Often these patients have four to eight watery, nonbloody

stools each day, accompanied by nausea and anorexia. Dehydration and D-xylose and fat malabsorption also

may develop. These patients tend to be severely immunodeficient, with a CD4 count almost always below 200

cells/mm3 and often below 100 cells/mm3. Mixed infections with E. bieneusi and E. intestinalis have also been

reported. E. bieneusi infection has been implicated in AIDS-related sclerosing cholangitis. However,

demonstration of E. bieneusi spores in extraepithelial tissues does not always appear to be associated with

subsequent development of systemic infection.

E. bieneusi spores have been identified in sputum and bronchoalveolar lavage fluid in addition to stool

specimens. E. bieneusi can colonize the respiratory tract, and clinical specimens from these specimens may

reveal the presence of spores. Multiorgan microsporidiosis caused by E. bieneusi has been diagnosed in patients

infected with HIV; organisms have been recovered in stools, duodenal biopsy specimens, nasal discharge, and

sputum. Infection with E. bieneusi has also been reported in immunocompetent individuals; symptoms were

selflimited, and diarrheal disease resolved within 2 weeks. E. bieneusi may be more commonly associated with

sporadic diarrheal disease than was previously suspected, and the immune system may play a role in the control

of this intestinal infection. It is also quite possible that E. bieneusi may persist as an asymptomatic infection in

immunocompetent individuals.

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Encephalitozoon spp.:

Both Encephalitozoon cuniculi and Encephalitozoon hellem have been isolated from human infections.

The spectrum disease in patients with AIDS, organ transplant recipients, and otherwise immunocompromised

patients includes keratoconjunctivitis, intraocular infection, sinusitis, bronchiolitis, pneumonitis, nephritis,

ureteritis, cystitis, prostatitis, urethritis, hepatitis, sclerosing cholangitis, peritonitis, diarrhea, and encephalitis.

Clinical manifestations may vary, ranging from an asymptomatic carrier state to organ failure.

Encephalitozoon (Septata) intestinalis:

 Encephalitozoon (Septata) intestinalis infects primarily small intestinal enterocytes, but infection does not

remain confined to epithelial cells. E. intestinalis is also found in lamina propria macrophages, fibroblasts, and

endothelial cells. Dissemination to the kidneys, lower airways, and biliary tract appears to occur through

infected macrophages.

Fortunately, these infections tend to respond to therapy with albendazole, unlike infections caused by E.

bieneusi.

Other Microsporidia

 Different microsporidial species have been isolated from immunocompetent individuals who presented with

keratoconjunctivitis, severe keratitis, or corneal ulcers. Also, keratoconjunctivitis has been found in an

immunocompetent contact lens wearer.

In immunocompromised patients, myositis has been seen in infections caused by Pleistophora sp., Pleistophora

ronneafiei, Trichomonas hominis, Anncaliia vesicularum, and Anncaliia algerae. Trachipleistophora

anthropophthera has been identified at autopsy in cerebral, cardiac, renal, pancreatic, thyroid, hepatic, splenic,

lymphoid, and bone marrow tissue of patients with AIDS. Disseminated infection caused by Anncaliia connori

was found at autopsy in a 4-month-old athymic male infant.

Laboratory diagnosis:

 The most commonly used stains are chromotrope-based stains (modified trichrome) and chemofluorescent

optical brightening agents, including calcofluor white and other chemofluorescent stains. Regardless of the

staining technique selected, the use of positive control material is highly recommended. Detection of the small

microsporidial spores requires adequate illumination and magnification (i.e., magnification using the oil

immersion objective [×100] for a total magnification of ×1000).

 Microsporidia do not tend to stain predictably, if at all, in tissues. However, spores occasionally can be seen

very well with use of the periodic acid-Schiff (PAS) stain, silver stains, or acid-fast stains. Modified Gram

stains also have proved sensitive. The spore has a small, PAS-positive posterior body; the spore coat stains with

silver, and the spores are acid-fast variable. Tissue examination by electronmicroscopy (EM) techniques is still

considered the best approach for differentiation of genera; however, this option is not available to all

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laboratories, and the sensitivity of EM may not be equal to that of other methods when examining stool or

urine.

