BLOOD AND TISSUE FLAGELLATES

Trypanosoma cruzi
 Etiologic agent of Chagas disease or American tryponosomiasis
 Group: Stercoraria
 Intracellular parasite
 Heavily infected cells: myocytes (myocardial tissue) and reticuloendothelial system
 Other tissues infected: skin, gonads, intestinal mucosa and placenta
 Vector: reduviid bugs (Triatoma, Panstrongylus, Rhodnius)
o These arthropods thrive under squalid house conditions such as thatched roofs and mud walls (poor rural areas)
 Reservoir hosts: domestic animals, armadillos, raccoons, rodents, marsupials, some primates
 Stages
o In humans- trypomastigotes found in the bloodstream, amastigotes in tissue cells
o Vector- amastigote, epimastigote and promastigote are found in midgut, trypomastigote found in hindgut
1. Amastigote
 Round/ovoid shape; 1.5-4 μm diameter
 Found in small groups of cyst like collections in tissues
2. Trypomastigote
 15-20 μm in length; posterior end is pointed
 Some are long and slender, others are short and stumpy
 Narrow undulating membrane with 2-3 undulations
 Single thread-like flagellum
 C-shaped in stained specimens
 U/S-shaped with a prominent kinetoplast (characteristic of specie)
 Infective stage: trypomastigotes
 Pathogenesis and Clinical Manifestations
o Life Cycle
 Reduviid bug takes a blood meal then it will pass the metacyclic trypomastigotes in
feces, then the trypomastigotes enter bite wound through mucosal membranes such
as conjunctiva.
 Trypomastigotes from reduviid bug entering the human will penetrate various cells
at bite wound site and inside the cells, they transform into amastigotes.
 Amastigotes multiply by binary fission in cells of infected tissues.
 Intracellular amastigotes transform into trypomastigotes, then burst out of the cell
and enters the bloodstream.
 Trypomastigotes can either enter the bloodstream wherein they are ready to replicate
again once they enter another cell, or they are ingested by an insect vector. They can
either be eaten when the reduviid bug takes a blood meal, or they can either stay or circulate in the bloodstream and wait to be
engulfed by the macrophages of the reticuloendothelial system and multiply again through binary fission as amastigotes.
 Once the trypomastigotes are ingested by the intermediate host, they will pass through the posterior portion of the insect’s midgut
and will then transform into epimastigotes, where they will multiply via longitudinal fission.
 Infective metacyclic trypomastigotes will then appear in the insect’s rectum 8 to 10 days after infection, and are passed in the
reduviid bug’s feces. The metacyclic trypomastigotes gain entry into the body through broken skin, or through mucous
membranes that are rubbed with fingers contaminated with the bug’s feces.
o Acute
 Focal or diffuse inflammation mainly affecting the myocardium
 Nonspecific signs and symptoms: fever, malaise, nausea, vomiting, generalized lymphadenopathy, cutaneous manifestations (may
also be present during this phase and is usually associated with a localized inflammatory reaction at or near the site of inoculation)
 Chagomas- furuncle like lesions associated with induration, central edema, regional lymphadenopathy
 This lesion represents site of entry of parasite
 Romana’s sign- eyelid swelling caused by the parasite penetrating through the conjunctiva.
 It is characterized by unilateral painless bipalpebral edema and conjunctivitis and may involve the lacrimal gland
and surrounding lymph nodes.
 After 1 or 2 months, symptoms resolve, and the patient goes into a latent or indeterminate phase.
o Latent/Indeterminate
 Usually asymptomatic in this stage
 Still capable of transmitting through insect vectors, blood transfusion, and organ transplantation.
 About 1/3 develop to chronic stage
o Chronic
 Multifactorial and dependent on the interaction between parasite and host; megasyndrome.
 Fibrotic reactions that cause injury to the myocardium, cardiac conduction network, and enteric nervous system.
 Heart: primary affected organ, may lead to cardiomegaly, congestive heart failure (CHF), thromboembolism, and arrhythmias.
