pathology

1. RECENT ADVANCES IN
HEMOPARASITES
Dr Parul Agrawal
Lt Col N Hazra

2. RECENT ADVANCES IN
INFECTIOUS DISEASES
Three key areas of study-
 Newer approaches to overall clinical
management & diagnosis
 A clearerunderstanding of basic
microbiology, pathogenesis and
host defence mechanisms
 Better ways of prevention including
vaccines

3.  INTRODUCTION TO HEMOPARASITES
 PLASMODIUM
 BABESIA
 LEISHMANIA
 TRYPANOSOMA
 MICROFILARIA
 RECENT ADVANCES IN
 PATHOGENESIS
 DIAGNOSIS
 TREATMENT
 PREVENTION (VACCINES)

4. PLASMODIUM

5. INTRODUCTION & LIFECYCLE
SPOROZOITES
Plasmodium includes 172
species
Four commonly infects man
P. falciparum
P. vivax
P. ovale
P. malariae
APICOMPLEXA GROUP
OF PROTOZOA
Apical complex consists of-
Polar ring,
Rhoptries and
Micronemes
Intestine of
Mosquito
LIVER
ANOPHELES
MOSQUITO

6. SPOROZOITE

7. RECENT ADVANCES PATHOGENESIS
SPOROZOITE HOST INTERACTION
 Sporozoite surface proteins involved
in invasion –
 Circumsporozoite (CS)
 Thrombospondin related anonymous
protein (TRAP)
 Membrane apical erythrocyte binding-like
surface protein (MAEBL)
 Sporozoite threonine and asparagine rich
protein (STARP)

8. RECENT ADVANCES PATHOGENESIS
SPOROZOITE HOST INTERACTION
 Hepatic heparan sulfate proteoglycans
(HSPGs)
 Sporozoite and liver stage antigen (SALSA)
 Liver stage antigen- 1 (LSA-1)
 Liver stage antigen- 3 (LSA-3)
 Sporozoite microneme protein essential for
cell transversal (SPECT)

9. RECENT ADVANCES PATHOGENESIS
STEPS IN SEQUESTRATION
1
23

10. RECENT ADVANCES PATHOGENESIS
Endothelial Cytoadherence Receptors
 CD 31
 CD 36
 ICAM-1
 Thrombospondin (TSP)
 Chondroitin sulfate A (CSA)
 E Selectin
 VCAM-1

11. RECENT ADVANCES PATHOGENESIS
Rosetting Receptors on RBC Surface
Blood group antigens A & B
CD36
Complement Receptor 1(CR1)
Heparan sulfate like
glycosaminoglycans
Serum proteins

12. RECENT ADVANCES PATHOGENESIS
Pf EMP1 - ADHESIVE LIGAND
 Central to malaria pathogenesis
 Invoved in cytoadherence and rosetting
 Major antigenically variant protein encoded
by multicopy gene family “var”
 60 different var genes exist
 Single variant dominates on surface of one
infected erythrocyte
 There may be several variants circulating in
one host

13. RECENT ADVANCES PATHOGENESIS
STRUCTURE OF var PfEMP1
DBL = Duffy binding like domains
CIDR = Cysteine rich interdomain
regions
ATS = Acidic terminal sequence

14. RECENT ADVANCES IN DIAGNOSIS
 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
 Immunochromat
ographic tests
(ICT) for
candidate
enzymes

15. DETECTION OF SPECIFIC NUCLEIC
ACID SEQUENCES
SPECIES SPECIFIC TARGETS FOR PCR
 Small subunit 18S rRNA
 Circumsporozoite (CS) genes
Nested PCR & RT PCR enable all four species
identification
GENUS SPECIFIC TARGET
 Large subunit RNA gene - extensively
conserved among Plasmodium spp.
Sensitivity ≤5 parasites/µl

16. NON MICROSCOPIC RAPID
DIAGNOSTIC TESTS
FEATURES -
 Results as accurate as microscopy
 Sensitivity - 100 parasites/µl
 Principle -
Immunochromatographic tests
Malaria antigens suitable as targets are
 HRP-2 (histidine rich protein)
 pLDH
 Aldolase

