentric /banana shape larger than red cell.

14. Plasmodium ovale RBC – enlarged with fimbriated margins. All stages. Single parasite. Prominent Schuffner’s dots.

15. Plasmodium malariae RBC – normal size. All stages seen. Single parasite . Low parasite density.

16. Parasite density index Mainly done for P.Falciparum infection. Thick smear: Total leukocyte count/µl X No. of parasites/200 No. of parasites/ μ l of blood : No. of parasites counted/No. of WBCs counted X 8000 Thin smear : No. of Parasites in 1 µL blood = RBC count X Parasite % *Parasite % = No. of parasites per 1000 RBC Only asexual forms are included, gametocytes are excluded.

17. Parasite density index Quantification of Parasites Counting of parasites is done on an approximate number by a thick smear. + → 1-10 parasites /100 thick film fields ++ → 11-100 parasites /100 thick film fields +++ → 1-10 parasites/thick film field ++++ → > 10 parasites/thick film field

18. Fluorescent dyes like acridine orange or benzothiocarboxy purine kawamoto technique. Stain the film with acridine orange. Stains DNA as fluorescent green & cytoplasmic RNA as red FLUROSCENCE MICROSCOPY

19. Quantitative Buffy Coat Developed by Becton-Dickinson Principle: QBC centrifugal hematology analysis utilises the density gradient layering of stained QBC cells, together with mechanical expansion of the microhaematocrit buffy coat. It is based on  acridine orange staining of centrifuged peripheral blood samples in a microhematocrit tube (QBC)  and examination under UV light source.

20. Reagents: Each QBC tube is internally coated with acridine orange, potassium oxalate, sodium heparin & dipotassium EDTA. Procedure : used with capillary blood collected from the finger or heel or venous blood. Small quantity blood is spun on QBC centrifuge at 1200 rpm for 5 mins . RBC containing parasites are less dense than normal RBC & get concentrate below buffy coat of leucocytes at top of RBC column. Pre coating tube with acridine orange induces florescence on parasites. Faster & more sensitive than thick blood smear Quantitative buffy coat

21. Peripheral smear QBC Method Cumbersome Easy Time 60 – 90 min 15 – 20 min Sensitivity 5 parasites/ μ l (thick) 200/ μ l (thin) More sensitive 1-2 parasite/ μ l Specificity Gold standard Artifacts Species identification Accurate Difficult Cost Inexpensive Costly Acceptability 100% Not so Availability Everywhere Limited

22. Rapid Antigen Detection Tests Based on immunochromatographic methods. Seen in forms such as dipstick , cards , & cassette bearing monoclonal antibody. Detection of Histidine -rich protein 2 (HRP-2) Parasite Lactate Dehydrogenase(PLDH)

23. HRP-2 PLDH

24. Simple procedure , Minimum skill No electricity requirement or special equipment or training Detect circulating Ag Expensive Cannot differentiate vivax / ovale / malariae HRP-2 : not secreted in gametogony stage. In “carriers”, band may be absent. False-positive responses due to the persistence of malaria antigens in the blood for up to 2 weeks Advantages Disadvantages Antigen Tests

25. Serodiagnosis Helpful in sero epidemiological survey. Not much helpful in clinical diagnosis. To identify infected donors in transfusion malaria. Tests → Indirect immunofluorescence Indirect hemagglutination tests ELISA

26. Molecular diagnosis DNA probe : Highly sensitive method diagnosis Based on the use of (32)P as a radioactive marker. S o this test cannot be used in situations where there is no well-equipped laboratory. It detects less than 10 parasite /µ Lof blood

27. FLOW CYTOMETRY Detection of hemozoin pigment in monocytes & neutrophils by depolarization of laser light as cell pass through a flow cytometer channel. Helpful for clinically unsuspected cases Not specific, species identification not possible.

28. Recent Diagnostic Modalities Identification of Diagnostic Biomarkers for Malaria. Non blood based assays Blood based assays PET PCR Loop mediated isothermal amplification(LOOP-LAMP) Non-instrumented Nucleic acid (NINA-LAMP) 4. Application of biosensing technology Quartz crystal microbalance biosensor Colorimetric biosensors Aptamers 5. Surface Enhanced Raman Spectroscopy

29. Recent Diagnostic Modalities 1 . Identification of Diagnostic Biomarkers for Malaria Biomarkers can be genomic, transcriptomic, proteomic or metabolomic markers. Blood and/or serum-based. Recent advances in proteomics have also focused on other body fluids such as saliva and urine. Advantage - determination of parasite species, estimation of parasitemia , intensity of the immune response and prognostic information.