 Less severe signs and symptoms: chest pain, palipations, dizziness, syncope, abnormal ECG findings, achalasia (associated with
megaesophagus), and chronic constipation (megacolon)
 The majority of symptomatic, chronic patients manifest with the cardiac form, while the rest develop the gastrointestinal form.
o Diagnosis
 A complete patient history is the primary tool for diagnosing Chagas disease. Possible exposure to T. cruzi should be established,
and risk factors such as place of residence or work, recent blood transfusion in an endemic area, and contact or exposure to T. cruzi
intermediate host should be evaluated.
 Acute
 Definitive diagnosis: direct visualization of the parasites in thick and thin blood smears using Giemsa stain.
 CSF, tissue samples, lymph nodes can also be used for parasite visualization
 Trypomastigotes can only be detected in the first 2 months of acute disease by direct examination.
 Other diagnostic techniques: Concentration methods (microhematocrit), Blood culture, PCR, Xenodiagnosis, wherein
laboratory-reared triatomine bugs are allowed to feed on suspected patients and are later examined for the presence of T. cruzi
metacyclic trypomastigotes
 Chronic (serologic tests)
 Serologic tests like ELISA, Indirect hemagglutination, Indirect immunofluorescence, and PCR
 recommends at least 2 techniques show a positive result
 Cardiac form- ECG and echocardiography
 GI form- barium esophagogram (detects esophageal dilatation), barium enema (detects megacolon of the sigmoid and rectum)
o Treatment
 Two drugs are recommended for the treatment of acute phase: Nifurtimox and Benznidazole
 Symptom-specific management is used to treat patients with chronic manifestations.
 Cardiac manifestations are controlled by pacemakers and antiarrhythmic drugs, such as amiodarone.
 Megasyndromes are managed with special diets, laxatives, and surgical procedures.
o Epidemiology
 Estimated to have infected more than 10M people worldwide
 Most cases in Latin America; Endemic areas include Central and South America
 In 2003, ranked 3rd as the leading cause of parasitic infection worldwide
o Prevention and Control
 Vector control and blood transfusion regulations
 Spraying of insecticides, use of insecticide-treated bed nets, and house improvements to prevent vector infestation have been proven
cost-effective.

Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense

 They are known to cause human African trypanosomiasis (HAT), also known as African sleeping sickness, a highly fatal disease
caused by two subspecies of Trypanosoma brucei: T. brucei gambiense and T. brucei rhodesiense.
 A third subspecies, T. brucei brucei- affects wild and domestic animals; collectively, the three subspecies represent the Trypanosoma
brucei complex.
 Primarily in sub-Saharan Africa
 Family: Salivaria
 Vector: tsetse fly
 Mode of Transmission
o Bite of the blood sucking tsetse fly (Glossina spp.) feeding from an infected mammalian host– most common,
o Mechanical methods (needle pricks)
o Vertical transmission (mother to child via placenta)
T. brucei gambiense T. brucei rhodesiense
Western and central region of sub-Saharan Africa East Africa
Primarily affects humans but utilizes dogs, pigs and Primarily a zoonosis of cattle and wild animals
sheep as reservoir hosts Humans are accidental hosts
Chronic type of sleeping sickness (months/years) More rapid and acute fatal form of sleeping sickness (weeks)
95% cases of HAT 5% cases of HAT
 Stages
o Only the epimastigote and trypomastigote forms are exhibited by the T. brucei complex.
o Trypomastigote
 Polymorphic: slender forms, and short, stumpy forms. Flattened and fusiform in shape
 14-33 μm length, 1.5-3.5 μm width
 Body tapers anteriorly, blunt posteriorly
 Central nucleus with large central karyosome
 Undulating membrane
 Single flagellum runs along the edge of the undulating membrane and becomes free anteriorly
 Infective stage: metacyclic trypomastigote
 Diagnostic stage: trypomastigote
 Pathogenesis and Clinical Manifestations
o Life Cycle
 Once trypomastigotes are ingested by the intermediate host (tsetse
fly). They will undergo several developmental changes from
trypomastigote into procyclic forms in the insect’s midgut.
 Tsetse fly takes a blood meal, trypomastigotes are ingested, and
transforming into procyclic trypomastigotes in the vector’s
midgut which will multiply by binary fission.
