Chapter 51: Apicomplexa and Microsporidia

GROUP CHARACTERISTICS

The Apicomplexa are obligate intracellular protozoan parasites. The name of this group of parasites derives from the complex of organelles located at the apical end of parasite life cycle stages that are involved in penetrating cells. These organelles include the rhoptries, micronemes, and associated microtubular complexes located in this region of the parasite. The Apicomplexa have alternating cycles of sexual and asexual reproduction. Asexual multiplication within the host occurs by a process of multiple fission termed schizogony. The nucleus of a trophozoite divides into several parts, forming a multinucleated schizont. The cytoplasm then condenses around each nuclear portion to form new daughter cells, or merozoites, which burst from their intracellular location to invade new host cells. After the completion of one or more of these asexual cycles, some merozoites differentiate into male and female gametocytes, initiating the sexual phase of the life cycle. In the case of malaria, the gametocytes reach maturity in the mosquito host and effect fertilization, forming a zygote, or motile ookinete. In other Apicomplexa, this process may occur in intestinal cells. The zygote then becomes an oocyst for these parasites. Sporozoites are formed within the oocyst by an asexual process of sporogony and when released, penetrate host tissue cells, and begin another asexual cycle as trophozoites. The only phase of this life cycle that is diploid is when the zygote is formed. All other stages in the life cycle are haploid. The general apicomplexan cell plan is illustrated in Figure 51–1.

Two sporozoan infections, malaria and toxoplasmosis, are common diseases in humans; together, they affect more than one-third of the world’s population and kill or deform perhaps a million neonates and children each year. Cryptosporidium, Cyclospora, and Isospora are Apicomplexa that can cause diarrhea, particularly in the immunocompromised. Babesia, a relative of malaria, and transmitted by ticks, is also a member of this group.

PARASITOLOGY

The plasmodia are Apicomplexa in which the sexual and asexual cycles of reproduction are completed in different host species. The sexual phase occurs within the gut of mosquitoes and results in the formation of a motile zygote, the ookinete. These arthropods subsequently transmit the parasite as sporozoites while feeding on a vertebrate host. Within the vertebrate, the plasmodia reproduce asexually, first in the liver and then in erythrocytes; they eventually burst from the erythrocyte and invade other uninvolved RBCs. This event produces periodic fever and anemia in the host, a disease process known as malaria. Of the many species of plasmodia, five are known to infect humans and are considered here: Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium knowlesi, and Plasmodium falciparum.

LIFE CYCLE OF MALARIAL PARASITES

The life cycle in the female Anopheles mosquito begins with the ingestion of male and female gametocytes from the circulation of a malaria-infected individual. In the gut of the mosquito, the gametocytes reach full maturation, are released from infected erythrocytes, and effect fertilization. The resulting zygote, an ookinete, is the only stage in the life cycle that is diploid, is motile, and penetrates the mosquito’s gut wall, lodges beneath the basement membrane facing the mosquito’s hemocoel, undergoes a postzygotic reduction division, and vacuolates to form an oocyst. Within this structure, thousands of sporozoites are formed by asexual division. The enlarging cyst eventually ruptures, releasing the sporozoites into the body cavity of the mosquito. Some penetrate the salivary glands, rendering the mosquito infectious for humans. The time required for the completion of the cycle in mosquitoes varies from 1 to 3 weeks, depending on the species of insect and parasite as well as on the ambient temperature and humidity.

Sporozoites from the mosquito’s salivary glands are injected into the human’s subcutaneous capillaries when the female mosquito feeds. Within minutes and up to 1 hour they attach to and invade liver cells (hepatocytes), a process mediated by a ligand present in the outer protein coat of the sporozoites (circumsporozoite protein). In P vivax and P ovale infections, some of the sporozoites enter a dormant state immediately after cell invasion to become hypnozoites. These stages are responsible for the relapse phenomenon seen in malarial infections caused by these species. In all malarial infections, the remaining sporozoites initiate exoerythrocytic schizogony, each producing about 2000 to 40 000 daughter cells, or merozoites, depending on the infecting species. After 1 to 2 weeks, the infected hepatocytes rupture, releasing merozoites into the general circulation.

