1. BLOOD AND TISSUE PROTOZOONS
◊ Leishmania
◊ Toxoplasma gondii
◊ Trypanasoma
◊ Babesia
◊ Plasmodium
Doç.Dr.Hrisi BAHAR

2. Taxonomy of Leishmania
Phylum
Order
Family
Genus
Sarcomastigophora
Kinetoplastida
Trypanosomatidae
Leishmania

3. Leishmania donovani ♦ Zoonotic parasite
♦ It is one of the 5 major parasitic diseases
♦ It is endemic in northern to Yangtse river
♦ 0.5 million patients before 1949
♦ Basically eradicated in 1958

4. Morphology of Leishmania
Digenetic Life Cycle
1-Promasitogte
*Insect stage
Motile
Midgut
2-Amastigote
*Mammalian stage
Non-motile
Intracellular

5. Life cycle
Life cycle
♦ The organism is transmitted by the bite of several species of blood-feeding sand flies (Phlebotomus) which carries the promastigote in the anterior gut and pharynx.
Promastigote of Leishmania sp
Promastigote of Leishmania sp Promastigote of Leishmania sp

6. Life cycle
♦ Promastigote gains access to mononuclear phagocytes where it transform into amastigotes and divides until the infected cell ruptures. The released organisms infect other cells.
♦ Promastigote gains access to mononuclear phagocytes where it transform into amastogotes and divides until the infected cell ruptures. The released organisms infect other cells.

7. *Phlebotomus sendfly is the
Life cycle
♦ The sandfly acquires the organisms during the blood meal, the amastigotes transform into flagellate promastigotes and multiply in the gut until the anterior gut and pharynx are packed. Dogs and rodents are common reservoirs.
*Phlebotomus sendfly is the
VECTOR
of Leishmania

8. Clinical Disease Visceral Cutaneous Generally Self- healing
Fatal (90% untreated)
Liver
Spleen
Bone marrow
Cutaneous
Generally Self- healing
Skin
Mucous membranes
Muco cutanous
Muco cutanous
mucosal involvement may occur if a skin lesion near the mouth or nose is not treated

9. Life cycle of Leishmania

10. Initial Infection of Leishmania
Similar in all species
Inoculation of promastigotes
Inflammation & chemotaxis
Receptor mediated phagocytosis
Promastigote Amasitgote
Transformation

11. Parasite Spread Macrophage lysis & parasite release Lymphatic spread
Blood spread
Target organs
Skin/lymph nodes/spleen/liver/
bone marrow

12. Main Points of Life Cycle
♦ Host: man and sandfly
♦ No sexual development in the host
♦ Residing site: macrophage
♦ Infective stage: promastigote
♦ Infective route: inoculation of sandfly
♦ Reservoir host: dog
♦ Infection could also via transfusion

13. Clinical Manifestation
Variable - Incubation weeks
Lowgrade fever
Hepato-splenomegaly
Bone marrow hyperplasia
Anemia, Leucopenia & Cachexia
Hypergammaglobulinnemia
Proteinuria, Hematuria

14. Cutaineous leishmaniasis
Cutaneous leıshmaniasis

15. Cutaineous Leishmaniasis
Clinical manifestation
♦ Most common form
♦ Characterized by one or more sores, papules or nodules on the skin
♦ Sores can change in size and appearance over time
♦ Often described as looking somewhat like a volcano with a raised edge and central crater
Some sores are covered by a scab or have not yet ulcerated so they may look like red raised plaques- sometimes with dry crust/scale

16. Cutaineous Leishmaniasis Clinical manifestation
♦ Sores are usually painless but can become painful if secondarily infected
♦ Swollen lymph nodes may be present near the sores (under the arm if the sores are on the arm or hand…)

17. Cutaineous Leishmaniasis Clinical manifestation
♦ Most sores develop within a few weeks of the sandfly bite, however they can appear up to months later
♦ Skin sores of cutaneous leishmaniasis can heal on their own, but this can take months or even years

18. Cutaineous Leishmaniasis
Clinical manifestation
♦ Sores can leave significant scars and be disfiguring if they occur on the face
♦ If infection is from L. tropica it can spread to contiguous mucous membranes (upper lip to nose)

19. Cutaineous leishmaniasis
Causative organism
L. tropica
L. major
L. aethiopica
L. mexicana
Location
Mediterranean basin, Afghanistan
Middle East, W. and N. Africa, Kenya
Ethiopia
Central America and Amazon.