 Procyclic trypomastigotes will then leave the midgut and
transform into epimastigotes. The epimastigotes will multiply in
the salivary gland of the vector and will transform into metacyclic
trypomastigotes. Once the tsetse fly takes another blood meal,
the metacyclic trypomastigotes is then injected into the human.
 In humans (Trypanosoma brucei), trypomastigotes will transform
into bloodstream trypomastigotes which are carried to other sites
of the body. They usually stay in the reticular tissue of lymph and spleen, and in the CSF. They multiply by binary fission in various
body fluids and circulating trypomastigotes in blood during acute phase. They can keep circulating within the bloodstream unt il
they are then eaten by the tsetse fly once the tsetse fly takes another blood meal and the cycle will repeat itself.
o Human African trypanosomiasis has two types, acute and chronic, depending on the subspecies causing the disease.
o Trypanosoma brucei gambiense sleeping sickness manifests months or years after initial infection, while symptoms of Trypanosoma
brucei rhodesiense sleeping sickness may appear just weeks after infection.
o Chancre- initial lesion of African trypanosomiasis which begins as a local, painful, pruritic, erythematous lesion located at the bite
site, progressing into a central eschar, and resolving after 2 to 3 weeks. More common in Gambian (Trypanosoma brucei gambiense)
sleeping sickness. 3 to 10 days after the development of the chancre, the next stages of the disease would manifest.
o Early Phase (Hemolymphatic) (both gambiense and rhodesiense has early and late phase)
 lasts for 1-6 months
 Due to tissue damage from paratoxins or immune complex reactions
 Proliferate in the bloodstream and lymphatics
 Irregular bouts of fever, headache, joint and muscle pain, malaise
 Anemia, myocardial inflammation, DIC, renal insufficiency may also occur
 Axillary and supraclavicular lymph node involvement present in both gambiense and rhodesiense
 Winterbottom’s sign- for T. gambiense – posterior cervical lymph nodes are enlarged, nontender, rubbery in consistency
o Late Phase (Meningoencephalitic)
 Involvement of CNS (brain and meninges) as the parasites find their way to the CNS through the bloodstream
 T. gambiense- occurs 3-10 months after initial infection; T. rhodesiense- manifest after few weeks
 Signs and Symptoms: apathy, behavioral changes, headache, sleep pattern changes, convulsions, tremors, speech defects, speech
and reflex disturbances, paralysis
 Kerandel’s sign- deep, delayed hyperesthesia (delayed bilateral pain out of proportion to the extent of tissue injury)
 Later stages: somnolence➜ deep coma➜ death
 CNS areas involved: frontal lobes, pons, medulla, perivascular area
 Antigenic variation- ability of trypanosomes to continuously change its surface coat so that the host’s antibodies can’t recognize
the parasite in subsequent waves of parasitemia
o Diagnosis
 Demonstration of highly motile trypomastigotes in fluid from chancre, LN aspirate, CSF
 Giemsa stain for thin and thick blood film
 Buffy coat concentration recommended to detect parasites in low numbers
 Examination for trypomastigotes is usually done during the hemolymphatic stage; more useful for the diagnosis of T. brucei
rhodesiense due the relative higher levels of parasitemia with rhodesiense.
 Other diagnostic techniques: ELISA, immunofluorescence (IF), indirect hemagglutination test, mini-anion exchange
centrifugation technique, PCR
 CSF examination- mandatory to detect CNS involvement
 Abnormal CSF findings include: increase in cell count, opening pressure, protein concentration and IgM levels
 Card agglutination test (CATT) for trypanosomiasis- detect circulating antigens in persons infected with T. brucei complex
 Available commercially, can be used in the field setting to screen at-risk population
 Rapid and highly specific method for screening
 Low sensitivity for certain strains of T. gambiense in Western Africa
o Treatment
 Early Phase
 Intravenous Suramin sodium- T. gambiense/ rhodesiense; Side effects: fever, rash, renal insufficiency, muscle pain
 Intramuscular pentamidine- T. gambiense; Side effects: tachycardia, hypotension, hypoglycemia
 both do not cross the blood-brain barrier- not used for late phase of disease
  Late Phase
 Intravenous melarsoprol- drug of choice for both types of sleeping sickness during late phase
 Nitrofurazone- 2nd line drug; used in cases of melarsoprol treatment failure.