30. BIOMARKERS Biomarker Parasite species Infection Stage Diagnostic Method P. falciparum Histidine-Rich Protein 3 (PfHRP3) P. falciparum Asexual stages and gametocytes of P. falciparum , expressed on red blood cell membrane surface Immunochromatographic assays Plasmodium aldolase P. vivax and P. falciparum Asexual blood-stag Immunochromatographic assays Hemozoin All Plasmodium spp. Intra- erythrocytic stage Magneto-Optical Detection Glutamate dehydrogenase (GDH) P. falciparum Intra- erythrocytic development Western blotting, immunochromatographic assays

31. BIOMARKERS Biomarker Parasite species Infection Stage Diagnostic Method Lactate dehydrogenase (LDH) All plasmodium species Trophozoite stage Immunochromatographic assays P. falciparum Histidine-Rich Protein 1 (PfHRP1) P. falciparum Asexual stages and gametocytes of P. falciparum , expressed on red blood cell membrane surface (Knob positive strains) Immunochromatographic assays P. falciparum Histidine-Rich Protein 2 (PfHRP2) P. falciparum (Knob-positive and negative strains) Immunochromatographic assays, Enzyme-Linked Immunosorbent Assay (ELISA)

32. Recent Diagnostic Modalities 2. Non-blood Based Assays: Nwakanma et al reported sensitivity and specificity of 73% and 97% respectively, when nested PCR ( nPCR ) amplification of the multicopy 18S rRNA gene in saliva was compared with blood-film microscopy. The sensitivity further increased to 82% at high parasitemia (≥1000 parasites/µL).

33. Recent Diagnostic Modalities 2.Non blood based assays: A recently developed Urine Malaria Test™(UMT) dipstick that detects P.falciparum Histidine -Rich Protein 2 (PfHRP2) showed moderate level of sensitivity when compared with blood-film microscopy in a normal field setting with a sensitivity of 83.75% and specificity of 83.48%. The UMT showed an improved sensitivity with a detection limit of 120 parasites/µL. Few reports have also suggested the use of first void morning urine as it might contain higher antigen titers than samples taken at later times.

34. Recent diagnostic modalities 3. Blood-Based Assays: New molecular approaches to parasite detection seek to reduce cost, Therapeutic Turnaround Time (TTAT) and labor intensity. Conventional nested PCR assay Real-time PCR Loop-mediated isothermal amplification (LAMP) Photo-induced electron transfer (PET)-PCR

35. 3.Blood based assays: A . Conventional nested 18S rRNA PCR assay Minimal clinical application Highly sensitive and specific nPCR laborious and time consuming Provides only qualitative data. B . Real-time PCR short assay preparation and analysis time better suited for large-scale studies than nPCR assays. relatively less expensive. Recent Diagnostic Modalities P. falciparum, P. vivax Target: mitochondrial cytochrome b gene ( cytb ) Limit of Detection: 10 parasites/µL

36. C. Photo-induced electron transfer (PET)-PCR: Principle: Self-quenching primers for the detection of Plasmodium Advantage: robust and much more cost-effective than nPCR Disadvantage: not ideal for high throughput screening of samples as nested PCR is senitive than PET-PCR Target: 18S rRNA gene Limit of Detection: P.falciparum-3.2 parasites/µL P.vivax-5 parasites/µL P.malariae-3.5parasites/µL P.ovale-5.8 parasites/µL Recent Diagnostic Modalities

37. D . Loop-mediated isothermal amplification (LAMP): Principle: By using species specific LAMP primers and use of only single temperature. Target: 18S ribosome RNA gene of P. falciparum. limit of detection 2.5 parasites/µL - whole blood 25.0 parasites/µL - dried blood spots Adavantage : simple and inexpensive Commercially available LoopampKit - detects P. falciparum and indirect detection of P. vivax Recent Diagnostic Modalities

38. D.LAMP: A LAMP assay for P. vivax α-tubulin was also developed using six primers that recognize the targeted gene at different regions . Limit of Detection: 100 copies of P.vivax α- tubulin gene per reaction. Recent Diagnostic Modalities

39. Recent Diagnostic Modalities PCR uses different temperatures LAMP uses single temperature of 65°C Detection threshold for both is 1-2parasites/ μL .   LAMP was used as the golden standard because LAMP more sensitive than PCR , as its sensitivity is not affected by left over hemoglobin like PCR.