20. Cutaineous Leishmaniasis
Small, raised lesion on trunk without significant oozing or scab.
Photograph provided by COL Naomi Aronson
Cutaineous Leishmaniasis

21. Cutaineous Leishmaniasis
Multiple lesions on arm with a variety of appearances.
Photograph provided by COL Naomi Aronson
Cutaineous Leishmaniasis

22. Cutaneous Leishmaniasis
Both lesions are leishmaniasis
Note the raised border and wet appearance of the sore on the back of the hand.
Sores over joints are very concerning as scarring with healing can lead to limited movement of joint.
Photograph provided by COL Charles Oster
Cutaneous Leishmaniasis

23. Cutaneous Leishmaniasis
Back of hand.
Note raised border and wet appearance. Patient has bacitracin ointment applied to lesion.
Photograph provided by COL Naomi Aronson
Cutaneous Leishmaniasis

24. Cutaneous Leishmaniasis
Upper Eyelid.
Note the dry, crusted/scabbed appearance which is different than previous sores shown.
Photograph provided by COL Naomi Aronson
Cutaneous Leishmaniasis

25. Cutaineous leishmaniasis of the face.

26. Visceral Leishmaniasis KALA-AZAR
Most severe form of the disease, may be fatal if left untreated
Usually associated with fever, weight loss, and an enlarged spleen and liver
Anemia (low RBC), leukopenia (low WBC), and thrombocytopenia (low platelets) are common
Lymphadenopathy may be present
Based on our experience with Desert Storm, well nourished American soldiers generally have a less symptomatic, relatively oligoparasitic infection that was not life threatening but posed some diagnostic challenges

27. Visceral Leishmaniasis
Causative organism
L.donovanii
L.d.donovani  
L.d.infantum
L.d chagasi
Location
China, India, Iran
Sudan, Kenya, Ethiopia
Mediterranean region
Brazil, Colombia, Venezuela, Argentina

28. Visceral Leishmaniasis in Desert Storm
The following symptoms were found in eight visceral leishmaniasis patients returning from Desert Storm
Fevers: 6 of 8
Weight loss: 2 of 8
Nausea, vomiting, low-grade watery diarrhea: 2 of 8
Lymphadenopathy: 2 of 8
Hepatosplenomegly: 2 of 8
Anemia: 3 of 8
Leukopenia or thrombocytopenia: 0 of 8
Elevated liver enzymes: 6 of 8
No symptoms: 1 of 8
Summary of the presenting symptoms of eight soldiers with visceral leishmaniasis from Desert Storm. The summary is from an New England Journal of Medicine article by COL Alan Magill and others from the Walter Reed Army Medical Center Infectious Disease Service.
Magill et al, NEJM 1993:328(19)

29. Visceral Leishmaniasis
♦ Symptoms usually occur months after sandfly bite
♦ Because symptoms are non-specific and often start after redeployment there is usually a delay in diagnosis
♦ Visceral leishmaniasis should be considered in any CHRONIC FEVER patient returning from an endemic area.
From the Desert Storm experience, would also consider visceral leishmaniasis in patients with low grade elevated temperature, chronically elevated liver function tests and mild anemia

30. ♦ Profile view of a teenage boy suffering from visceral leishmaniasis
♦ Profile view of a teenage boy suffering from visceral leishmaniasis. The boy exhibits splenomegaly, distended abdomen and severe muscle wasting. 

31. ♦ A 12-year-old boy suffering from visceral leishmaniasis
♦ A 12-year-old boy suffering from visceral leishmaniasis. The boy exhibits splenomegaly and severe muscle wasting.

32. ♦ Jaundiced hands of a visceral leishmaniasis patient. 

33. ♦ Enlarged spleen and liver in an autopsy of an infant dying of visceral leishmaniasis.

34. Mucocutaineous Leishmaniasis

35. Mucocutaineous Leishmaniasis
♦ Occurs with Leishmania species from Central and South America
♦ Very rarely associated with L. tropica which is found in the Middle East
- This type occurs if a cutaneous lesion on the face spreads to involve the nose or mouth
- This rare mucosal involvement may occur if a skin lesion near the mouth or nose is not treated
Much of the destruction is from a hyperimmune reaction to the Leishmania infection. There are not many parasites in the affected tissue so confirming the diagnosis can be difficult. This form of leishmaniasis would be unusual in soldiers infected in South West Asia.

