those caused

by P. falciparum, with fatal.

Laboratory diagnosis (ALL SPECIES):

 Examination of a single blood specimen is not sufficient to exclude the diagnosis of malaria, especially when the

patient has received partial prophylaxis or therapy and has a low number of organisms in the blood. Patients with a

relapse case or an early primary case may also have few organisms in the blood smear. Regardless of the presence or

absence of any fever periodicity, both thick (Figure 23) and thin blood films should be prepared immediately, and at

least 200 to 300 oil immersion fields should be examined on both films before a negative report is issued. If the

initial specimen is negative, additional blood specimens should be examined over a 36-hour time frame.

 Although Giemsa stain is recommended for all parasitic blood work, the organisms can also be seen with other

blood stains, such as Wright’s stain. Using any of the blood stains, the white blood cells (WBCs) serve as the builtin quality control; if the WBCs look good, any parasites present will also look good. compares the multinucleated

stages (schizont) of Plasmodium malariae and Plasmodium vivax. Fluorescent nucleic acid stains, such as acridine

orange, may also be used to identify organisms in infected RBCs. However, this may be more difficult to interpret

because of the presence of white blood cell nuclei or RBC Howell-Jolly bodies.

Figure 22 Plasmodium falciparum. A, Ring forms; B, oocyte; and C, sporozoites.

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Serologic Methods:

 Several rapid malaria tests (RMTs) are now commercially available.

Molecular Diagnostics:

Other methods include direct detection of the five species by using a specific DNA probe after PCR amplification

of target DNA sequences.

Therapy:

Antimalarial drugs are classified according to the stage of malaria against which they are targeted. These drugs

are referred to as tissue schizonticides (which kill tissue schizonts), blood schizonticides (which kill blood

schizonts), gametocytocides (which kill gametocytes), and sporonticides (which prevent formation of sporozoites

within the mosquito). It is important for the clinician to know the species of Plasmodium involved in the

infection, the estimated parasitemia, and the geographic and patient travel history to assess the possibility of drug

resistance related to the organism and geographic area.

Babesia spp.:

 The genus Babesia includes approximately 100 species transmitted by ticks of the genus Ixodes. In addition to

humans, these blood parasites infect a variety of wild and domestic animals.

General characteristics:

 Organism Although the life cycle of Babesia spp. is similar to that of Plasmodium spp., no exoerythrocytic

stage has been described; also, sporozoites injected by the bite of an infected tick invade erythrocytes directly.

Once inside the erythrocytes, the trophozoites reproduce by binary fission rather than schizogony. Once the tick

begins to take a bloo meal; the sporozoites are injected into the host with the tick’s saliva.

Figure 23 A, Plasmodium malariae schizont. B, Plasmodium viax schizon

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The trophozoites of Babesia can mimic P. falciparum rings; however, there are differences that can help

differentiate the two organisms (Figure 24). Babesia trophozoites vary in size from 1 to 5 μm; the smallest are

smaller than P. falciparum rings. Also, ring forms outside of the RBCs and two to three rings per RBC are much

more common in Babesia. The ring forms of Babesia tend to be very pleomorphic and range in size, even within

a single RBC. The diagnostic tetrads, the Maltese Cross, though not seen in every specimen or species, may be

present (see Figure 24).

Pathogenesis and spectrum of disease:

 Babesiosis is clinically similar to malaria, and symptoms include high fever, myalgias, malaise, fatigue,

hepatosplenomegaly, and anemia.

Laboratory diagnosis:

 Examination of thick and thin stained blood films is the most direct approach t diagnosis.

Molecular Diagnostics:

Although rare, molecular methods such as PCR are available in some laboratories.

Therapy:

Mild cases caused by B. microti usually resolve spontaneously, and in more serious cases, treatment with

clindamycin and quinine or atovaquone and azithromycin is used.

Prevention:

 Personal protective measures, such as long pants, longsleeved shirts, and insect repellant, may reduce the riskof

infection when outdoors in endemic areas for the tick vectors.

Figure 24 Babesia in red blood cells.

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Trypanosoma spp.:

 Trypanosoma spp. are hemoflagellate protozoa that live in the blood and tissue of the human host ( Figures 25).