The erythrocytic phase of malaria starts with the attachment of a released hepatic merozoite to a specific receptor on the RBC surface. After attachment, the merozoite releases substances from its apical organelles, the rhoptries, which affects red cell membrane fluidity resulting in invagination of the cell membrane and entry of the parasite into a parasitophorous vacuole. The intracellular parasite initially appears as a small ring-shaped trophozoite, which enlarges and becomes more active and irregular in outline. Within a few hours, nuclear division occurs, producing the multinucleated schizont. The cytoplasm eventually condenses around each nucleus of the schizont to form an intraerythrocytic cluster of 6 to 24 merozoite daughter cells. About 24 (P knowlesi), 48 (P vivax, P ovale, and P falciparum) to 72 (P malariae) hours after initial invasion, infected erythrocytes rupture, releasing the merozoites and producing the first clinical manifestations of disease. The newly released merozoites invade other RBCs, where most repeat the asexual cycle. Other merozoites are transformed into sexual forms or gametocytes. These latter forms do not produce RBC lysis and continue to circulate in the peripheral vasculature until ingested by an appropriate mosquito. The recurring asexual cycles continue, involving an ever-increasing number of erythrocytes until the development of host immunity helps contain the erythrocytic cycle. The dormant hepatic sporozoites of P vivax and P ovale survive the host’s immunologic attack and may, after a latent period of months to years, resume intrahepatic multiplication. This leads to a second release of hepatic merozoites and the initiation of another erythrocytic cycle, a phenomenon known as relapse. The life cycle of malarial parasites is summarized in Figure 51–2 and variations in the differential characteristics of the parasites infecting humans are summarized in Table 51–1.

MORPHOLOGY OF ERYTHROCYTIC PARASITES

The morphology of the stained intraerythrocytic Plasmodium parasites is shown in Figure 51–3. In stained smears, three characteristic features aid in the identification of plasmodia: Red nuclear chromatin; blue cytoplasm; and brownish-black malarial pigment, or hemozoin, consisting largely of a hemoglobin degradation product, ferriprotoporphyrin IX. The change in the shape of the cytoplasm and the division of the chromatin at different stages of parasite development are obvious. Gametocytes can be differentiated from the asexual forms by their large size and lack of nuclear division. Some of the infected erythrocytes develop membrane invaginations or caveolae–vesicle complexes, which are thought to be responsible for the appearance of the dark Schüffner dots or granules (see following text).

The appearance of each of the five species of plasmodia that infect humans is sufficiently different to allow their differentiation in stained smears, although some similarities in some stages exist between the different species. The parasitized erythrocyte in P vivax and P ovale infections is pale and enlarged and contains numerous Schüffner dots. All asexual stages (trophozoite, schizont, merozoite) may be seen simultaneously. Cells infected by P ovale are elongated and frequently irregular or fimbriated in appearance. In P malariae infections, the RBCs are not enlarged and contain no granules. The trophozoites often present as “band” forms, and the merozoites are arranged in rosettes around a clump of central pigment. In P falciparum infections, the rings are very small and may contain two chromatin dots rather than one. There is often more than one parasite per cell, and parasites are frequently seen lying against the margin of the cell. Intracytoplasmic granules known as Maurer dots may be present, but are often cleft shaped and fewer in number than Schüffner dots. Schizonts and merozoites of P falciparum are not present in the peripheral blood as they are sequestered in postcapillary venules. Gametocytes are large and banana shaped. Plasmodium knowlesi shares many of the morphologic characteristics of P malariae, but can be distinguished from the latter by its fever cycle and diagnostically by using polymerase chain reaction (PCR). These characteristics are summarized in Table 51–1.

PHYSIOLOGY

Species of plasmodia differ significantly in their ability to invade subpopulations of erythrocytes; P vivax and P ovale attack only immature cells (reticulocytes), whereas P malariae attacks only senescent cells. During infection with these species, therefore, no more than 1% to 2% of the cell population is involved. Plasmodium falciparum, in contrast, invades RBCs, regardless of age, and may produce very high levels of parasitemia and particularly serious disease. In part, these differences may be related to the known differences in the RBC receptor sites available to the individual Plasmodium species. In the case of P vivax, the site is closely related to the Duffy blood group antigens (Fy^a and Fy^b). Duffy-negative individuals, who constitute most people of West African ancestry, are therefore resistant to vivax malaria. RBC sialoglycoproteins, particularly glycoprotein A, has been implicated as the P falciparum receptor site.