40. E . Non-Instrumented Nucleic Acid (NINA)-LAMP: Principle : Uses an exothermic chemical reaction between saline and a magnesium iron alloy to generate energy for amplification of parasite mitochondrial DNA . Uses electricity-free heater Target : DNA Limit of Detection : 5 DNA copies/test Adavantage :- high sensitivity 96.8% in P.Falciparum 100% differentiation of non-falciparum species. Recent Diagnostic Modalities

41. 4. Application of Bio-Sensing Technology :- Biosensors are self-contained analytical devices which can analyze blood and urine without the need for additional processing steps or reagent addition. A monoclonal antibody reactive to P. falciparum infected red blood cells was immobilized onto the surface of a gold nanoparticle-modified screen-printed electrode. It showed good sensitivity with a linearity ranging from 10 2 cells/mL to 10 8 cells/mL Recent Diagnostic Modalities

42. Recent Diagnostic Modalities A.Quartz Crystal Microbalance (QCM) Biosensor: Principle: Quartz crystal microbalances (QCM) are sensitive to nanogram -scale changes in mass and biomechanical properties and are increasingly used in biomedical research. Specimen: blood Target: CD36 and chondroitin sulfate A (CSA) Limit of Detection: 200 ng of target DNA

43. B . Aptamers : Principle: A class of nucleic acid molecules recognize and bind target molecules. Single-strand DNA aptamers for parasite lactate dehydrogenase ( pLDH ) are in use. Types: Aptasensors , based on electrochemical impedance spectroscopy (EIS) detect both P.vivax lactate dehydrogenase ( PvLDH ) and P.falciparum lactate dehydrogenase ( PfLDH ). Limit of detection - 1 parasite/µL . Advantage:- simple and rapid alternative methods Recent Diagnostic Modalities

44. C . Biosensor (colorimetric aptasensors ) Principle : Colorimetric biosensors can be used to detect a particular analyte through color changes easily by naked eyes or simple portable optical detectors for quantitative measurement.   Specimen: Blood Target: Recombinant PvLDH & PfLDH Limit of Detection : 1.25 pM ( PvLDH ), 2.94 pM ( PfLDH ) Recent Diagnostic Modalities

45. 5.Surface enhanced Raman spectroscopy (SERS): Applied in probing the hemozoin content of red blood cells (RBCs) and was found to be suitable for the diagnosis of malaria at low parasitemia and hemozoin concentrations in blood. Ultrasensitive detection limit for hemozoin at 0.00005% parasitemia level in the ring stage (2.5 parasites/µL) Recent Diagnostic Modalities

46. MICROFILARIA

47. Filaria Wucheraria bancrofti Brugiya malayi Brugiya timori Other species: Loa loa Oncocerca volvulus Mansonella streptocerca , Mansonella ozzardi , Mansonella perstans Lymphatic filariasis

48. filariasis Wuchereria bancrofti Brugia malayi Brugia timori Life cycle Human → definitive host Mosquitoes( culex , anopheles, aedes ) → intermediate host

49. Fialria -Diagnostic methods Microscopic examination of blood Concentration techniques Detection of filarial antigen in blood by rapid immunochromatographic test Detection of parasite DNA by PCR Demostration microfilaria hydrocele fluid / chylous urine Demonstration adult worms lymph node biopsy

50. Filaria -Diagnostic methods Microscopic examination : Microfilaria exhibits periodicity Loa loa exhibits →diurnal periodicity W.bancrofti,B.malayi → noctural periodicity Capillary blood is collected between 10pm-4am Direct wet mount – serpentine movement Giemsa stained thick smear film

51. Filaria - Diagnostic methods Concentration techniques: 1.Membrane filtration 2.Microhematocrit tube or capillary tube method 3.Lysed venous blood method 4.Lysed capillary blood method

52. Filaria – Diagnostic methods 1.Membrane filtration method: Sensitive method 10 ml of anticoagulated blood Polycarbonate membrane filter – pore size 3micro m 10 ml of methylene blue solution

53. 2.Microhematocrit tube or Capillary tube method: Filaria – Diagnostic methods

54. Filaria – Diagnostic methods 3.Lysed venous or capillary blood method:- 10 ml of venous blood or 0.1 ml of capillary blood is obtained . Add saponin -saline solution that cause red cell lysis . Centrifuge the solution and supernatant is removed and a drop of methylene blue is added. Examined under microscope.