African trypanosomiasis:

The primary area of endemic infection with T. brucei gambiense (West African trypanosomiasis) coincides with

the vector tsetse fly belt through the heart of Africa, where 300,000 to 500,000 people may be infected in Western

and Central Africa. T. brucei rhodesiense (which causes Rhodesian trypanosomiasis or East African sleeping

sickness) is more limited in distribution than T. brucei gambiense, being found only in central East Africa, where

the disease has been responsible for some of the most serious obstacles to economic and social development of

Africa. Within this area, the tsetse flies prefer animal blood, which therefore limits the raising of livestock. The

infection in humans has a greater morbidity and mortality than does T. brucei gambiense infection, and game

animals, such as the bushbuck, and cattle are natural reservoir hosts.

A unique feature of African trypanosomes is their ability to change the antigenic surface coat of the outer

membrane of the trypomastigote, helping to evade the host immune response. The trypomastigote surface is

Figure 25Trypanosoma cruzi trypomastigote

Figure 26 A, Trypanosoma cruzi in blood film (1600×). B, Trypanosoma cruzi parasites in cardiac muscle (2500×).

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covered with a dense coat of variant surface glycoprotein (VSG). There are approximately 100 to 1000 genes in

the genome, responsible for encoding as many as 1000 different VSGs. More than 100 serotypes have been

detected in a single infection. It is postulated that the trypomastigote changes its antigenic coat about every 5 to 7

days (antigenic variation). This change is responsible for successive waves of parasitemia every 7 to 14 days and

allows the parasite to evade the host humoral immune response.

Each time the antigenic coat changes, the host does not recognize the organism and must mount a new

immunologic response. The sustained high immunoglobulin M (IgM) levels are a result of the parasite producing

variable antigen types, and in an immunocompetent host, the absence of elevated IgM levels in serum rules out

trypanosomiasis.

General Characteristics:

Trypanosomal forms are ingested by the tsetse fly (Glossina spp.) when a blood meal is taken. The organisms

multiply in the lumen of the midgut and hindgut of the fly. After approximately 2 weeks, the organisms migrate

back to salivary glands where the organisms attach to the epithelial cells of the salivary ducts and then transform to

their epimastigote forms. Multiplication continues within the salivary gland, and metacyclic (infective) forms

develop from the epimastigotes in 2 to 5 days.

While feeding, the fly introduces the metacyclic trypanosomal forms into the next victim in saliva injected into the

puncture wound. The entire developmental cycle in the fly takes about 3 weeks, and once infected, the tsetse fly

remains infected for life.

In fresh blood, the trypanosomes move rapidly among the red blood cells. An undulating membrane and flagellum

may be seen with slower moving organisms. The trypomastigote forms are 14 to 33 μm long and 1.5 to 3.5 μm

wide , With a blood stain, the granular cytoplasm stains pale blue. The centrally located nucleus stains reddish. At

the posterior end of the organism is the kinetoplast, which also stains reddish, and the remaining intracytoplasmic

flagellum (axoneme), which may not be noticeable. The flagellum arises from the kinetoplast, as does the

undulating membrane.

The flagellum runs along the edge of the undulating membrane until the undulating membrane merges with the

trypanosome body at the anterior end of the organism. At this point, the flagellum becomes free to extend beyond

the body.

Pathogenesis and Spectrum of Disease:

 Trypanosoma brucei gambiense. African trypanosomiasis caused by T. brucei gambiense (West African sleeping

sickness) has a long, mild, chronic course that ends in death with central nervous system (CNS) involvement after

several years’ duration. This is unlike the disease caused by T. brucei rhodesiense (East African sleeping

sickness), which has a short course and ends fatally within 1 year. After the host has been bitten by an infected

tsetse fly, a nodule or chancre at the site may develop after a few days. Usually, this primary lesion will resolve

spontaneously within 1 to 2 weeks, and is rarely seen in patients living in an endemic area. Trypomastigotes may