Certain RBC genetic polymorphisms may also affect parasitism. The altered hemoglobin (hemoglobin S) associated with the sickle cell trait limits the intensity of the parasitemia caused by P falciparum, and thereby provides a selective advantage to individuals who are heterozygous for the sickle cell gene. Thus, the sickle cell gene, which would otherwise be disadvantageous, is very common in populations living in malarious areas. Parasite growth appears to be retarded in RBCs heterozygous for hemoglobin S (SA) when they are exposed to conditions of reduced oxygen tension such as those which might be present in the visceral capillaries. These conditions cause the hemoglobin in infected cells to polymerize, rendering it unusable by the parasite. In essence, the parasite starves to death. Sickling may also render the erythrocyte more susceptible to phagocytosis or directly damage the parasite. A similar protective effect may be exerted by hemoglobins C, D, and E; thalassemias; and glucose-6-phosphate dehydrogenase (G6PD) or pyridoxal kinase deficiencies, because these abnormalities have also been found more frequently in malarious areas. The protection in these conditions may be related to the increased susceptibility of such RBCs to oxidant stress. In thalassemia, the protection may also be related in part to the production of fetal hemoglobin, which retards maturation of P falciparum, as well as an increased binding of antibodies to modified parasitic antigens (neoantigens) presenting on the surface of the erythrocytes.

Once invasion has occurred, malaria parasites may induce several changes in the erythrocytic membrane. These include alteration of its lipid concentration, modification of its osmotic properties, and incorporation of parasitic neoantigens, rendering the RBCs susceptible to immunologic attack. Plasmodium vivax and P ovale stimulate the production of caveolae–vesicle complexes, which are visualized as Schüffner dots in stained smears. In P falciparum infections, electron-dense elevated knobs or excrescences form on the RBC surface. These produce a strain-specific, high–molecular-weight adhesive protein (PfEMP1), which mediates binding to receptors on the endothelium of capillaries and postcapillary venules of the brain, placenta, and other organs, where they can produce obstruction and microinfarcts.

Malarial parasites generate energy by the anaerobic metabolism of glucose. They appear to satisfy their protein requirements by the degradation of hemoglobin within their acidic food vacuoles, resulting in the formation of the malarial pigment (hemozoin) mentioned previously. It has been estimated that the average plasmodium destroys between 25% and 75% of the hemoglobin of its host erythrocyte. Unlike their vertebrate hosts, malarial parasites synthesize folates de novo. Thus, antifolate antimicrobials such as pyrimethamine are effective antimalarial agents.

GROWTH IN THE LABORATORY

Continuous in vitro cultivation of plasmodia in human erythrocytes was first achieved in 1976. More recently, the sporogonic cycle has been propogated in laboratory reared mosquites. These twin developments provide new opportunities for studying the biology, immunology, and chemotherapy of human malaria. The most immediate impact of these advances has been on the introduction of methods for testing the sensitivity of P falciparum to chemotherapeutic agents. Ultimately, these agents will play critical roles in the development of effective antimalarial vaccines.

MALARIA

EPIDEMIOLOGY

Malaria has a worldwide distribution between 45°N and 40°S latitude, generally at altitudes below 1800 m. Plasmodium vivax is the most widely distributed of the four species, and together with the uncommon P malariae, is found primarily in temperate and subtropical areas. Plasmodium falciparum is the dominant organism of the tropics. Plasmodium ovale is rare and found principally in Africa. Plasmodium knowlesi, first recognized in humans in 1965, accounts for up to 70% of the infections recorded in some areas of Southeast Asia. It is also a zoonotic species, with long-tailed macaques being the dominant reservoir.

The intensity of malarial transmission in an endemic area depends on the density and feeding habits of suitable mosquito vectors and the prevalence of infected humans, who serve as parasite reservoirs. In hyperendemic areas (areas where more than half of the population is parasitemic), transmission is usually constant, and disease manifestations are moderated by the development of immunity. Mortality is largely restricted to infants and to nonimmune adults who migrate into the region and is primarily caused by P falciparum. When the prevalence of disease is lower, transmission is typically intermittent. In this situation, solid immunity does not develop and the population suffers repeated, often seasonal, epidemics, the impact of which is shared by people of all ages.

Presently, an estimated 2 billion people live in malaria-endemic areas in 103 of the poorest countries of Africa, Asia, Latin America, and Oceania. Within these areas, malaria transmission has been reduced significantly over the last 15 years, largely as the result of the compliant use of insecticide impregnated bed nets (Figure 51–4). Still, many within these areas are thought to be carrying the malaria parasite at any given time. Approximately 700 000 individuals, primarily African children, die of malaria annually. Although endemic malaria disappeared from the United States three decades ago, imported cases continue to be reported. An increase in international travel has resulted in an increase in the number of US cases to approximately 1500 to 2000 annually as reported by the CDC. Forty-five percent of the patients with imported malaria have acquired the disease in Africa, 30% in Asia, and 10% in the Caribbean or Latin America. Fifty percent of recent infections have involved American travelers: Nearly 60% of these acquired their infection in Africa. Climatologists and epidemiologists warn that global warming could enhance mosquito and therefore malaria transmission into areas where malaria was once endemic. Current vector distribution for malaria is shown in (Figure 51–5).