Therapy:

 Albendazole therapy can result in clinical cure of HIVassociated infection with Encephalitozoon spp., along

with elimination of spore shedding. Albendazole is not effective for Enterocytozoon infections, although

clinical improvement occurs in some patients. Oral purified fumagillin appears to eradicate Enterocytozoon

bieneusi in many patients, but serious adverse events and parasitic relapse have been seen. Antiretroviral

combination therapy results in complete clinical response with elimination of intestinal microsporidia.

Prevention:

 The presence of infective spores in human clinical specimens suggests that taking precautions when handling

body fluids and following personal hygiene measures,such as hand washing, may be important in preventing

primary infections in the health care setting.

Blood and Tissue Protozoa:

Sporozoa, Flagellates (Blood, Tissue):

Sporozoa (Malaria and Babesiosis):

Plasmodium vivax

Plasmodium ovale

Plasmodium malariae

Plasmodium falciparum

Plasmodium knowlesi

Babesia spp.

Flagellates (Leishmaniae, Trypanosomes):

Leishmania tropica complex

Leishmania mexicana complex

Leishmania braziliensis complex

Leishmania donovani complex

Leishmania peruviana

Trypanosoma brucei gambiense

Trypanosoma brucei rhodesiense

Trypanosoma cruzi

Trypanosoma rangeli

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Plasmodium spp.:

 Malaria has been well documented as an ancient disease in Egyptian and Chinese writing beginning in 2700

bc. By 200 bc, malaria was identified in Rome, spread throughout Europe during the twelfth century, and

arrived in England by the fourteenth century. By the early 1800s malaria was found worldwide.

Malaria has played a tremendous role in world history, influencing the outcome of wars, the movement of

populations, and the development and decline of various nations. Before the American Civil War, malaria was

found as far north as southern Canada; but it was no longer endemic within the United States by the 1950s. It is

estimated that more than 500 million individuals worldwide are infected with Plasmodium spp., and as many as

2.7 million people a year, most of whom are children, die from the infection. Malaria is endemic in more than

90 countries with a population of 2400 million people, representing 40% of the world’s population. At least

90% of deaths caused by malaria occur in Africa.

 The vector for malaria is the female anopheline mosquito. When the vector takes a blood meal, sporozoites

contained in the salivary glands of the mosquito are discharged into the puncture wound Within an hour, these

infective sporozoites are carried via the blood to the liver, where they penetrate hepatocytes and begin to grow,

initiating the preerythrocytic or primary exoerythrocytic cycle. The sporozoites become round or oval and

begin dividin repeatedly. Schizogony results in large numbers of exoerythrocytic merozoites. Once these

merozoites leave the liver, they invade the red blood cells (RBCs), initiating the erythrocytic cycle. A dormant

schizogony may occur in P. vivax and P. ovale organisms,which remain quiescent in the liver. These resting

stages have been termed hypnozoites and lead to a true relapse, often within 1 year or up to more than 5 years

later.

Delayed schizogony does not occur in P. falciparum, P. malariae, or P. knowlesi. Once the RBCs and

reticulocytes have been invaded, the parasites grow and feed on hemoglobin. Within the RBC, the merozoite (or

young trophozoite) is vacuolated, ring shaped, more or less ameboid, and uninucleate. The excess protein and

hematin present from the metabolism of hemoglobin combine to form malarial pigment. Once the nucleus begins

to divide, the trophozoite is called a developing schizont. The mature schizont contains merozoites (whose

number depends on the species), which are released into the bloodstream. Many of the merozoites are destroyed

by the immune system, but others invade RBCs and initiate a new cycle of erythrocytic schizogony. After

several erythrocytic generations, some of the merozoites begin to undergo development into the male and female

gametocytes. Although malaria is often associated with travelers to endemic areas, other situations resulting in

infection include blood transfusions, use of contaminated hypodermic needles, bone marrow transplantation,

congenital infection, and transmission within the United States by indigenous mosquitoes that acquired the

parasites from imported infections.

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PLASMODIUM VIVAX (BENIGN TERTIAN MALARIA):

General Characteristics:

 P. vivax infects only the reticulocytes; thus, the parasitemia is limited to approximately 2% to 5% of the available RBCs (

Figures 21).