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be detected in fluid aspirated from the ulcer. The trypomastigotes enter the bloodstream, causing a low-grade

parasitemia that may continue for months with the patient remaining asymptomatic. This is considered stage I

disease, where the patient can have systemic trypanosomiasis without CNS involvement. During this time,

theparasites may be difficult to detect, even by thick blood film examinations. The infection may self-cure during

this period without development of symptoms or lymph node invasion. Symptoms may occur months to years after

infection. When the lymph nodes are invaded, the first symptoms appear and include remittent, irregular fevers

with night sweats. Headaches, malaise, and anorexia may also be present. The febrile periods of up to 1 week

alternate with afebrile periods of variable duration. Many trypomastigotes may be found in the circulating blood

during fevers, but few are seen during afebrile periods. Lymphadenopathy is a consistent feature of Gambian

trypanosomiasis, and the enlarged lymph nodes are soft and painless. In addition to lymph node involvement, the

spleen and liver become enlarged. With Gambian trypanosomiasis, the blood lymphatic stage may last for years

before the sleeping sickness syndrome occurs. When the organisms finally invade the CNS, the sleeping sickness

stage of the infection is initiated (stage II disease). Behavioral and personality changes are seen during CNS

invasion. This stage of the disease is characterized by steady progressive meningoencephalitis, apathy, confusion,

fatigue, loss of coordination, and somnolence (state of drowsiness). In the terminal phase of the disease, the patient

becomes emaciated and progresses to profound coma and death, usually from secondary infection. Thus, the

typical signs of true sleeping sickness are seen in patients with Gambian disease.

Trypanosoma brucei rhodesiense. T. brucei rhodesiense produces a more rapid, fulminating disease than does T.

brucei gambiense. Fever, severe headaches, irritability, extreme fatigue, swollen lymph nodes, and aching muscles

and joints are typical symptoms. Progressive confusion, personality changes, slurred speech, seizures, and difficulty in

walking and talking occur as the organisms invade the CNS. The early stages of the infection are like those of T.

brucei gambiense infections. However, CNS invasion occurs early, the disease progresses more rapidly, and death

may occur before there is extensive CNS involvement. The incubation period is short, often within 1 to 4 weeks,

with trypomastigotes being more numerous and appearing earlier in the blood. Lymph node involvement is less

pronounced. Febrile episodes are more frequent, and the patients are more anemic and more likely to develop

myocarditis or jaundice. Some patients may develop persistent tachycardia, and death may result from arrhythmia and

congestive heart failure. Myocarditis may develop in patients with Gambian trypanosomiasisbut is more common and

severe with the Rhodesian form.

Laboratory Diagnosis (All Species):

Routine Methods. Blood can be collected from either finger stick or venipuncture (use EDTA anticoagulant).

Multiple thick and thin blood films should be made for examination, and multiple blood examinations should be

done before trypanosomiasis is ruled out. Parasites will be found in large numbers in the blood during the febrile

period and in small numbers when the patient is afebrile. In addition to thin and thick blood films, a buffy coat

concentration method is recommended to detect the parasites. Parasites can be detected on thin blood films with a

detection limit at approximately 1 parasite/200 microscopic fields (high dry power magnification, ×400) and thick

blood smears when the numbers are greater than 2000/mL, and when they are greater than 100/mL with hematocrit

capillary tube concentration.

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Antigen Detection. A simple and rapid test, the card

indirect agglutination trypanosomiasis test (TrypTect CIATT), is available, primarily in areas of endemic

infection, for the detection of circulating antigens in persons with African trypanosomiasis. The sensitivity of the

test (95.8% for T. brucei gambiense and 97.7% for T. brucei rhodesiense) is significantly higher than those for

lymph node puncture, micro hematocrit centrifugation, and cerebrospinal fluid examination (CSF) after single and

double centrifugation. Its specificity is excellent, and it has a high positive predictive value.

Antibody Detection. Serologic techniques that have been widely used for epidemiologic screening include

indirect fluorescent antibody assays (enzyme-linked immunosorbent assay [ELISA]), the indirect

hemagglutination test, and the card agglutination trypanosomiasis test. A major problem in endemic areas is that

individuals have elevated antibody levels attributable to exposure to animal trypanosomes that are noninfectious to

humans.

Serumand CSF IgM concentrations are of diagnostic value. However, CSF antibody titers should be interpreted

with caution because of the lack of reference values and the possibility that the CSF will contain serum as the

result of a traumatic tap.

Molecular Diagnostics:

Referral laboratories have used molecular methods to detect infections and differentiate species, but these

methods are not routinely used in the field.

Therapy:

All drugs used in the therapy of African trypanosomiasis are toxic and require prolonged administration.

Treatment should be started as soon as possible, and the antiparasitic drug selected depends on whether the CNS

is infected.