Clinical manifestations of malaria typically develop within weeks to months of arrival of cases in the United States; however, 25% of cases caused by P vivax are delayed beyond that time. Approximately 40% of imported cases and almost all associated fatalities have been caused by the virulent P falciparum. Tragically, most of these cases could have been prevented or successfully treated. Congenital malaria in infants born in the United States of mothers from malarious areas is occasionally observed. Infections transmitted by transfusions of whole blood, leukocytes, or platelets, or by organ transplantation are, fortunately, now unusual in this country due to the improved screening procedures of blood banks.

Anopheline mosquitoes capable of transmitting malaria are present throughout much of the United States. On rare occasions, malaria is transmitted from an imported case to individuals who have never traveled outside of the country.

PATHOGENESIS

The fever, anemia, circulatory changes, and immunopathologic phenomena characteristic of malaria are all the result of the erythrocytic cycle of the plasmodia. There are no clinical signs of infection associated with the liver phase of infection.

Fever

Fever, the hallmark of malaria, appears to be initiated by the process of RBC rupture that leads to the liberation of a new generation of merozoites. To date, all attempts to detect the factor(s) mediating the fever have been unsuccessful. It is possible that parasite-derived pyrogens are released at the time of red cell rupture; alternatively, the fever might result from the release of interleukin-1 (IL-1) and/or tumor necrosis factor (TNF) from macrophages involved in the ingestion of parasitic or erythrocytic debris. Early in malaria, RBCs appear to be infected with malarial parasites at several different stages of development, each inducing erythrocyte destruction at a different time. The resulting fever is irregular and hectic. Because temperatures higher than 40°C destroy mature parasites, a single population eventually emerges, parasite replication is synchronized, and fever occurs in distinct paroxysms at 24 hour (P knowlesi), 48 hour (P falciparum, P vivax, P ovale) or, in the case of P malariae, 72 hour intervals. Periodicity is seldom seen in patients who are rapidly diagnosed and treated. Periodicity is also not always a hallmark of P falciparum infections. Fever-induced modifications to membrane architecture and infected-cell sequestration events are thought to play a role in disrupting periodicity in these infections. Sometimes, the fever can more or less be continuous.

Anemia

Parasitized erythrocytes are phagocytosed by a stimulated reticuloendothelial system or are destroyed at the time of parasite-induced cell rupture, releasing toxic products. This not only results in destruction of infected cells, but noninfected ones as well, resulting in an anemia that may be disproportionate to the degree of parasitism. Depression of marrow function, sequestration of erythrocytes within the enlarging spleen, and accelerated clearance of nonparasitized cells all appear to contribute to the anemia. So too might cytokine imbalances brought about by overstimulation of innate immune responses. Such imbalances can influence erythropoiesis. Intravascular hemolysis, though uncommon, may occur, particularly in P falciparum malaria. When hemolysis is massive, hemoglobinuria develops, resulting in the production of dark urine. This process in conjunction with malaria is known as blackwater fever.

Circulatory Changes

The high fever results in significant vasodilatation. In falciparum malaria, vasodilatation leads to a decrease in the effective circulating blood volume and hypotension, which may be aggravated by other changes in the small vessels and capillaries. The intense parasitemias of P falciparum is capable of producing comas and the adhesion of infected RBCs to the endothelium of visceral capillaries can impair the microcirculation and precipitate tissue hypoxia, lactic acidosis, and hypoglycemia. Although all deep tissues are involved, the brain is the most intensely affected resulting in what has been described as cerebral malaria (Figure 51–6).

Cytokines

Elevated levels of IL-1 and TNF are consistently found in patients with malaria. Probably released at the time of parasite rupture from erythrocytes, these proteins are certainly an essential part of the host’s immune response to malaria. By modulating the effects of endothelial cells, macrophages, monocytes, and neutrophils, they may play an important role in the destruction of the invading parasite. However, TNF levels increase with parasite density, and high concentrations appear harmful. TNF has been shown to cause upregulation of endothelial adhesion molecules; high concentrations might precipitate cerebral malaria by increasing the sequestration of P falciparum-parasitized erythrocytes in the cerebral vascular endothelium. Alternatively, excessive TNF levels might precipitate cerebral malaria by directly inducing hypoglycemia and lactic acidosis.