17. NON MICROSCIPIC RAPID
DIAGNOSTIC TESTS………..
 Detect antigens
derived from
malaria parasites
 Immuno-
chromatographic
tests
 Dipstick or cassette
format
 Results within 10
minutes :rapid
result

19. DETECTION OF HRP-2
 HRP-2 is a water soluble
protein produced by
asexual stages & young
gametocytes of
P.falciparum
 Immuno chromatographic
test in dipstick format-
monoclonal Ab captures
the HRP-2 Ag
 Two HRP-2 based tests :
(a) Para Sight F test
(b) ICT Malaria Pf test

20. ADVANTAGE OF HRP – 2
DETECTION SYSTEM
 Sensitivity - 96.5%-100%
 Specificity - 95%-98%
 Advantage - rapid
- sensitive
- result straight forward
- can be taught to village
health workers
- no special equipment
required
 Disadvantage - high cost
- not quantitative

21. DETECTION OF PARASITE LDH
 pLDH is an enzyme produced by sexual &
asexual stages of the parasite
 Different isomers of pLDH exist for different
Plasmodium spp.
 No cross reaction with human LDH
 Advantage - Sensitivity 99.9%
- Falciparum or non
falciparum
- Prognostic value
- Rapid- 15-20 min

22. DETECTION OF PARASITE LDH
OptiMAL- Test

23. RECENT ADVANCES TREATMENT
 CHLOROQUINE
 MEFLOQUINE
 QUININE/QUINIDINE
 SULFADOXINE-PYRIMETHAMINE
 DOXYCYCLINE
 ARTEMISININ DERIVATIVES
 TAFENAQUINE (NEWER 8
AMINOQUINOLINE)

24. RECENT ADVANCES TREATMENT
FOR UNCOMPLICATED MULTIDRUG
RESISTANT MALARIA -
 QUININE/QUINIDINE + DOXYCYCLINE
 MEFLOQUINE + ARTESUNATE
FOR COMPLICATED SEVERE MALARIA
 PARENTERAL ARTEMETHER/ARTESUNATE
 PARENTERAL QUININE/QUINIDINE
 TAFENAQUINE

25. ANTIMALARIAL MECHANISM OF
ACTION
MECHANISM OF
RESISTANCE
Chloroquine Forms toxic
complex with
haem
Mutation in Pf
CRT
Sulfadoxine –
Pyrimethamine
Inhibitor of folic
acid synthesis
(DHFR & DHPS)
Point mutation in
DHFR & DHPS
Atovaquone –
proguanil
A binds to
Cytochrome b,
P inhibits DHFR
Mutation in Cyt b
Point mutation in
DHFR
Doxycycline Protein synthesis
inhibition
Resistance not
known
Mefloquine
Quinine
Quinidine
Binds to haem
forming toxic
complexes
Mutations in
gene pfmdr1 and
Pf CRT

26. RECENT ADVANCES MALARIA
VACCINE DEVELOPMENT
 Biological basis for malaria vaccine
 Persons subjected to long term
exposure to malaria develop partial
protection
 Immune persons generally have fewer
& less dense parasitemia

27. VACCINE STRATEGIES
 Induce antibody production against P.
falciparum circumsporozoite (CS)
protein
 Eliminate asexual stages of parasite
from blood
 Induce immunity against sexual
reproduction of parasite

28. VACCINE STRATEGIES………..
 Transmission blocking vaccine –
Blocks sexual reproduction
 Anti-disease vaccine – neutralizing
antibodies to parasite products
 Antibodies to components of
parasitized RBCs required for
endothelial attachment

29. VACCINE ANTIGENS
 Pre-erythrocytic antigens –
 Circumsporozoite protein (CS)
 Sporozoite surface protein 2 (SSP 2)
 Liver stage specific antigen 1 (LSA-1)
 Liver stage specific antigen 2 (LSA-2)