American trypanosomiasis:

 American trypanosomiasis (Chagas’ disease) is a zoonosis occurring throughout the American continent and

involves reduviid bugs/kissing bugs (vectors) living in close association with human reservoirs (dogs, cats,

armadillos, opossums, raccoons, and rodents). Transmission to humans depends on the defecation habits of the

insect vector.

humans vary with the geographic area. A very serious problem is disease acquisition through blood transfusion

and organ transplantation. A large number of patients with positive serologic results can remain asymptomatic.

Patients can present with either acute or chronic disease.

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Trypanosoma cruzi:

General Characteristics:

 Trypomastigotes are ingested by the bug as it obtains a blood meal. The trypomastigotes transform into

epimastigotes that multiply in the posterior portion of the bug’s midgut. After 8 to 10 days, trypomastigotes

develop from the epimastigotes. Humans contract Chagas’ disease when the bug defecates while taking a blood

meal and the parasites in the feces are rubbed or scratched into the bite wound or onto mucosal surfaces.

In humans, T. cruzi is found in two forms: amastigotes and trypomastigotes ,The trypomastigote form is present in

the blood and infects the host cells. The amastigote form multiplies within the cell, eventually destroying the cell,

and both amastigotes and trypomastigotes are released into the blood.

 The trypomastigote is approximately 20 μm long, and it usually assumes a C or U shape in stained blood films.

Trypomastigotes occur in the blood in two forms: a long slender form and a short stubby one. The nucleus is

situated in the center of the body, with a large oval kinetoplast located at the posterior extremity. A flagellum

arises from the kinetoplast and extends along the outer edge of an undulating membrane until it reaches the

anterior end of the body, where it projects as a free flagellum. When the trypomastigotes are stained with any of

the blood stains, the cytoplasm stains blue and the nucleus, kinetoplast, and flagellum stain red or violet.

When the trypomastigote penetrates a cell, it loses its flagellum and undulating membrane and divides by binary

fission to form an amastigote .

The amastigote continues to divide and eventually fills and destroys the infected cell. Both amastigote and

trypomastigote forms are released from the cell. The amastigote is indistinguishable from those found in

leishmanial infections. It is 2 to 6 μm in diameter and contains a large nucleus and rod-shaped kinetoplast that

stains red or violet with blood stains. The cytoplasm stains blue. Only the trypomastigotes are found free in the

peripheral blood.

Pathogenesis and Spectrum of Disease:

The clinical stages associated with Chagas’ disease are categorized as acute, indeterminate, and chronic. The acute

stage represents the initial encounter of the patient with the parasite, whereas the chronic phase is the result of late

sequelae. In children under the age of 5, the disease is seen in its acute form, whereas in older children and adults,

the disease is milder and is commonly diagnosed in the subacute or chronic form. The incubation period in

humans is about 7 to 14 days but is somewhat longer in some patients.

Acute symptoms occur 2 to 3 weeks after infection and include high fevers, enlarged spleen and liver, myalgia,

erythematous rash, acute myocarditis, lymphadenopathy, keratitis, and subcutaneous edema of the face, legs, and

feet. There may be symptoms of CN involvement, which carry a very poor prognosis. Myocarditis is confirmed by

electrocardiographi changes, tachycardia, chest pain, and weakness. Amastigotes proliferate within the cardiac

muscle cells and destroy the cells, leading to conduction defects and a loss of heart contractility (see Figure 26).

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Death may occur due to myocardial insufficiency or cardiac arrest. In infants and very young children, swelling of

the brain can develop, causing death.

The chronic stage may be initially asymptomatic (indeterminate stage), and even though parasites are rarely seen

in blood films, transmission by blood transfusion is a serious problem in endemic areas. Chronic Chagas’ disease

may develop years after undetected infection or after the diagnosis of acute disease. Approximately 30% of

patients may develop chronic Chagas’ disease, including cardiac changes and enlargement of the colon and

esophagus. Megacolon results in constipation, abdominal pain, and the inability to discharge feces. There may be

acute obstruction leading to perforation, septicemia, and death. However, the most frequent clinical signs of

chronic Chagas’ disease involve the heart, where enlargement of the heart and conduction changes are commonly

seen.

Laboratory Diagnosis:

Routine Methods. Trypomastigotes may be detected in blood by using thick and thin blood films or the buffy coat

concentration technique. Any of the blood stains can be used for both amastigote and trypomastigote stages.

