Blood and Tissue Protozoa

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Blood and Tissue Protozoa

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. Plasmodium falciparum is the major species associated with deadly infections throughout the world. Unfortunately, prevention remains a complex problem, and no drug is universally effective for all Plasmodium species.

In addition, of the five species that infect humans, P. vivax and P. falciparum cause 95% of infections. P. vivax may be responsible for 80% of the infections, because this species has the widest distribution in the tropics, subtropics, and temperate zones. P. falciparum is generally confined to the tropics, P. malariae is sporadically distributed, and P. ovale is confined mainly to central West Africa and some South Pacific islands. The fifth human malaria, Plasmodium knowlesi, a malaria parasite of long-tailed macaque monkeys, has been confirmed in human cases from Malaysian Borneo, Thailand, Myanmar, and the Philippines.

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 (Figure 49-1). 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 dividing 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.

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 (Tables 49-1 to 49-3, Figures 49-2 and 49-3). 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.

TABLE 49-1

Plasmodium spp.: Clinical Characteristics of the Five Human Infections

Infection P. vivax P. ovale P. malariae P. falciparum P. knowlesi Comments
Incubation period 8-17 days 10-17 days 18-40 days 8-11 days 9-12 days All may be extended for months to years
Prodromal symptoms
 Severity
 Initial fever pattern
Mild to moderate
Irregular (48 hr)
Mild
Irregular (48 hr)
Mild to moderate
Regular (72 hr)
Mild
Continuous remittent
Mild to moderate
Regular (24 hr)
All may mimic influenza symptoms
Early symptoms may reflect lack of regular periodicity
Symptom periodicity 48 hr 48 hr 72 hr 36-48 hr 24-27 hr  
Initial paroxysm
 Severity
 Mean duration
Moderate to severe
10 h
Mild
10 h
Moderate to severe
11 h
Severe
16-36 h
Moderate to severe
Not available
P. knowlesi might increase/lose virulence on passage in humans
Duration of untreated primary attack 3-8+ wk 2-3 wk 3-24 wk 2-3 wk Not available  
Duration of untreated infection 5-7 yr 12 mo 20+ yr 6-17 mo Not available  
Parasitemia limitations Young RBCs Young RBCs Old RBCs All RBCs All RBCs  
Anemia Mild to moderate Mild Mild to moderate Severe Moderate to severe P. knowlesi can be as dangerous as P. falciparum
CNS involvement Rare Possible Rare Very common Possible  
Nephrotic syndrome Possible Rare Very common Rare Probably common  

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TABLE 49-2

Plasmodia in Giemsa-Stained Thin Blood Smears

  Plasmodium vivax Plasmodium malariae Plasmodium falciparum Plasmodium ovale Plasmodium knowlesi
Persistence of exoerythrocytic cycle Yes No No Yes No
Relapses Yes No, but long-term recrudescence is recognized No long-term relapses Possible, but usually spontaneous recovery No
Time of cycle 44-48 hr 72 hr 36-48 hr 48 hr 24 hr
Appearance of parasitized RBCs; size and shape 1.5-2 times larger than normal; oval to normal; may be normal size until ring fills half of cell Normal shape; size may be normal or slightly smaller Both normal 60% of cells larger than normal and oval; 20% have irregular, frayed edges Normal shape, size
Schüffner’s dots (eosinophilic stippling) Usually present in all cells except early ring forms None None; occasionally comma-like red dots are present (Maurer’s dots) Present in all stages including early ring forms; dots may be larger and darker than in P. vivax No true stippling; occasional faint dots
Color of cytoplasm Decolorized, pale Normal Normal, bluish tinge at times Decolorized, pale Normal
Multiple rings/cell Occasional Rare Common Occasional Common
All developmental stages present in peripheral blood All stages present Ring forms few, since ring stage brief; mostly growing and mature trophozoites and schizonts Young ring forms and no older stages; few gametocytes All stages present All stages present
Appearance of parasite; young trophozoite (early ring form) Ring is image diameter of cell, cytoplasmic circle around vacuole; heavy chromatin dot Ring often smaller than in P. vivax, occupying image of cell; heavy chromatin dot; vacuole at times “filled in”; pigment forms early Delicate, small ring with small chromatin dot (frequently 2); scanty cytoplasm around small vacuoles; sometimes at edge of red cell (appliqué form) or filamentous slender form; may have multiple rings per cell Ring is larger and more ameboid than in P. vivax; otherwise similar to P. vivax Rings image to image diameter of RBC; double chromatin dots; appliqué forms rare; multiple rings per RBC
Growing trophozoite Multishaped irregular ameboid parasite; streamers of cytoplasm close to large chromatin dot; vacuole retained until close to maturity; increasing amounts of brown pigment Non-ameboid rounded or band-shaped solid forms; chromatin may be hidden by coarse dark brown pigment Heavy ring forms; fine pigment grains Ring shape maintained until late in development; non-ameboid compared to P. vivax Slightly ameboid and irregular; band forms seen; very little pigment
Mature trophozoite Irregular ameboid mass; 1 or more small vacuoles retained until schizont stage; fills almost entire cell; fine brown pigment Vacuoles disappear early; cytoplasm compact, oval, band shaped, or nearly round and almost filling cell; chromatin may be hidden by peripheral coarse dark brown pigment Not seen in peripheral blood (except in severe infections); development of all phases following ring form occurs in capillaries of viscera Compact; vacuoles disappear; pigment dark brown, less than in P. malariae Denser cytoplasm (slightly ameboid) band forms seen; little to no malaria pigment (scattered, fine brown grains)
Schizont (pre-segmenter) Progressive chromatin division; cytoplasmic bands containing clumps of brown pigment Similar to P. vivax except smaller; darker, larger pigment granules peripheral or central Not seen in peripheral blood (see above) Smaller and more compact than P. vivax Between 2 and 5 divided nuclear chromatin masses; abundant pigment granules occupy image of RBC
Mature schizont 16 (12-24) merozoites, each with chromatin and cytoplasm, filling entire red cell, which can hardly be seen 8 (6-12) merozoites in rosettes or irregular clusters filling normal-sized cells, which can hardly be seen; central arrangement of brown-green pigment Not seen in peripheral blood image of cells occupied by 8 (8-12) merozoites in rosettes or irregular clusters RBCs normal size; distorted/fimbriated RBCs very rare; occupy whole RBC; maximum of 16 merozoites; no rosettes; grapelike clusters
Macrogametocyte Rounded or oval homogeneous cytoplasm; diffuse delicate light brown pigment throughout parasite; eccentric compact chromatin Similar to P. vivax, but fewer in number; pigment darker and more coarse Gender differentiation difficult; “crescent” or “sausage” shapes characteristic; may appear in “showers” with black pigment near chromatin dot, which is often central Smaller than P. vivax Occupy most of RBC; bluish cytoplasm; dense pink chromatin at periphery of parasite
Microgametocyte Large pink to purple chromatin mass surrounded by pale or colorless halo; evenly distributed pigment Similar to P. vivax, but fewer in number; pigment darker and more coarse Same as macrogametocyte (described above) Smaller than P. vivax Occupy most of RBC; cytoplasm pinkish purple; early forms similar to mature trophozoite
Main criteria Large pale red cell; trophozoite irregular; pigment usually present; Schüffner’s dots not always present; several phases of growth seen in one smear; gametocytes appear as early as third day Red cell normal in size and color; trophozoites compact, stain usually intense, band forms not always seen; coarse pigment; no stippling of red cells; gametocytes appear after a few weeks Development following ring stage takes place in blood vessels of internal organs; delicate ring forms and crescent-shaped gametocytes are only forms normally seen in peripheral blood; gametocytes appear after 7-10 days Red cell enlarged, oval, with fimbriated edges; Schüffner’s dots seen in all stages; gametocytes appear after 4 days or as late as 18 days Ring forms compact; single/double chromatin dots, appliqué forms, multiple rings/RBC (mimic P. falciparum); overall RBCs not enlarged; developing stages mimic P. malariae (band forms, 16 merozoites in mature schizont, but no rosettes)