30. VACCINE ANTIGENS…………….
 Merozoite and Erythrocytic Antigens -
 Erythrocyte binding antigen (EBA-175)
 Merozoite surface protein 1 (MSP-1)
 Merozoite surface protein 2 (MSP-2)
 Ring infected erythrocyte surface antigen
(RESA)
 Serine repeat antigen (SERA)

31. VACCINE ANTIGENS………
 Transmission blocking and
pathogenicity antigens
 Pfs25- most promising transmission
blocking vaccine, immunogen found on
the surface of P. falciparum zygotes &
ookinetes
 Cytoadherence antigens
 Anti-disease antigens- two glycoproteins
released upon rupture of shizont

32. MULTIPLE-COMPONENT MALARIA
VACCINE
 SPf66 – A synthetic polymer consists
of three peptides from P. falciparum
merozoite & one peptide from CS
protein
 Safe
 Immunogenic
 Protective efficacy – 39%

33. BABESIA

34. INTRODUCTION
 Emerging zoonotic disease of humans
 Transmitted by Ixodid ticks
 Piroplasms – pear shaped appearance
inside RBCs of vertebrate hosts

35. LIFECYCLE OF BABESIA
STRAHLENKORPERIXODID TICK VERTEBRATE HOST
SPOROBLAST
IN SALIVARY GLAND
RING FORMS
TICK GUT

36. MORPHOLOGICAL &
PHYLOGENETIC CLASSIFICATION
 Based on the size of intraerythrocyic form
(trophozoite) Babesia species has two
groups-
Small Babesia Large Babesia
 B.microti B.bovis
 B.gibsoni B.canis
 WA1 B.divergans
Size distinction is generally consistent with
phylogenetic classification based on ss-rDNA

37. BABESIA
TRENDS IN PHYLOGENETIC TREE
 B.divergens small in diameter but
genetically related to large Babesia cluster
 Small Babesia are more related to Theileria
than to large Babesia species
 Unlike Babesia, Theileria piroplasm first
undergoes asexual division in lymphocytes
before invading erythrocytes
 Thus B.equi is reclassified as T. equi
 New species WA1 isolated in 1991 from a
immunocompetent patient

38. BABESIA RECENT ADVANCES
PATHOGENESIS
 Antibody based depletion of CD4 T cells
results in increased susceptibility to
B.microti/WA1
 Depletion of CD8 T cells increases
resistance to B.microti
 Successful immune response is associated
with release of TNF-α and IFN-γ
 Phagocytosis of parasitized erythrocytes by
macrophages is essential to control
parasitemia

39. BABESIA RECENT ADVANCES
DIAGNOSIS
 Appropriate clinical manifestations
 Descriptive history –
 Travel to endemic area
 Tick bite
 Blood transfusion
 Splenectomy
 Blood smear examination
 Serology- IFATs
 PCR

40. PCR FOR BABESIA

41. PCR FOR BABESIA
Detection of
Babesia microti
By PCR
Journal of Clin
Microbiol Aug 1992

42. BABESIA RECENT ADVANCES
TREATMENT
 Clindamycin + Oral Quinine
 Chloroquine & other antimalarials not
effective
 Atovaquone + Azithromycin
 Pentamidine + Trimethoprim-
sulphamethoxazole
 In HIV positive –
 Clindamycin + Doxycycline + Azithromycin

43. BABESIA
PREVENTION - VACCINES
 At present no human vaccine
 Development of vaccines for cattles
 Live attenuated vaccines
 Recombinant vaccines- vaccine from
major surface antigen of sporozoite
form particularly apical complex
proteins
 Rhoptry associated protein (RAP-1)

44. LEISHMANIA

45. WORLD DISTRIBUTION

47. INTRODUCTION
 Leishmaniasis has surged as a reactivating
infection in AIDS pateints
 Vector- Phlebotomine sandflies
 Leishmaniasis is a disease complex caused
by 17 different species
 L.donovani
 L.major
 L.tropica
 L.braziliensis complex