Molecular Diagnostics. Referral laboratories have used molecular methods to detect infections with as few as one

trypomastigote in 20 mL of blood, but these methods are not routinely used in the field.

Antigen Detection. Immunoassays have been used to detect antigens in urine and sera in patients with congenital

infections and those with chronic Chagas’ disease.

Antigen detection can also be valuable for early diagnosis and for diagnosis of chronic cases in patients with

conflicting serologic test results.

Antibody Detection. Serologic tests for antibody detection include complement fixation, indirect fluorescent

antibody, indirect hemagglutination tests, and ELISA.

Histology. In tissue, amastigotes can be differentiated from fungal organisms because they will not stain positive

with periodic acid-Schiff, mucicarmine, or silver stains.

Therapy:

Nifurtimox (Lampit) and benznidazole (Radamil) reduce the severity of acute Chagas’ disease. In some cases,

surgery has been successfully used to treat cases of chagasic heart disease, megaesophagus, and megacolon.

Leishmania spp:.

Leishmaniasis is caused by more than 20 species of the protozoan genus Leishmania, with a disease spectrum

ranging from self-healing cutaneous lesions to debilitating mucocutaneous infections, subclinical viscerotropic

dissemination, and fatal visceral involvement. Publisheddisease burden estimates place leishmaniasis second in

mortality and fourth in morbidity among all tropical diseases.

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General characteristics:

 The parasite has two distinct phases in its life cycle: amastigoteand promastigote ( Figure27 ). The amastigote

form is an intracellular parasite in the cells of the reticuloendothelial system and is oval, measuring 1.5 to 5 μm,

and contains a nucleus and kinetoplast. Leishmania spp. exist as the amastigote in humans and as the

promastigote in the insect host. As the vector takes a blood meal, promastigotes are introduced into the human

host. Depending on the species, the parasites then move from the bite site to the organs within the

reticuloendothelial system (bone marrow, spleen, liver) or to the macrophages of the skin or mucous

membranes.Depending on the species involved, infection with Leishmania spp. can result in cutaneous, diffuse

cutaneous, mucocutaneous, or visceral disease. In endemic areas with leishmaniasis, co-infection with human

immunodeficiency virus (HIV)-positive patients is common. If co-infected patients are severely

immunocompromised, up to 25% will die shortly after being diagnosed. The use of highly active antiretroviral

therapy (HAART) has dramatically improved the prognosis of these co-infected patients.

Pathogenesis and spectrum of disease:

The first sign of cutaneous disease is a lesion (generally a firm, painless papule) at the bite site. Although a

single lesion may appear insignificant, multiple lesions or disfiguring facial lesions may be devastating for the

patient.

 Usually, the lesions will have a similar appearance and will progress at the same speed. The original lesion

may remain as a flattened plaque or may progress to a shallow ulcer. As the ulcer enlarges, it produces exudate

and often becomes secondarily infected with bacteria or other organisms.

 In mucocutaneous leishmaniasis, the primary lesions are similar to those found in cutaneous leishmaniasis.

Untreated primary lesions may develop into the mucocutaneous form in up to 80% of the cases. Dissemination to

the nasal or oral mucosa may occur from the active primary lesion or may occur years later after the original

lesion has healed. These mucosal lesions do not heal spontaneously, and secondary bacterial infections are

common and may be fatal. Also, untreated visceral leishmaniasis will lead to death; secondary bacterial and viral

infections are also common in these patients. The incubation period ranges from 10 days to 2 years,

usually being 2 to 4 months. Common symptoms include fever, anorexia, malaise, weight loss, and, frequently,

diarrhea.

Figure 27, Leishmania donovani parasites in Kupffer cells of liver (2000×). B, Leishmania sp.

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Clinical signs include nontender enlarged liver and spleen, swollen lymph nodes, and occasional acute

abdominal pain. Darkening of facial, hand, foot, and abdominal skin (kala-azar) is often seen in light-skinned

persons in India. Death may occur after a few weeks or after 2 to 3 years in chronic cases. The majority of

infected individuals will be asymptomatic or have very few or minor symptoms that will resolve without therapy.

Since 1990, an increase in leishmaniasis in organ transplant recipients has been documented. Most of these cases

have been visceral leishmaniasis.