36. Muco cutaineous Leishmaniasis
Causative organism
Location
Brazil, Peru, Ecuador, Columbia, Venezuela
L. braziliensis complex

37. Photograph provided courtesy of COL Donald Skillman

38. Control ♦ Vector control ♦ Reservoir control
♦ Treatment of active cases
♦ Vaccination

39. Diagnosis ♦ Clinical signs & symptoms ♦ Hypergammaglobulinemi
♦ ELISA/Formol gel
♦ Bone marrow biopsy
♦ Spleen or liver biopsy
♦ Culture & Histology

40. Diagnosis: Cutaneous Leishmaniasis
Patients with any of the following findings should be referred early to avoid long term complications:
Big lesions (greater than an inch in size)
Many lesions (3 or more)
Sores on the face
Sores on the hands and feet
Sores over joints

41. ♦ Pentavalent antimony
Treatment
♦ Good nursing
♦ Diet
♦ Antibiotics
♦ Pentavalent antimony
♦ Pentamidine

42. Treatment Cutaneous and Mucocutaneous
Antimony (Pentostam®, Sodium stibogluconate) is the drug of choice
20 days of intravenous therapy
Fluconazole may decrease healing time in L. major infection
Biopsy and culture to determine species is required
Six weeks of therapy is needed
Since L tropica is of more concern in the SWA theatre (because of potential visceralizing infection, rare mucocutaneous involvement, and chronic (recidivans) skin infection) , would not advocate use of azoles routinely as there is not data to support their use in L.tropica and speciation can not be reliably made using clinical appearance.
Mucocutaneous infection is treated with a longer treatment course (28 days)

43. Treatment Visceral Leishmaniasis
♦ Liposomal amphotericin-B (AmBisome®) is the drug of choice
3 mg/kg per day on days 1-5, day 14 and day 21
♦ Pentostam® is an alternative therapy
28 days of therapy is required
♦ Although AmBisome® is widely available,

44. J Med Entomol May;36(3):
Sandflies (Diptera: Psychodidae) associated with epidemic cutaneous leishmaniasis in Sanliurfa, Turkey.
Alptekin D, Kasap M, Luleyap U, Kasap H, Aksoy S, Wilson ML.
Department of Medical Biology, Faculty of Medicine, Cukurova University, Balcali, Adana, Turkey.
As part of a project to study the possible impact of environmental change on health in southeastern Turkey, we evaluated sandfly species diversity, abundance, and habitat associations in an urban area where cutaneous leishmaniasis was undergoing epidemic re-emergence. Houses and caves in and around the city of Sanliurfa, Turkey, were sampled using mechanical aspirators, sticky papers, and CDC light traps. Of 1,649 sandflies captured, including 6 Phlebotomus and 1 Sergentomyia species, nearly all were P. papatasi (Scopoli) (967) or P. sergenti Parrot (674). Sandflies were active during June-September (hot dry season), but not during January (cool rainy season). Resting phlebotomines were abundant inside houses. Houses sampled in 3 neighborhoods with a high cutaneous leishmaniasis incidence (9-65 cases per 1,000 population) had > 10 times more flies than at a comparison site where few cases (0.2 per 1,000) have been reported. Results indicated that P. sergenti or P. papatasi were the probable vectors of cutaneous leishmaniasis during this outbreak and that control of these sandflies may eliminate transmission.

45. BLOOD AND TISSUE PROTOZOONS
TOXOPLASMA

46. Toxoplasmosis Agent: Toxoplasma gondii Taxonomy: Phylum Apicomplexa
Distribution: Worldwide
Definitive Host: Any member of the Felidae
Intermed. Hosts: Any warm blooded animal
Invertebrates: Coprophagous (“feces-eating”)
Insects can be transport hosts

47. Human toxoplasmosis Inapparent or mild flu-like illness (most cases)
Fetal death and mental retardation,
blindness,
Epilepsy…. May not manifest for years
~ congenital infections/year
Ocular toxoplasmosis
Severe encephalitis in
İmmunocompromised persons.

48. Life Stages of Toxoplasma
• Tachyzoites = proliferative form in blood or CSF,acute or recurrent
• Bradyzoites = lifelong “tissue cysts”, any host
• Oocysts = (with sporozoites) shed in feces after completion of sexual phase in feline gut epithelium . Infectious after 48 hours or more environmental incubation.
Oocysts
Survive months to years despite freezing, heat, dehydration
Thousands-to-millions shed per cat
Oocysts are shed from cats for 1-2 weeks only.
Only ~1% of cats are shedding oocysts at a given time

49. Life Cycle Toxoplasma

50. The only known definitive hosts for Toxoplasma gondii are members of family Felidae (domestic cats and their relatives). 
1-Unsporulated oocysts are shed in the cat’s feces
2- Although oocysts are usually only shed for 1-2 weeks, large numbers may be shed.  Oocysts take 1-5 days to sporulate in the environment and become infective.  Intermediate hosts in nature (including birds and rodents) become infected after ingesting soil, water or plant material contaminated with oocysts . 
3- Oocysts transform into tachyzoites shortly after ingestion.  These tachyzoites localize in neural and muscle tissue and develop into tissue cyst bradyzoites .
4-  Cats become infected after consuming intermediate hosts harboring tissue cysts .  Cats may also become infected directly by ingestion of sporulated oocysts.  Animals bred for human consumption and wild game may also become infected with tissue cysts after ingestion of sporulated oocysts in the environment . 
5-Humans can become infected by any of several routes:

51. Toxoplasma gondii
Toxoplasmosis. Toxoplasma gondii tachyzoites (Giemsa stain).

52. Tachysoids of t.gondii and macrophages

53. Bradysoits of Toxoplasma

54. Toxoplasma gondii
Tachyzoites are the motile, asexually reproducing form of the parasite. Unlike the bradyzoites, the free tachyzoites are usually efficiently cleared by the host's immune response, although some manage to infect cells and form bradyzoites, thus maintaining the infection.