 Eflornithine- may also be used during early phase, less toxic than melarsoprol, only effective against T. gambiense
o Epidemiology
 Affects around 300,000-500,000 people in 36 countries in sub-Saharan Africa
 Tsetse flies live near the banks of rivers and streams, therefore transmission can readily occur when people frequent these areas to
swim and do their laundry
 T. rhodesiense is an occupational hazard for persons working in game reserves/game parks
 Cattle and antelopes can serve as reservoir hosts
o Prevention and control
 Vector control (primary method)
 Tsetse fly trapping (main strategy to decrease vector population)
 Insecticides, protective clothing (recommended to prevent contact with the insect vector)
 Regulation and treatment of reservoir hosts such as cattle and game animals are also being looked upon as an effective means of
preventing disease transmission.
Leishmania spp.
 It causes an infection known as leishmaniasis.
 Divided into 2 subgenera, differentiated from one another by the location of their development inside the
insect vector and the area which they are endemic.
 Currently, about 15 species of leishmania that causes clinical manifestations in humans.
Old world New world
L. tropica (Asia, Eastern Europe) Mexico, Central America, South America, Amazon rain forest
L. aethiopica (Africa) L. Mexicana, L. amazonensis, L. guyanensis, L. braziliensis, L. chagasi
L. major
Arthropod: sandfly (Phlebotomus) Arthropod: sandfly (Lutzomyia)
 Vector: Sandfly
 Dogs- primary reservoir in urban areas; Rodents- reservoir in both urban and rural areas
 Stages
o Leishmania spp. produces amastigotes (intracellularly) and promastigotes (hindgut, midgut and proboscis of insect vectors)
1. Amastigotes
 Ovoid/rounded bodies; 2-3 μm length
 Lives intracellularly in monocytes, polymorphonuclear (PMN) leukocytes, or endothelial cells
 Nucleus is large; Axoneme arises from kinetoplast and extends to anterior tip
2. Promastigotes
 Single free flagellum arising from the kinetoplast at the anterior end
 15-20 μm length, 1.5-3.5 μm width
 It depends on the subgenera. It is produced in the hindgut (Viannia subgenus), midgut (Vianna and Leishmania subgenera), and
proboscis (Vianna and Leishmania subgenera) of insect vectors.
 Infective stage: Promastigotes (human), amastigotes (sandfly)
 Pathogenesis and Clinical Manifestations
o Life Cycle
 Promastigotes present in the proboscis of the sandfly are then injected into the host’s skin
during feeding.
 Once a sandfly takes a blood meal, the promastigotes are then injected into the
bloodstream.
 The promastigotes are phagocytized by macrophages or other types of mononuclear
phagocytic cells. They would invade the cells of the reticuloendothelial system and will
transform into amastigotes.
 Amastigotes will multiply in cells of various tissues and infect other cells. When the
parasitized cell ruptures, the amastigotes released can either invade new cells or they
can be taken out by the sandfly during breathing.
 When the sandfly takes a blood meal, it ingests macrophages infected with amastigotes.
 Amastigotes in the sandfly will transform into promastigote stage in the gut which will then divide and migrate to the proboscis of
the sandfly and the sandfly would take a blood meal and the cycle repeats itself.
o Clinically, leishmaniasis can be divided into four categories
A. Cutaneous Leishmaniasis (most common)
 Caused by several species of Leishmania, including L. tropica (dry or urban oriental sore), L. major (moist
or rural oriental sore), and L. mexicana (chiclero ulcer, usually affecting the ears).
 Incubation period: 2 weeks to several months
 Oriental button- erythematous papule or nodule that is produced at the inoculation site (left picture).
 The lesion has raised edges and a central crater.
  During the course of several weeks, the papule forms a violaceous ulcer as it enlarges in size (right picture). This may heal after a
few months, leading to a disfiguring scar
 In the case of New World leishmaniasis, cutaneous form may progress to other forms of leishmaniasis.
B. Diffuse Cutaneous Leishmaniasis
 Also known as anergic or lepromatous leishmaniasis.