Figure 21 The morphology of malaria parasites. Plasmodium vivax: 1, Early trophozoite (ring form). 2, Late trophozoite with Schuffner’s dots

(note enlarged red blood cell). 3, Late trophozoite with ameboid cytoplasm (very typical of P. vivax). 4, Late trophozoite with ameboid cytoplasm.

5, Mature schizont with merozoites (18) and clumped pigment. 6, Microgametocyte with dispersed chromatin. 7, Macrogametocyte with compact

chromatin. Plasmodium malariae: 1, Early trophozoite (ring form). 2, Early trophozoite with thick cytoplasm. 3, Early trophozoite (band form). 4,

Late trophozoite (band form) with heavy pigment. 5, Mature schizont with merozoites (9) arranged in rosette. 6, Microgametocyte with dispersed

chromatin. 7, Macrogametocyte with compact chromatin. Plasmodium ovale: 1, Early trophozoite (ring form) with Schuffner’s dots. 2, Early

trophozoite (note enlarged red blood cell). 3, Late trophozoite in red blood cell with fimbriated edges. 4, Developing schizont with irregularly

shaped red blood cell. 5, Mature schizont with merozoites (8) arranged irregularly. 6, Microgametocyte with dispersed chromatin. 7,

Macrogametocyte with compact chromatin. Plasmodium falciparum: 1, Early trophozoite (accole or applique form). 2, Early trophozoite (one ring

is in headphone configuration/double chromatin dots). 3, Early trophozoite with Maurer’s dots. 4, Late trophozoite with larger ring and Maurer’s

dots. 5, Mature schizont with merozoites (24). 6, Microgametocyte with dispersed chromatin. 7, Macrogametocyte with compact chromatin. Note:

Without the appliqué form, Schuffner’s dots, multiple rings/cell, and other developing stages, differentiation among the species can be difficult. It

is obvious that the early rings of all four species can mimic one another very easily. Remember: One set of negative blood films cannot rule out a

malarial infection.

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Splenomegaly occurs during the first few weeks of infection, and the spleen will progress from being soft and

palpable to hard, with continued enlargement during a chronic infection. If the infection is treated during the

early phases, the spleen will return to its normal size. A secondary or dormant schizogony occurs in P. vivax

and P. ovale, which remain quiescent in the liver. These resting stages have been termed hypnozoites. After a

few days of irregular periodicity, a regular 48-hour cycle is established. An untreated primary attack may last

from 3 weeks to 2 months or longer. Over time, the paroxysms (symptomatic period) become less severe and

more irregular in frequency and then cease altogether. In approximately 50% of patients infected with P. vivax,

relapses occur after weeks, months, or even after 5 years or more. The RBCs tend to be enlarged (young

RBCs), there may be Schüffner’s dots (exclusively found in P. vivax and P. ovale) after 8 to 10 hours, the

developing rings are ameboid, and the mature schizont contains 12 to 24 merozoites .

Pathogenesis and Spectrum of Disease:

 In patients who have never been exposed to malaria, symptoms such as headache, photophobia, muscle aches,

anorexia, nausea, and sometimes vomiting may occur before organisms can be detected in the bloodstream. In

other patients with prior exposure to the malaria, the parasites can be found in the bloodstream several days

before symptoms appear. Severe complications are uncommon in P. vivax infections, although coma and

sudden death or other symptoms of cerebral involvement have been reported, particularly in patients with

varying degrees of primaquine resistance. These patients can exhibit cerebral malaria, renal failure, circulatory

collapse, severe anemia, hemoglobinuria, abnormal bleeding, acute respiratory distress syndrome, and jaundice.

Acute cerebral malaria involves changes in mental status and if untreated may result in fatality within 3 days.

Plasmodium ovale:

General Characteristics

Although P. ovale and P. vivax infections are clinically similar, P. ovale malaria is usually less severe, tends to

relapse less frequently, and usually ends with spontaneous recovery, often after no more than 6 to 10 paroxysms

, Like P. vivax, P. ovale infects only the reticulocytes, so that the parasitemia is limited to approximately 2% to

5% of the available RBCs. For many years the literature has stated that as with P. vivax, a secondary or dormant

schizogony occurs in P. ovale, which remain quiescent in the liver.