55. Filaria - Diagnostic Methods Immunodiagnosi s Antigen detection:- Immunochromatographic test:- Detected by monoclonal antibody AD12.1 Recently filarial streak test also available ELISA :-uses monoclonal antibody against Og4C3 antigen

56. Filaria - Diagnostic Methods Antibody detection :- detection of anti- microfilarial immunoglobulin IgG4 antibody 3 recombinant antigens :-Bm14,WbSXP,BmR1 Target detected Field assay Confirmatory assay Microfilaria Blood film PCR Filarial antigen ICT Og4C3 ELISA Antifilarial antibody Brugia rapid test Bm14 ELISA

57. Microfilaria in the lymphnode biopsy

58. LEISHMANIA

59. Leishmanias Transmitted→infected female sandfly Life cycle of L.donovani has 2 forms Promastigote :-flagellated form found in sandfly Amastigote :-Non flagellated found in mammalian tissue

60. Leishmania -Diagnosis Examination of splenic, bone marrow aspirate, buffy coat preparation of peripheral blood for amastigote forms Detection of anti- leishmania antibodies Intradermal skin test DNA studies Animal inoculation Culture of parasite

61. Leishmania - Diagnostic methods 1.Examination of splenic aspirate , bone marrow aspirate, lymphnode aspirate or buffy coat prepartaion of peripheral blood for amastigote forms: Splenic aspirate has the high sensitivity. Aspirate is smeared and stained with geimsa or buffy coat is made.

62. Amastigote forms small , round to oval with nucleus and rod shaped kinetoplast . Pale blue cytoplasm. Arranged in groups inside macrophages Lye freely between cells.

63. Bone marrow aspiration

64. Leishmania - Diagnostic modalities Tests that detect raised nonspecific immunoglobulins : In Visceral Lieshmaniasis , increased amounts of polyclonal IgG & IgM produced Formol gel(Aldehyde test) Antimony test Formol gel test

65. Leishmania – Diagnostic Methods Chopra’s antimony test:- 1-2ml of patient serum in test tube & 4%urea stibamine solution added Positive test:- Formation of profuse flocculant precipitate is seen.

66. Leishmania - Diagnostic Methods Tests that detect specific anti- Leishmania antibodies in serum: Complement fixation test Indirect immunofluorescence test Concurrent immunoelectrophoresis Enzyme linked assay Direct agglutination test Latex agglutination test

67. Leishmania - Diagnostic Methods Specific antibodies test :- Direct agglutination test : Antigen-trypsin –treated promastigotes , fixed in formalin & stained with coomasie briliant blue & pt.serum incubated with this antigen Agglutination noted

68. Rapid immunochromatographic test Detects specific IgG anti- Leshmania antibodies against rk39 antigen High sensitivity &specificity Leiahmania - Dagnostic methods

69. Katex test:- Detects leishmanial antigens in urine of patients with leishmaniasis . Latex particles sensitized with antibodies against L.donovani antigen. Agglutination indicates positive test. Leishmania - Diagnostic Methods

70. Leishmania - Diagnostic Methods Skin Test: The Montenegro skin test Delayed type of hypersensitivity 0.5ml of killed promastigotes injected intradermally and read after 75 hrs. Positive in patients recovered from kala-azar . Negative in active infection.

71. Leishmania - Diagnostic Methods Parasite culture :- NNN ( Novy -McNeal Nicolle ) medium After inoculation with 1-2 drops of splenic /bone aspirate incubate at 22-28 c Examine weekly for 4 weeks

72. Leishmania - Diagnostic Methods Animal inoculation :- Golden hamsters inoculated with infected tissue via intraperitoneal,intrasplenic , intradermal .