 Localized, non-ulcerating papule, eventually developing numerous diffuse satellite lesions that affect the face and
extremities.
 This type of leishmaniasis may be initially diagnosed as lepromatous leprosy.
C. Mucocutaneous Leishmaniasis
 Develops in 2-5% of people infected with L. braziliensis
 May also be due to spread of cutaneous type caused by L. tropica
 Involvement of the mucous membranes of the nasal and oral cavities results in nasal stuffiness, discharge, epistaxis, destruction of
nasal septum. This disfiguration is often called espundia.
 Progression into the pharynx and larynx may threaten the airway passage, and may lead to dysphonia, dysphagia, aspiration
pneumonia
 Lesions usually manifest with few parasites.
 Systemic Th1 response is strong, increased levels of peripheral mononuclear cells in blood.
D. Visceral Leishmaniasis (“kala azar”)
 Disseminated parasitosis caused by L. donovani complex: L. donovani, chagasi and infantum
 Incubation period: 2-8months
 Spread of parasites in bone marrow, spleen, liver
 Acute- fever with chills
 Subacute and chronic- fever, weakness, loss of appetite, weight loss, hemorrhage, abdominal enlargement associated with
hepatosplenomegaly
 Phagocytosed amastigotes present in small amounts in blood; numerous in reticuloendothelial cells of spleen, liver, lymph nodes,
bone marrow, intestinal mucosa, and other organs
 Th1 response low or absent
 Untreated: >95% mortality rate, amastigotes accumulating in the tissue cells\
 Post-kala azar dermal leishmaniasis (PKDL)- sequela of visceral leishmaniasis seen in endemic areas
 Cutaneous eruption➜ hypopigmented macules, malar erythema, nodules & ulcerations
 These lesions usually manifest a few months to several years after treatment.
 Diagnosis
o Microscopic demonstration of Leishmania (amastigotes) from lesion and tissue scrapings, aspirate or biopsy
 Giemsa and H&E stains often used are often used in microscopic and histologic samples, and the demonstration of amastigotes
confirms the diagnosis of leishmaniasis
o Cultures- unreliable due to the difficulty of isolating the parasites
o Leishmanin skin test (Montenegro skin test)- used to identify exposure to the parasite
 (+) in Cutaneous Leishmaniasis and Mucocutaneous Leishmaniasis and (-) in Diffuse Cutaneous Leishmaniasis and kala-azar
o ELISA, rk39 antigen dipstick test- high specificity and sensitivity for Visceral Leishmaniasis
o Direct agglutination, urine antigen assays, flow cytometry, and molecular diagnostic modalities (PCR, RFLP analysis)
 Treatment
o Sodium stibogluconate and N-methyl-glucamine- primary pharmacologic treatment
 Requires daily intramuscular or intravenous administration for up to 4 weeks, and hospital confinements are necessary.
o Amphotericin B- used in cases of treatment failure of the first two drugs especially in patients with AIDS, areas with high resistance;
high cure rate
 Side effects: abdominal pain, nausea, arthralgia, and even fatal arrhythmias
o Miltefosine- is the only oral drug currently given to Visceral Leishmaniasis patients.
o Pentamidine- 2nd line for Cutaneous Leishmaniasis and Visceral Leishmaniasis
o Topical paromomycin- has shown efficacy in certain areas or in patients with Cutaneous Leishmaniasis
 Epidemiology
o Global disease, 88 countries in four continents, affects more than 12 million people worldwide, and more than 350 million are at risk
for the disease
o Primarily a disease of poverty
o Affects people living in squalid conditions- associated with poor housing, malnutrition, weak immune system, lack of resources
o Visceral Leishmaniasis is an opportunistic infection in AIDS patients (Visceral Leishmaniasis and HIV co-infection)➜ accelerates
HIV replications and progression to AIDS
o In the Philippines, there have been imported cases of cutaneous lesions referred to the University of the Philippines-College of Public
Health, where amastigotes were identified from the patients.
 Prevention and control
o Insect repellants containing DEET and permethrin, insecticide treated clothing, fine-mesh bed nets
o Regulation of reservoir hosts (insecticide treated dog collars, mass testing of domestic dogs).