Laboratory diagnosis:

After the cutaneous lesion exudate is removed, these lesions should be thoroughly cleaned with 70% alcohol.

Specimens can be collected from the margin of the lesion by aspiration, scraping, or punch biopsy or by making

a slit with a scalpel blade. Smears can be prepared from the material obtained and stained with any of the blood

stains; biopsy specimens should also be submitted for routine histologic examination. Specimens for visceral

disease include lymph node aspirates, liver biopsy specimens, bone marrow specimens, and buffy coat

preparations of venous blood. Amastigotes with reticuloendothelial cells have been detected in a number of

different specimens from HIV-positive patients.

Stained smears can be examined for the presence of the amastigotes. Although the specimens can be cultured

using special techniques, these procedures are not routinely available.

PCR methods have excellent sensitivity and specificity for direct detection, for identification of causative

species, and for assessment of treatment efficacy; although not routinely available, they can be performed at

some reference centers. A rapid immunochromatographic dipstick test using the recombinant K39 antigen has

become available for the qualitative detection of total anti–Leishmania immunoglobulins.

 In patients with severe visceral leishmaniasis (kalaazar), there is a characteristic hypergammaglobulinemia,

including both IgG and IgM. In highly suspect patients for the diagnosis of visceral leishmaniasis (assuming they

are immunocompetent), if hypergammaglobulinemia is not present, this may be used to rule out the original

diagnosis. Although serologic testing is available from some reference centers such as CDC, serologic assays are

not very useful for the diagnosis of mucocutaneous and visceral leishmaniasis.

THERAPY:

 In simple cutaneous leishmaniasis, lesions usually heal spontaneously, although treatment options include

cryotherapy heat, photodynamic therapy, surgical excision of, lesions, and chemotherapy. Standard therapy

consists of injections of antimonial compounds; however, relapse is quite common and the patient response

varies depending on the Leishmania species and type of disease.

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Other Protozoa:

Amebae, Flagellates (Other Body Sites):

 Amebae:

Naegleria fowleri

Acanthamoeba spp.

Balamuthia mandrillaris

 Flagellates:

Trichomonas vaginalis

Trichomonas tenax

 Coccidia (Other Body Sites):

 Coccidia:

Toxoplasma gondii

Free-living amebae:

 Infections caused by small, free-living amebae belonging to the genera Naegleria, Acanthamoeba, and

Balamuthia are generally not very well-known or recognized clinically.

Also, methods for laboratory diagnosis are unfamiliar and not routinely offered by most laboratories.

However, approximately 310 cases of primary amebic meningoencephalitis (PAM) caused by Naegleria

fowleri and more than 150 cases of granulomatous amebic encephalitis (GAE) caused by Acanthamoeba spp.

and Balamuthia mandrillaris (including several cases in patients with acquired immunodeficiency syndrome

[AIDS]) have been documented.Other infections caused by these organisms result in Acanthamoeba keratitis,

now numbering more than 750 cases and related primarily to poor lens care in contact lens wearers.

Additionally, both Acanthamoeba spp. and B. mandrillaris can cause cutaneous infections in humans.

Sappinia pedata, a free-living ameba normally found in soil contaminated with the feces of elk and buffalo,

was identified in an excised brain lesion from a 38-year-old immunocompetent man who developed a frontal

headache, blurry vision, and loss of consciousness following a sinus infection. Additionally,

Paravahlkampfia francinae, a new species of the free-living ameba genus Paravahlkampfia, was recently

isolated from the cerebrospinal fluid (CSF) of a patient with a headache, sore throat, and vomiting, symptoms

typical of primary amebic meningoencephalitis (PAM) caused by Naegleria fowleri from the environment.

NAEGLERIA FOWLERI:

General characteristics

There are both trophozoite and cyst stages in the life cycle, with the stage present primarily dependent on

environmental conditions. Trophozoites can be found in water or moist soil and can be maintained in tissue

culture or other artificial media. The amebae may enter the nasal cavity by inhalation or aspiration of water,

dust, or aerosols containing the trophozoites or cysts. N. fowleri is incapable of survival in clean, chlorinated

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water. Following inhalation or aspiration, the organisms then penetrate the nasal mucosa, probably through

phagocytosis of the olfactory epithelium cells, and migrate via the olfactory nerves to the brain.

