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

Taxonomy of Leishmania Phylum Order Family Genus Sarcomastigophora Kinetoplastida Trypanosomatidae Leishmania

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

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

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

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.

*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

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

Life cycle of Leishmania

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

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

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

Clinical Manifestation Variable - Incubation 3-100+ weeks Lowgrade fever Hepato-splenomegaly Bone marrow hyperplasia Anemia, Leucopenia & Cachexia Hypergammaglobulinnemia Proteinuria, Hematuria

Cutaineous leishmaniasis Cutaneous leıshmaniasis

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

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…)

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

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)

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.

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

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

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

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

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

Cutaineous leishmaniasis of the face.

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

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

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)

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

♦ 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. 

♦ 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.

♦ Jaundiced hands of a visceral leishmaniasis patient. 

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

Mucocutaineous Leishmaniasis

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.

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

Photograph provided courtesy of COL Donald Skillman

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

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

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

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

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)

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,

J Med Entomol. 1999 May;36(3):277-81. 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.

BLOOD AND TISSUE PROTOZOONS TOXOPLASMA

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

Human toxoplasmosis Inapparent or mild flu-like illness (most cases) Fetal death and mental retardation, blindness, Epilepsy…. May not manifest for years ~ 400-4000 congenital infections/year Ocular toxoplasmosis Severe encephalitis in İmmunocompromised persons.

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

Life Cycle Toxoplasma

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:

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

Tachysoids of t.gondii and macrophages

Bradysoits of Toxoplasma

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.

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

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

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

♦ 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.

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.

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.

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

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

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

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.

Toxoplasmosis Treatment of toxoplasmosis in immunocompetent patients is usually unnecessary. In immunocompromised patients, the recommended treatment is a combination of pyrimethamine given at 25-100 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.

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.