Other Pathogenic Phenomena

Thrombocytopenia is common in malaria and appears to be related to both splenic pooling and a shortened platelet lifespan. Both direct parasitic invasion and immune mechanisms may be responsible. There may be an acute transient glomerulonephritis in falciparum malaria and progressive renal disease in chronic P malariae malaria. These phenomena probably result from the host immune response, with deposition of immune complexes in the glomeruli.

IMMUNITY

Once infected, the host quickly mounts a stage-, species-, and strain-specific immunologic response that typically limits parasite multiplication and moderates the clinical manifestations of disease, without eliminating the infection. A prolonged recovery period marked by recurrent exacerbations in both symptoms and number of erythrocytic parasites follows. Recrudescences are marked by periods in which the parasitemia drops below the threshold of detection, only to surge again. Fluctuations in immunity probably account for this phenomenon. With time, these recrudescences become less severe and less frequent, and eventually may stop altogether.

The exact mechanisms involved in this recovery are uncertain. In simian and probably in human malaria, recovery is known to require the presence of both T and B lymphocytes. It is probable that the T lymphocytes act partially through their helper effect on antibody production. Some authorities have suggested that they also play a direct role through lymphokine production by stimulating effector cells to release nonspecific factors capable of inhibiting intraerythrocytic multiplication. The B lymphocytes begin production of stage- and strain-specific antiplasmodial antibodies within the first 2 weeks of parasitemia. With the achievement of high levels of antibodies, the number of circulating parasites decreases. The infrequency with which malaria occurs in young infants has been attributed to the transplacental passage of such antibodies. It is uncertain whether they are directly lethal, act as opsonizing agents, or block merozoite invasion of RBCs. Antibody responses are also detectable against sporozoites and, because of this, much attention has been given to develop a vaccine against this parasite stage. Because sporozoites clear so quickly from the peripheral circulation, however, they may escape immune detection and all it would take is one to initiate hepatic schizogony resulting in blood stage infection. Antibodies against sporozoites have no effect on erythrocytic stages of infection.

In simian malaria, the parasite can undergo antigenic variation and thereby escape the suppressive effect of the antibodies. This antigenic variation leads to cycles of recrudescent parasitemia, but ultimately to production of specific antibodies to the variants, and cure. In P falciparum malaria, chronic infection is maintained through the insertion of highly polymorphic variant antigens that are inserted into the infected erythrocyte membrane. With P falciparum, the disease typically does not exceed 1 year, but with P malariae the erythrocytic infection can be extremely persistent, lasting in one case up to 53 years. How erythrocytic parasites circulating in numbers too small to be detected on routine blood films escape immunologic destruction remains a puzzle. In a closely related simian malaria, splenectomy results in rapid cure, suggesting that suppressor T lymphocytes in the spleen may play a protective role. In infection with P vivax and P ovale, latent hepatic infection may result in the discharge of fresh merozoites into the bloodstream after the disappearance of erythrocytic forms. This phenomenon, known as relapse, can maintain infection for 3 to 5 years or longer.

In almost all cases, immunity to malaria is usually short lived and does not result in a sterile immunity. Many individuals, living in areas where transmission is sporadic, can be infected multiple times by the same species of parasite. A question often asked: If natural infection with malaria does not result in a lasting or sterile immunity, can a vaccine be developed that will?

MALARIA: CLINICAL ASPECTS

MANIFESTATIONS

The incubation period between the bite of the mosquito and the onset of disease is approximately 2 weeks. With P malariae and with strains of P vivax in temperate climates, however, this period is often more prolonged. Individuals who contract malaria while taking antimalarial suppressants may not experience illness for many months. In the United States, the interval between entry into the country and onset of disease exceeds 1 month in 25% of P falciparum infections and 6 months in a similar proportion of P vivax cases.

The clinical manifestations of malaria vary with the species of plasmodia but typically include chills, fever, splenomegaly, and anemia. The hallmark of disease is the malarial paroxysm. This manifestation begins with a cold stage, which persists for 20 to 60 minutes. During this time, the patient experiences continuous rigors and feels cold. With the consequent increase in body temperature, the rigors cease and vasodilatation commences, ushering in a hot stage. The temperature continues to rise for 3 to 8 hours, reaching a maximum of 40°C to 41.7°C before it begins to fall. The wet stage consists of a decrease in fever and profuse sweating. It leaves the patient exhausted but otherwise well until the onset of the next paroxysm.