 However, newer findings indicate that hypnozoites have never been demonstrated by biologic experiments.

After a few days of irregular periodicity, a regular 48-hour cycle is established. Over time, the paroxysms

become less severe and more irregular in frequency and then stop altogether. In some patients infected with P.

ovale, relapses occur after weeks, months, or up to 1 year or more. The RBCs tend to be enlarged (young

RBCs), Schüffner’s dots (also known as James stippling) are present from the beginning of the cycle, the

developing rings are less ameboid than those of P. vivax, and the mature schizont contains an average of eight

merozoites.

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Pathogenesis and Spectrum of Disease:

The incubation period is similar to that for P. vivax malaria, but the frequency and severity of the symptoms are

much less, with a lower fever and a lack of typical rigors. The geographic range is usually described as being

limited to tropical Africa, the Middle East, Papu New Guinea, and Irian Jaya in Indonesia. However, P. ovale

infections in Southeast Asia may cause benign and relapsing malaria in this area. In both Southeast Asia and

Africa, two different types of P. ovale circulate in humans. Human infections with variant-type P. ovale are

associated with a higher level of parasitemia.

Plasmodium malariae (QUARTAN MALARIA):

General Characteristics

 P. malariae invades primarily the older RBCs, limiting the number of infected cells ( Figures 21). The

incubation period between infection and symptoms may be much longer than that for P. vivax or P. ovale

malaria, ranging from about 27 to 40 days. A regular periodicity is seen from the beginning, with a more severe

paroxysm, including a longer cold stage and more severe symptoms during the hot stage. Collapse during the

sweating phase is not uncommon. A regular periodicity of 72 hours is seen from the beginning of the

erythrocytic cycle. The infection may end with spontaneous recovery, or there may be a recrudescence or series

of recrudescence (recurrence of symptoms) over many years. These patients are left with a latent infection and

persisting low-grade parasitemia for many, many years. The RBCs tend to be normal to small (old RBCs), there

is no true stippling, the RBCs may have fimbriated edges, the developing rings tend to demonstrate “band”

forms, and the mature schizont contains an average of 6 to 12 merozoites.

Pathogenesis and Spectrum of Disease:

 Proteinuria is common in P. malariae infections and may be associated with clinical signs of nephrotic

syndrome. With a chronic infection, kidney problems result from deposition within the glomeruli of circulating

antigenantibody complexes. A membrane proliferative type of glomerulonephritis is the most common lesion

seen in quartan malaria. Because chronic glomerular disease associated with P. malariae infections is usually

not reversible with therapy, genetic and environmental factors may play a role in the disease, as well. The

patient may have a spontaneous recovery, or there may be a recrudescence or series of recrudescence over

many years (>50 years).

In these cases, patients are left with a latent infection and persisting low-grade parasitemia.

Plasmodium falciparum (MALIGNANT TERTIAN MALARIA):

General Characteristics

 Plasmodium falciparum invades all ages of RBCs, and the number of infected cells may exceed 50% ,

Schizogony occurs in the spleen, liver, and bone marrow rather than in the circulating blood. Ischemia caused

by the obstruction of vessels within these organs by parasitized RBCs will produce various symptoms,

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depending on the organ involved. A decrease in the ability of the RBCs to change shape when passing through

capillaries or the splenic filter may lead to plugging of the vessels Also, only P. falciparum causes

cytoadherence, a feature that is associated with severe malaria. The asexual and sexual forms circulate in the

bloodstream during infections by four of the Plasmodium species. However, in P. falciparum infections, as the

parasite grows, the RBC membrane becomes sticky and the cells adhere to the endothelial lining of the

capillaries of the internal organs. Thus, only the ring forms and the gametocytes (occasionally mature

schizonts) normally appear in the peripheral blood. Periodicity of the cycle will not be established during the

early stages, and the presumptive diagnosis may be totally unrelated to a possible malaria infection. If the fever

does develop a synchronous cycle, it is usually a cycle of 36 to 48 hours. Because P. falciparum infects young

and old RBCs, very heavy parasitemia can occur. The RBCs are all sizes; there is no true stippling, but

Maurer’s dots (coarse granulation in the cytoplasm of RBCs) are sometimes present; often there are multiple

rings per RBC and the rings are delicate and often have two dots of chromatin, appliqué or accolé forms (ring

forms identified within the marginal regions of the erythrocytes); and the gametocytes are crescent-shaped.