48. INTRODUCTION……….
 CLINICAL FORMS OF LEISHMANIASIS -
 Visceral (VL)
 Cutaneous (CL)
 Mucocutaneous (MCL)
 Diffuse cutaneous (DCL)
 Post kala-azar dermal (PKDL)

49. LIFE CYCLE

50. Morphological forms of Leishmania
AMASTIGOTE
PROMASTIGOTE

51. RECENT ADVANCE PATHOGENESIS
ESCAPE FROM HOST IMMUNE SYSTEM
 Lieshmania alters the macrophage signal
transduction machinery
 Lipophosphoglycan (LPG) on the surface of
promastigote prevents the attachment of
C5b-C9 (MAC)
 Inhibition of macrophage functions
 Inhibition of JAK2/STAT1 signalling due to
defective phosphorylation on IFN-γ
stimulation
 Inhibition of MAP Kinases

53. LEISHMANIA
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

54. LEISHMANIA
TREATMENT
 VISCERAL LEISHMANIASIS
 Pentavalent antimony compounds -
 Stibogluconate sodium
 Meglumine antimoniate
 Liposomal Amphotericin B (AmBisome)
 Amphotericin B deoxycholate
 Miltefosine- oral phosphocholine analogue
 Sitamaquine
 Aminosidine (Paromomycin)
 Pentamidine isethionate
 Imidazoles (Fluconazole, Ketoconazole)

55. LEISHMANIA
TREATMENT………….
 CUTANEOUS LEISHMANIASIS –
Treatment depends upon site & extent
 In addition to drugs listed in VL
 Cryotherapy/ Hyperthermic therapy
 Surgical excision of lesion
 Topical application of 15% paromomycin+
12% methyl benzethonium chloride
 Immunotherapy (BCG+ Promastigote)
 Azithromycin
 GM-CSF

56. RESISTANCE TO ANTIMONY COMPOUNDS

57. LEISHMANIA VACCINES
 Ancient ways of immunization-
 To expose the bottoms of babies to
sandfly bite
 Use of thorn to transfer infectious
material from lesions to uninfected
individuals
 Vaccination with live organisms and
problems associated -
 Large uncontrolled skin lesions
 Exacerbation of psoriasis
 Immunosuppression

58. LEISHMANIA VACCINES………
 Killed promastigotes
 Killed promastigotes with BCG
 Killed promastigotes with IL-12
 Irradiated promastigotes
 Recombinant or native gp63
 Recombinant or native gp46/M2/PSA-2
 Recombinant LACK (Leishmania homologue
of the receptor for activated C kinase)
 LACK with IL-12

59. LEISHMANIA VACCINES………
 Flagellar antigen Icr1
 Amastigote specific vaccine targets A2, P4
and P8
 Elution of antigenic peptides from antigen
presenting cells (subunit vaccine)
 Synthetic peptides
 Non protein antigens – Leishmania
lipophosphoglycan (LPG)
 Naked DNA vaccines

60. TRYPANOSOMA
American Trypanosomiasis
(Chagas’ disease)
African Trypanosomiasis
(Sleeping sickness)

61. EPIMASTIGOTE
TRYPOMASTIGOTE
AMASTIGOTE

62. AMERICAN TRYPANOSOMIASIS
CHAGAS’ DISEASE
 Zoonosis caused by Trypanosoma cruzi
 Vector – triatomine insects (kissing bugs)
 Other ways of transmission – blood
transfusion, organ transplantation etc
 Acute disease with systemic symptoms
 Chronic disease localized organ damage-
cardiomegaly, megaesophagus
 In humans T.cruzi found in two forms-
amastigotes & trypomastigotes

63. CHAGAS’ DISEASE AND
AUTOIMMUNITY
 Substantial lymphocyte activation during
acute phase of disease
 Both B & T lymphocytes are involved (CD4
as well as CD8)
 Humoral immune response is polyclonal
 Large amount of immunoglobulin are
produced (IgG2)
 Administration of anti-CD4 antibodies
suppress polyclonal antibody
production