Typical paroxysms first appear in the second or third week of fever, when parasite replication within erythrocytes becomes synchronized. In falciparum malaria, synchronization may never take place, and the fever may remain hectic and unpredictable. The first attack is often severe and may persist for weeks in the untreated patient. Eventually the paroxysms become less regular, less frequent, and less severe. Symptoms finally cease with the disappearance of the parasites from the blood.

In falciparum malaria, capillary blockage can lead to several serious complications. When the central nervous system is involved (cerebral malaria), the patient may develop delirium, convulsions, paralysis, coma, and rapid death. Acute pulmonary insufficiency frequently accompanies cerebral malaria, killing about 80% of those involved. When splanchnic capillaries are involved, the patient may experience vomiting, abdominal pain, and diarrhea with or without bloody stools. Jaundice and acute renal failure are also common in severe illness. These pernicious syndromes generally appear when the intensity of parasitemia exceeds 100 000 organisms per cubic millimeter of blood. Most deaths occur within 3 days.

DIAGNOSIS

Malarial parasites can be demonstrated in stained smears of the peripheral blood in virtually all symptomatic patients. Typically, capillary or venous blood is used to prepare both thin and thick smears, which are stained with Wright or Giemsa stain and examined for the presence of erythrocytic parasites. Thick smears, in which erythrocytes are lysed with water before staining, concentrate the parasites and allow detection of very mild parasitemia. Nonetheless, it may be necessary to obtain several specimens before parasites are seen. Artifacts are numerous in thick smears, and correct interpretation requires experience. The morphologic differences among the five species of plasmodia may allow their speciation on the stained thin smear by the skilled observer.

Several attempts have been made to improve the standard thin and thick smear method. One such procedure involves acridine orange staining of centrifuged parasites in quantitative buffy coat (QBC) tubes. Although it is expensive, this requires a fluorescence microscope and permits less reliable parasite speciation; its rapidity and ease of use make it attractive to laboratories that are only occasionally called on to identify patients with malaria. Simple, specific card antigen detection procedures are now available. The most widely used test, ParaSight F, detects a protein (HRP2) excreted by P falciparum within minutes. The test can be performed under field conditions and has a sensitivity of more than 95%. A second rapid test, OptiMAL, detects parasite lactate dehydrogenase, and, unlike ParaSight F, can distinguish between P falciparum and P vivax. Numerous PCR assays have also been developed for the laboratory diagnosis of malaria.

Serologic tests for malaria are offered at a few large reference laboratories but are used primarily for epidemiologic purposes. They are occasionally helpful in speciation and detection of otherwise occult infections. The recently completed sequencing of the malaria genome will lead to newer diagnostic methods.

TREATMENT

The indications for treatment rests on several factors. These include the severity of disease, the infecting species of Plasmodium, and the part of the world in which the infection was acquired. The immune status of the afflicted patient may also factor into this equation. The species and area of infection acquisition are likely to help determine if the parasite is resistant to any antimalarials or not. Falciparum malaria is potentially lethal in nonimmune individuals, such as new immigrants or travelers to a malarious area, and immunosuppressed indigenous individuals, such as pregnant women. These individuals must receive urgent treatment.

The complete treatment of malaria requires the destruction of erythrocytic schizonts, hepatic schizonts, and erythrocytic gametocytes. The first terminates the clinical attack, the second prevents relapse, and the third renders the patient noninfectious to Anopheles and thus breaks the cycle of transmission. Unfortunately, no single drug accomplishes all three goals. The present strategy of chemotherapy is shown in Table 51–2.

Termination of Acute Attack

Several agents can destroy asexual erythrocytic parasites. Chloroquine, a 4-aminoquinoline, has been the most commonly used. It acts by inhibiting the degradation of hemoglobin, thereby limiting the availability of amino acids necessary for growth. It has been suggested that the weak basic nature of chloroquine also acts to raise the pH of the food vacuoles of the parasite, inhibiting their acid proteases and effectiveness. When originally introduced, it was rapidly effective against all four species of plasmodia and, in the dosage used, free of serious side effects. However, chloroquine-resistant strains of P falciparum are now widespread in Africa and Southeast Asia; they are also found, though less frequently, in other areas of Asia and in Central America and South America. Chloroquine-resistant strains of P vivax have been reported from Papua New Guinea, India, and Pakistan, but overall remains poorly defined worldwide.

Other schizonticidal agents include quinine/quinidine, antifolate–sulfonamide combinations, mefloquine, halofantrine, and the artemisinins. Unfortunately, resistance to these agents is increasing, particularly in Southeast Asia. The artemisinins are also unique in their capacity to reduce transmission by preventing gametocyte development. Resistance to this latter first-line drug is increasing in areas of Southeast Asia.