55. Routes of Transmission of Toxoplasma
• Foodborne (third leading cause of all types)
• Waterborne
• Contaminated soil
• Transplacental
• Organ transplants
• Blood transfusion
• Laboratory accidents

56. Life Stages of T.gondii in Cats
Types A-E are sexual
phases, all completed
in feline gut epithelium.
Prepatent periods:
Tissue Cysts: 3-10 days
Oocysts: > 18 days
Tachyzoites: > 13 days

57. T. Gondii Oocysts Unsporulated TransmissionElectron
Microscopy - showing
sporocysts each
containing sporozoites

58. ♦ Accidental ingestion of contaminated cat
feces. For example,accidental touching of
hands to mouth after gardening, cleaning a
cat’s litter box, or touching anything that has
come into contactwith cat feces.
♦ Ingestion of raw or partly cooked meat,
especially pork, lamb, or venison, or by
touching hands to mouth after handling
undercooked meat.

59. Sources of T. gondii exposure
♦ Contamination of knives, utensils, cutting
boards and other foods that have had contact
with raw meat.
♦ Drinking water contaminated with Toxoplasma.
♦ Although extremely rare, by receiving an infected organ transplant.

60. Human Risks from Cat Contact
♦ Oocysts sporulate in 48 hours+ at room
temperatures.
♦ Most cats do not leave feces on their fur for two days,
so it is unlikely that humans become infected from
direct contact with cats themselves.
♦ Because cats usually exhibit no signs of illness while
passing oocysts, it is difficult to determine when a
particular cat's feces may be infectious to people or
other mammals.
♦ Most adult cats will not pass oocysts ever again after
recovering from an initial exposure to Toxoplasma.

61. Congenital Toxoplasmosis
♦ Manifestations of congenital toxoplasmosis may not
become apparent until the second or third decade of life.
♦ Serologic tests are used to diagnose acute infection in
pregnant women, but false-positive tests occur frequently,
therefore, serologic diagnosis must be confirmed at a
reference laboratory before treatment with potentially toxic
drugs should be considered.
Infant girl with T.gondii hydrocephalus

62. Congenital T. gondii Infections
Many false positives via some kits (8 brands)
• Send to reference lab for confirmation before treatment
• PCR of amniotic fluid useful for test confirmation/exclusion
• Pyrimethamine & sulfonamide for positive PCR-AF tests
• Spiramycin for negative PCR-AF tests

63. Toxoplasma encephalitis
The most frequent
cause of focal CNS
infections in AIDS
patients

64. Toxoplasma gondii and Schizophrenia
E. Fuller Torrey* and Robert H. Yolken†
*Stanley Medical Research Institute, Bethesda, Maryland, USA; (2003)
Recent epidemiologic studies indicate that infectious agents may
contributeto some cases of schizophrenia. In animals, infection with
Toxoplasmagondii can alter behavior and neurotransmitter function.
In humans, acuteinfection with T. gondii can produce psychotic
symptoms similar to thosedisplayed by persons with schizophrenia.
Since 1953, a total of 19 studies of T. gondii antibodies in persons
withschizophrenia and other severe psychiatric disorders and in
controls havebeen reported; 18 reported a higher percentage of
antibodies in theaffected persons; in 11 studies the difference was
statistically significant.
Two other studies found that exposure to cats in childhood
was a risk factor for the development of schizophrenia.

65. Toxoplasmosis
Treatment of toxoplasmosis in immunocompetent patients is usually unnecessary. In immunocompromised patients, the recommended treatment is a combination of pyrimethamine given at mg daily and trisulfapyrimidines given at 2-6 g daily, both for a month. This drug combination inhibits dihydrofolate reductase in T. gondii, preventing synthesis of DNA and protein.

66. Toxoplasmosis
In acutely infected pregnant women, the recommended treatment includes spiramycin if the fetus has not yet acquired toxoplasmosis.
Spiramycin is an antibiotic that localizes to the placenta and has been shown to reduce placental infection by 60%. It does have some teratogenic effects, which must be weighed against the risk of congenital infection. If the fetus is infected, the aforementioned drug combination is administered instead of spiramycin.