64. CHAGAS’ DISEASE
DIAGNOSIS
 Care while handling the sample as
trypomastigotes are highly infectious
 Demonstration of trypomastigotes in blood
& amastigotes in tissue
 Serology
 Culture
 Animal inocculation & xenodiagnosis
 PCR – can detect 1 trypomastigote in 20
ml of blood

65. CHAGAS’ DISEASE
DIAGNOSIS : TARGET FOR PCR
 Highly repetitive nuclear & kinetoplast DNA
(k DNA)
 Primer TCZ1-TCZ2 for nuclear repetitive
188bp sequence
 Primer S35-S36 against 330 bp kinetoplast
minicircle
PCR is useful in –
Persons with borderline serology results
Received specific treatment
Acute or congenital disease

66. CHAGAS’ DISEASE
XENODIAGNOSIS
TRYPANOSOME
FREE
REDUVID BUG
IS ALLOWED TO
FEED ON
INDIVIDUAL
SUSPECTED OF
HAVING
CHAGAS
DISEASE

67. CHAGAS’ DISEASE
TREATMENT
 Current therapy is unsatisfactory
 Nifurtimox (Nitrofuran derivative)
 Benznidazole (Nitroimidazole derivative)
 Cure rate is <10% with both drugs
 Posaconazole has activity against T.cruzi
 Fluconazole, Ketoconazole, Itraconazole &
Allopurinol has no activity against T.cruzi
 Recombinant IFN γ reduces the severity of
disease

68. AFRICAN TRYPANOSOMIASIS
SLEEPING SICKNESS
West & Central Africa – T.brucei
gambiense
South & East Africa – T.brucei
rhodesiense

69. AFRICAN TRYPANOSOMIASIS
SLEEPING SICKNESS
 Both are morphologically indistinguishable
 Limited to the Tsetse fly belt of Africa
 Prototype of a neglected disease
 Affecting the poorest people of poorest
continent
 Development of new diagnostic tests and
drugs severely affected

70. AFRICAN TRYPANOSOMIASIS
PATHOGENESIS
 Antigenic variation – Parasite changes
the antigenic structure of surface
glycoprotein every 5 days
 Variant antigen types (VATs)- In Tsetse
fly parasite synthesizes a surface coat
made up of about a dozen of antigenic
glycoproteins
 Evades the immune destruction indefinitely

71. AFRICAN TRYPANOSOMIASIS
DIAGNOSIS
 Diagnosis of Trypanosomiasis follows
a three step procedure-
 Screening
 Diagnostic confirmation
 Staging
 First stage – Haemolymphatic
 Second stage - Meningoencephalitic

72. AFRICAN TRYPANOSOMIASIS
DIAGNOSIS - SCREENING
 Antibody detection (IgM) –
 CATT
 LATEX/ IgM
 IFA
 ELISA
 Biological parameters –
 Raised ESR
 Low hematocrit
 Decreased albumin
 Increased Immunoglobulin (IgM)

73. AFRICAN TRYPANOSOMIASIS
DIAGNOSIS - SCREENING
Card Agglutination Test for Trypanosomiasis (CATT)
Antigen consists of lyophilized blood stream
forms of T.b. gambiense variable antigen type
LiTat 1.3

74. AFRICAN TRYPANOSOMIASIS
DIAGNOSIS - CONFIRMATION
 Demonstration of trypanosomes –
 Chancre aspirate
 Lymph node aspirate
 Wet & Thick blood films
 Microhaematocrit centrifugation technique
 Quantitative buffy coat
 Mini-anion exchange centrifugation

75. AFRICAN TRYPANOSOMIASIS
DIAGNOSIS - STAGING
 First stage – Haemolymphatic
 Second stage – Meningoencephalitis
 According to WHO Second stage is
defined by the presence in CSF of –
 Trypanosomes
 Raised WBC count (>5 cells/µl)
 Increased protein content (>370mg/lit)
NOTE – Examine the CSF immediately after LP as
trypanosomes in CSF start to lyse within 10 min