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Plasmodium malariae (quartan malaria) Plasmodium ovale Plasmodium falciparum (malignant tertian malaria) Plasmodium knowlesi (simian malaria)*

Early stages mimic P. falciparum; later stages mimic P. malariae

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*Preparation of thick and thin blood films within <60 min of collection.

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Figure 49-2 The morphology of malaria parasites. Plasmodium vivax: 1, Early trophozoite (ring form). 2, Late trophozoite with Schüffner’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 Schüffner’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 (accolé or appliqué 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, Schüffner’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. (Reprinted by permission of the publisher from Garcia LS: Diagnostic medical parasitology, ed 5, Washington, DC, 2007, Copyright by American Society for Microbiology.)
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Figure 49-3 Morphology of malaria parasites. Column 1 (left to right): Plasmodium vivax (note enlarged infected RBCs). (1) Early trophozoite (ring form) (note one RBC contains 2 rings—not that uncommon); (2) older ring, note ameboid nature of rings; (3) late trophozoite with Schüffner’s dots (note enlarged RBC); (4) developing schizont; (5) mature schizont with 18 merozoites and clumped pigment; (6) microgametocyte with dispersed chromatin. Column 2: Plasmodium ovale (note enlarged infected RBCs). (1) Early trophozoite (ring form) with Schüffner’s dots (RBC has fimbriated edges); (2) early trophozoite (note enlarged RBC, Schüffner’s dots, and RBC oval in shape); (3) late trophozoite in RBC with fimbriated edges; (4) developing schizont with irregular-shaped RBC; (5) mature schizont with 8 merozoites arranged irregularly; (6) microgametocyte with dispersed chromatin. Column 3: Plasmodium malariae (note normal or smaller than normal infected RBCs). (1) Early trophozoite (ring form); (2) early trophozoite with thick cytoplasm; (3) late trophozoite (band form); (4) developing schizont; (5) mature schizont with 9 merozoites arranged in a rosette; (6) microgametocyte with compact chromatin. Column 4: Plasmodium falciparum. (1) Early trophozoites (the rings are in the headphone configuration with double chromatin dots); (2) early trophozoite (accolé or appliqué form); (3) early trophozoites (note the multiple rings/cell); (4) late trophozoite with larger ring (accolé or appliqué form); (5) crescent-shaped gametocyte; (6) crescent-shaped gametocyte. Column 5: Plasmodium knowlesi—with the exception of image 5, these were photographed at a higher magnification (note normal or smaller than normal infected RBCs). (1) Early trophozoite (ring form); (2) early trophozoite with slim band form; (3) late trophozoite (band form); (4) developing schizont; (5) mature schizont with merozoites arranged in a rosette; (6) microgametocyte with dispersed chromatin. Note: Without the appliqué form, Schüffner’s dots, multiple rings per cell, and other developing stages, differentiation among the species can be very difficult. It is obvious that the early rings of all five species can mimic one another very easily. Remember: One set of negative blood films cannot rule out a malaria infection. (From Garcia LS: Malaria Clin Lab Med 30:93-129, 2010,with permission. Column 5 courtesy CDC.)

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 (Figure 49-3 [3]).

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 (see Tables 49-1 to 49-3, Figures 49-2 and 49-3). 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.

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