Strains of P malariae, P ovale, and P vivax (except for some acquired in the South Pacific and South America) remain sensitive to chloroquine and may be treated with this agent. Plasmodium vivax infections acquired in New Guinea and Sumatra, however, should be assumed to be chloroquine-resistant and managed with mefloquine alone or in combination with other agents. Plasmodium falciparum has now become variably resistant to all drug groups, including the artemisinin compounds.

There is a growing consensus that the most effective way to slow the further development of drug-resistant strains of P falciparum is to use one of the artemisinins in combination with quinine/quinidine, antifolate–sulfonamide compounds, mefloquine, or halofantrine.

Radical Cure

In P vivax and P ovale infections, hepatic schizonts persist and must be destroyed to prevent reseeding of circulating erythrocytes with consequent relapse. Primaquine, an 8-aminoquinoline, is used for this purpose. Some P vivax infections acquired in Southeast Asia and New Guinea fail initial therapy owing to relative resistance to this 8-aminoquinoline. Retreatment with a larger dose of primaquine is usually successful. Unfortunately, primaquine may induce hemolysis in patients with G6PD deficiency. Persons of Asian, African, and Mediterranean ancestry should thus be screened for this abnormality before treatment. Chloroquine destroys the gametocytes of P vivax, P ovale, and P malariae but not those of P falciparum. Primaquine and artemisinins, however, are effective for this latter species.

PREVENTION

Personal Protection

In endemic areas, mosquito contact can be minimized with the use of house screens, insecticide bombs within rooms, and/or insecticide-impregnated mosquito netting around beds. Those who must be outside from dusk to dawn, the period of mosquito feeding, should apply insect repellent and wear clothing with long sleeves and pants. In addition, it is possible to suppress clinical manifestations of infection, if they occur, with a weekly dose of chloroquine. In areas where chloroquine-resistant strains are common, an alternative schizonticidal agent should be used. Mefloquine, Malarone, or doxycycline are usually preferred. The antifolate pyrimethamine plus a sulfonamide can be taken as well. However, use of this combination is occasionally accompanied by serious side effects, so it is recommended only when mefloquine- and doxycycline-resistant strains are present in the area, and then only for individuals residing in areas of intense transmission for prolonged periods of time. On leaving an endemic area, it is necessary to eradicate residual hepatic parasites with primaquine before discontinuing suppressive therapy.

General

Malaria control measures have been directed toward reducing the infected human and mosquito populations to below the critical level necessary for sustained transmission of disease. The techniques used include those mentioned previously, treatment of febrile patients with effective antimalarial agents, chemical or physical disruption of mosquito breeding areas, and residual insecticide sprays. An active international cooperative program aimed at the eradication of malaria resulted in a dramatic decline in the incidence of the disease between 1956 and 1968. Eradication was not achieved, however, because mosquitoes became resistant to some of the chemical agents used, and today malaria still infects 200 to 300 million inhabitants of Africa, Latin America, and Asia. Tropical Africa alone accounts for 100 million of the afflicted and for most of the 1 million deaths that occur annually because of this disease. The long-term hope for progress in these areas now depends on the compliant use of existing and development of new technologies.

Vaccines

Three advances in the last decade have produced the hope for the first time that an effective malaria vaccine might be within reach of medical science. The establishment of a continuous in vitro culture system and the successful propogation of malaria in laboratory-raised mosquitos has provided the large quantities of parasite needed for antigenic analysis. Development of the hybridoma technique allowed the preparation of monoclonal antibodies with which antigens responsible for the induction of protective immunity could be identified. Finally, recombinant DNA procedures enabled scientists to clone and sequence the genes encoding such antigens, permitting the amino acid structure to be determined and peptide sequences suitable for vaccine development to be identified. In 2012, a phase III clinical trial consisting of a protein fragment from the outer surface of P falciparum, fused with a hepatitis B virus protein, and combined with an immune adjuvant reduced episodes of both clinical and severe malaria in children aged 5 to 17 months by approximately 50%. This vaccine targets the preerythrocytic stage of the disease. Studies are continuing, with development of new adjuvants that may be even more potent. Hopefully, this may lead to vaccine strategies that are sorely needed throughout the developing world. Other attenuated sporozoite vaccines are currently in clinical trial.

The genus Babesia is represented by species that are close relatives of malaria belonging to the order Piroplasmida. They are small parasites of the mammalian host RBCs and are transmitted by ticks. These parasites were the first shown to be transmitted by an arthropod intermediate host. The organism involved in this instance was B bigemina, the causative agent of redwater fever in cattle.