76. AFRICAN TRYPANOSOMIASIS
DIAGNOSIS – SECOND STAGE
 Demonstration of intrathecal synthesis of
immunoglobulins
 LATEX/IgM for CSF
 CSF - Antibodies against brain specific
components – neurofilaments and
galactocerebrosides (GalC) may be
promising markers
 These autoantibodies might result from the
CNS damage & immune activation triggered
by trypanosome invasion

77. AFRICAN TRYPANOSOMIASIS
TREATMENT
 FIRST STAGE – HEMOLYMPHATIC
 Pentamidine isoethionate (Lomidine)
 Suramin
 Eflornithine / Difluoromethylornithine
(DFMO)
 SECOND STAGE – MENINGOENCEPHALITIC
 Eflornithine
 Melarsoprol - highly toxic drug

78. TRYPANOSOMIASIS
PREVENTION
 No vaccine is available for T.cruzi and
T.brucei
 Chemoprophylaxis is not recommended
because of high toxicity of drugs

79. MICROFILARIA

80. MICROFILARIA
Tapered tail, with a
subterminal and a
terminal nuclei
Brugia malayi
The cells can be visualized
individually and do not
extend to the tip of the tail
W. bancrofti

81. LYMPHATIC FILARIASIS
PATHOGENESIS
 Lymphangiectasia but little inflammation
 Endothelial proliferation, fibrin deposition,
granulomatous inflammatory infiltrate of
eosinophils, lymphocytes & macrophages
 Molting & death of worms leads to
inflammation followed by fibrosis &
obstruction of lymph flow
 Secondary bacterial infections
 Complex immunological events

82. LYMPHATIC FILARIASIS &
WOLBACHIA
 Most filaria infected with endosymbiontic
rickettsial bacteria Wolbachia
 Chronic release of wolbachia may cause
progressive damage to infected lymphatics
and desensitization of immune system
 Antibiotic treatment of worms has shown
that clearance of bacteria results in
embryotoxicity, inhibition of molting &
eventually death of worms

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. LYMPHATIC FILARIASIS
RECENT ADVANCES TREATMENT
 No satisfactory treatment for filariasis
 Diethylcarbamazine (DEC) No effect
 Ivermectin on adults
 Albendazole + DEC/ Ivermectin
 No study on antibiotic therapy directed
against Wolbachia
 In 2003 it was suggested that the common
antibiotic doxycycline might be effective in
treating elephantiasis

85. LYMPHATIC FILARIASIS
PREVENTION / VACCINE
 DEC/ Ivermectin /both administered every
6-12 months may reduce transmission
 These drugs alone are not the answer in
eradicating the disease
 WHO has embarked on a campaign of mass
administration of Albendazole (donated by
GlaxoSmithKline) + DEC/ Ivermectin yearly
for five years
 Global elimination of lymphatic filariasis by
2020

86. LYMPHATIC FILARIASIS
PREVENTION / VACCINE
 Candidate antigen for vaccine –
 Stage specific microfilarial chitinase
(Prototype transmission blocking vaccine)
 Chitinase is essential for worm
development & exsheathment of
microfilaria

87. REFERENCES
 Manual of Clinical Microbiology, Patrick R
Murray; 8th
edition; Chapters 129 – 130.
 Principles and Practice of Infectious
Disease, Mandell, Douglas and Bennett, 6th
edition; Chapters 272-275, 279.
 Clinical Microbiology Reviews –
 Rapid diagnostic tests for malaria parasites;
Jan 2002 (15), 66-78.
 Malaria vaccine development; July 1994 (7)
p303-310

88. REFERENCES……..
 Babesiosis; July 2000(13), 451-469
 Drug resistance in leishmaniasis; Jan 2006
(19)111-126
 Leishmaniasis: Current status of vaccine
development; April 2001(14), 229-243
 Options for field diagnosis of human african
trypanosomiasis; Jan 2005(18), 133-146
 Chagas’ disease and the autoimmunity
hypothesis; April 1999, 210-223