73. Leishmania - Diagnostic Methods T r y pan o s o me free Reduvid bug is allowed to feed on individual suspected of having chagas disease. Xenodiagnosis

74. Babesiosis Ixodes ticks (deer tick) B.microti B.divergens B.duncani Co-infection with Lyme disease

75. Babesiosis -Diagnosis Microscopic examination Immunofluorescence assay or immunoblotting Polymerase chain reaction Animal innoculation Giemsa / wright stain →blood smears Rings forms are multiple &forms tetrads ( maltese cross)

76. Trypanosomiasis Trypanosoma brucei has 2 forms T . brucei rhodesience -East Africa T. brucei gambiense -Central Africa Caused by tse tse fly Chagas disease (American trypanosomiasis→T.cruzi transmitted by reduviid bug (kissing bug) Monomorphic trypanosomes Polymorphic

77. Diagnosis Peripheral blood smears ( thick,thin ) staines with giemsa show whip –like flagellum Hematocrit centrifugation technique for buffy coat examination Miniature anion –exchange centrifugation technique ,which filters RBC’S but not trypanosomes.

78. Trypanosomiasis - Diagnostic Methods Serological assay Card agglutination test Antigen detection by ELISA Recent → Isoenzyme analysis & restriction fragment length polymorphism used for subspecies detection.

79. Ehrlichiosis / anaplasmosis Small obligate intracellular gram negative parasites Affinity towards WBC particularly mononuclear phagocytes Species E.sennetsu E.caffeensis E.phagocytophilia Caused by ticks

80. Ehrlichiosis - Diagnosis Single elevated IgG antibody titre by IFA. Detection with PCR Difficult to cultures

81. Summary CONVENTIONAL Stained blood films: thick and thin “GOLD STANDARD” Sensitivity of thick blood film is about 50 parasites/µl Fluorescence microscopy- QBC Sensitivity - 100 parasites/µl RECENT Detection of specific nucleic acid sequences Non Microscopic: Rapid diagnostic tests Immunochromatographic tests (ICT) for candidate enzymes. Malaria Diagnostics

82. LE I S H M A N IA DIAGNOSIS CONVENTIONAL Direct demonstration of amastigotes in stained preparations Culture in NNN medium Aldehyde test Leishmanin test RECENT  Serologic tests- IFA ELISA Immunoblot test ICT (K39) PCR targeting the kinetoplast DNA. Western blot

83. LYMPHATIC FILARIASIS DIAGNOSIS CONVENTIONAL Demonstration of circulating microfilaria Concentration of blood with polycarbonate membrane filter. RECENT Serology – IFA, ELISA, ICT Use of monoclonal antibodies to detect W.bancrofti Ag PCR & DNA probes Ultrasonography Lymphoscintigraphy

84. Summary Conventional microscopic examination of peripheral thick and thin blood smears remains the gold standard for malaria diagnosis. Quick and convenient RDTs are currently implemented in many remote settings, but are costly and need improved quality control. Serological tests are useful for epidemiological surveys, but not suitable for the diagnosis of acute illness.

85. Summary Molecular-biological techniques are appropriate for research laboratories; they can be used to identify the development of drugresistance , species identification, quantifying parasite density with low parasitemia . Finally, the level of malaria endemicity , the urgency of diagnosis, experience of physician, effectiveness of healthcare workers, and budget resources, are all factors influencing the choice of malaria-diagnostic method.

86. References FrancisD.Krampa , YawAniweh,GordonA.Awandare,ProsperKanyong ; Recent Progress in the Development of Diagnostic Tests for Malaria; Diagnostics 2017, pg : 54-66. Newby, G.; Bennett, A.; Larson, E.; Cotter, C.; Shretta , R.; Phillips, A.A.; Feachem , R.G.A. The path to eradication: A progress report on the malaria-eliminating countries. Lancet 2016, 387, 1775–1784. O’Meara, W.P.; Mangeni , J.N.; Steketee , R.; Greenwood, B. Changes in the burden of malaria in sub-Saharan Africa. Lancet Infect. Dis. 2010, 10, 545–555. World Health Organization (WHO). World Malaria Report 2016; WHO: Geneva, Switzerland, 2016. Henry's Clinical Diagnosis and Management by Laboratory Methods Richard A. McPherson MD MSc  ,  Matthew R. Pincus MD PhD  23 rd edition.