Pathogenesis and Spectrum of Disease:

 The onset of a P. falciparum malaria attack occurs 8 to 12 days after infection and is characterized by 3 to 4

days of vague symptoms such as aches, pains, headache, fatigue,

anorexia, or nausea. This stage is followed by fever, a more severe headache, and nausea and vomiting, with

occasional severe epigastric pain. At the onset of fever, there may be a feeling of chilliness. As with the other

Plasmodium spp., periodicity of the cycle will not be established during the early stages.

Severe or fatal complications can occur at any time and are related to the obstruction of vessels in the internal

organs (liver, intestinal tract, adrenal glands, intravascular hemolysis/black water fever, and kidneys).

Blackwater fever is a complication of malaria that is a result of red blood cell lysis, releasing hemoglobin into

the bloodstream and urine, causing discoloration. The severity of the complications may not correlate with the

peripheral blood parasitemia, particularly in P. falciparum infections in a patient who has never been exposed

to malaria before (immunologically naïve).

Disseminated intravascular coagulation is a rare complication and is seen with a high parasitemia, pulmonary

edema, anemia, and cerebral and renal complications.

Vascular endothelial damage from endotoxins and bound parasitized blood cells may lead to clot formation in

small vessels. Cerebral malaria is more common in P. falciparum malaria, but can occur in the other species. If

the onset is gradual, the patient becomes disoriented or violent or may develop severe headaches and pass into

coma. However, some patients, including those with no prior symptoms, may suddenly become comatose.

Physical signs of central nervous system involvement vary, and there is no correlation between the severity of

the symptoms and the parasitemia.

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Extreme fevers, 41.7° C (107° F) or higher, may occur in an uncomplicated malaria attack or in cases of

cerebral malaria. Without vigorous therapy, the patient usually dies. Cerebral malaria is considered to be the

most serious complication and the major cause of death with P. falciparum; it occurs in up to 10% of all P.

falciparum patients admitted to the hospital and is responsible for 80% of fatal cases.

Plasmodium knowlesi

General Characteristics:

 P. knowlesi invades all ages of RBCs, and the number of infected cells can be significantly more than seen in

P. vivax, P. ovale, and P. malariae. P. knowlesi infection should be considered in patients with a travel history

to forested areas of Southeast Asia, especially if P. malariae is diagnosed, unusual forms are seen with

microscopy, or if a mixed infection with P. falciparum/P. malariae is diagnosed. Because the disease is

potentially fatal, proper identification to the species level is critical.

The early blood stages of P. knowlesi resemble those of P. falciparum, whereas the mature blood stages and

gametocytes resemble those of P. malariae.

Unfortunately, these infections are often misdiagnosed as the relatively benign P. malariae; however, infections

with P. knowlesi can be fatal. The RBCs are all sizes, there is no true stippling (fine, granular, blue stippling in

RBCs stained with Wright’s stain or red when using eosin hematoxylin as seen in Figure 21 (P. vivax photo,

third from the top), often there are multiple rings per RBC (there may be 2 to 3 rings), the rings ar delicate and

often have 2 to 3 dots of chromatin, band forms are typically seen with the developing trophozoites, and the

mature schizont contains 16 merozoites. The early stages mimic P. falciparum, whereas the later stages mimic

P. malariae. Because of different levels of parasitemia, low organism densities, and confusion among various

morphologic criteria for identification, detection of mixed infections can be quite difficult. Even if a mixed

infection is suspected, identification to the species level may not be possible using routine microscopy methods.

However, using polymerase chain reaction (PCR) methods, it is likely that higher detection and identification

rates of chronic and mixed malarial infections will be possible.

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Pathogenesis and Spectrum of Disease:

 Patients exhibit chills, minor headaches, and daily lowgrade fever. Patients who have been diagnosed with high numbers

of P. malariae organisms by microscopy should receive intensive management as appropriate for severe P. falciparum

malaria, assuming the infection is actually caused by P. knowlesi. Overall, these infections can beas severe as