The trophozoites can occur in two forms: ameboid and flagellate ( Figure 28). The size ranges

from 7 to 35 μm. The diameter of the rounded forms is usually 15 μm. There is a large, central karyosome

and no peripheral nuclear chromatin. The cytoplasm is somewhat granular and contains vacuoles. The

ameboid form organisms change to the transient, pear-shaped flagellate form when they are transferred from

culture or teased from tissue into water and maintained at a temperature of 27° to 37° C. These flagellate

forms do not divide, but when the flagella are lost, the ameboid forms resume reproduction. Cysts are

generally round, measuring from 7 to 15 μm with a thick double wall.

Pathogenesis and spectrum of disease

Primary amebic meningoencephalitis (PAM) caused by N. fowleri is an acute, suppurative infection of the brain

and meninges (Figure 29). With extremely rare exceptions, the disease is rapidly fatal in humans. The period

between organism contact and onset of symptoms such as fever, headache, and rhinitis varies from a few days

to 2 weeks. Early symptoms include vague upper respiratory tract distress, headache, lethargy, and occasionally

olfactory problems. The acute phase includes sore throat; a stuffy, blocked, or discharging nose; and severe

headache.

Progressive symptoms include pyrexia, vomiting, and stiffness of the neck. Mental confusion and coma

usually occur approximately 3 to 5 days before death, which is usually caused by cardiorespiratory arrest and

pulmonary edema.

PAM resembles acute bacterial meningitis, and these conditions may be difficult to differentiate. Unfortunately,

if the CSF Gram stain is interpreted incorrectly as a false positive, the resulting antibacterial therapy has no

impact on the amebae and the patient will usually die within a few days.

Figure 28 Naegleria fowleri, Acanthamoeba spp. Diagram of trophozoites and cysts . Flagellate and

cyst forms of Naegleria fowleri; (lower row) trophozoite and cyst of Acanthamoeba spp.

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Laboratory diagnosis

Routine Methods

Clinical and laboratory data usually cannot be used to differentiate pyogenic meningitis from PAM. A high

index of suspicion is often critical for early diagnosis.

Most cases are associated with exposure to contaminated water through swimming or bathing. There is

normally an incubation period of 1 day to 2 weeks, and then a course of 3 to 6 days, most often ending in death.

Analysis of the CSF will show decreased glucose and increased protein concentrations. The leukocyte count

will range from several hundred to >20,000 cells per mm3. Although Gram stains and bacterial cultures of CSF

will be negative, serious patient complications can occur as the result of incorrect therapy if false-positive Gram

stains are reported.

A confirmed diagnosis is made by the identification of amebae in the CSF or in biopsy specimens. CSF should

be placed on a slide, under a cover slip, and observed for motile trophozoites; smears can be stained with any of

the blood stains. It is important not to mistake leukocytes for actual organisms or vice versa. This type of

misidentification often occurs when using a counting chamber and the amebae sink to the bottom and round up,

hencethe recommendation to use just a regular slide and cover slip. Depending on the temperature and lag time

between specimen collection and examination, motility may vary.

Slides may be warmed slightly to improve motility. The most important differential characteristic is the

spherical nucleus with a large karyosome.

Specimens should never be refrigerated before examination, and CSF should be centrifuged at a slow speed

(250× g). If N. fowleri is the causative agent, only trophozoites are normally seen, whereas cysts and

trophozoites can be seen with Acanthamoeba spp.

Other Methods:

Most cases are diagnosed at autopsy; confirmation of tissue findings must include culture and/or special

staining with monoclonal reagents in indirect fluorescent antibody procedures.

Therapy:

Although many antimicrobial and antiparasitic drug have been screened for activity against N. fowleri, only a

few patients have recovered after receiving intrathecal and intravenous injections of amphotericin B or in

combination with miconazole.

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 Arranged by Sarah Mohssen

SectionIII– Parasitology By Nada Sajet

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Acanthamoeba spp.

General characteristics

Unlike N. fowleri, Acanthamoeba spp. do not have a flagellate stage in the life cycle, only the trophozoite and cyst.

Several species of Acanthamoeba cause granulomatous amebic encephalitis (GAE), primarily in immunosuppressed,

 Acanthamoeba spp. also cause amebic keratitis Motile organisms have spine-like pseudopods; there is a wide organism

size range (25 to 40 μm), with the average diameter of the trophozoites being 30 μm. The nucleus has the typical large