Babesia microti is one of the parasites of interest to human health. It was first reported from a patient on Nantucket Island, Massachusetts. Since then there have been hundreds of cases reported from New England and in the states of Wisconsin, Washington, and California. Hard ticks of the genus Ixodes are the principal vectors. These ticks are also capable of transmitting Lyme disease. Within the tick vector the disease can also be transmitted between stages of development (transstadial transmission) or across generations through the ova (transovarial transmission). Babesia divergens is the primary species infecting humans in Europe. It is also transmited by Ixodes ticks. Unlike malaria, Babesia only infects the RBCs of its human host. Resulting symptoms can be flu-like with attendant symptoms of fevers, chills, sweats, etc; not too unlike malaria. Diagnosis is affected by finding the small piroplasms in blood smears (Figure 51–7). Because they resemble malaria it is often necessary to send smears to a reference laboratory or to have serologic or PCR testing performed.

Patients usually respond well to treatment with quinine and clindamycin. Because these may be poorly tolerated, atovaquone and azithromycin can also be used. Preventive measures include avoidance of areas known to be tick infected, using appropriate insecticides, wearing appropriate clothing and performing daily tick inspections if one ventures into wooded areas where ticks live.

PARASITOLOGY

Like the plasmodia, T gondii, the cause of toxoplasmosis, is an obligate intracellular apicomplexan. It differs from Plasmodium in that both sexual and asexual reproductive cycles occur within the gastrointestinal tract of felines, the definitive host. The disease is transmitted to other host species by the ingestion of oocysts passed in the feces of infected felines, or through carnivorism from one infected host to another. The principal mode of transmission to humans is either via ingestion of oocysts from contaminated cat feces or via ingestion of meat products containing tissue cysts (bradyzoites). Transplacental transmission may also occur.

MORPHOLOGY

Toxoplasma gondii was first demonstrated in 1908 in the gondi, an African rodent, by Nicolle and Manceaux. Its name, derived from the Greek toxo (arc), is based on the characteristic shape of the organism. All strains of this parasite appear to be closely related antigenically. The major morphologic forms of the parasite are the oocyst, trophozoite, and tissue cyst (Figure 51–8).

Oocyst

The oocyst is ovoid, measures 10 to 12 μm in diameter, and possesses a thick wall that makes it resistant to most environmental challenges. It may be destroyed by heat higher than 66°C and by chemicals such as iodine and formalin. In its immature form, the center of the cyst lacks internal structure. With maturation, two sporocysts appear, and later four sporozoites may be discerned within each sporocyst. Sporulation does not occur at temperatures lower than 4°C or higher than 37°C. Complete sporulation may occur within 24 to 48 hours outside the host. This form is responsible for the fecal–oral route of transmission of the parasites from felines to other warm-blooded animals.

Tachyzoite (Trophozoite)

The term “trophozoite” is used in its broadest sense to refer to the asexual proliferative forms responsible for cell invasion and clinical disease. In different stages of the asexual cycle, it is referred to by several other terms, including merozoite and tachyzoite. It is crescent or arc shaped, measures 3 by 7 μm, and can invade all nucleated cell types. Although tachyzoites are obligate intracellular organisms, they may survive extracellularly in a variety of body fluids for periods of hours to days. They cannot, however, survive the digestive activity of the stomach and, therefore, are not infective on ingestion.

Tissue Cysts

Cysts measure 10 to 200 μm in diameter. The contained organisms, referred to as bradyzoites, are like tachyzoites, but are smaller and divide more slowly. Tissue cysts are resistant to digestive enzymes and, like oocysts, are infectious to the animal that ingests them. They survive normal refrigerator temperatures but are killed by freezing and thawing and by normal cooking temperatures.

LIFE CYCLE

Definitive Host

Sexual reproduction of T gondii occurs only in the intestinal tract of felines, most commonly in the domestic cat (FIGURE 51–9). Ingested parasites enter the epithelial cells of the ileum by mechanisms like that of other apicomplexan parasites. Intracellularly, the trophozoites reside within a membrane-bound vacuole and undergo schizogony. With cell rupture, merozoites are released. The merozoites infect adjacent epithelial cells; they then repeat another asexual cycle or eventually differentiate into gametocytes, initiating sexual reproduction. Fusion of the mature male and female gametes leads to the formation of an oval, thick-walled oocyst that is then shed in the feces. In the typical infection, millions of these structures are released daily for 1 to 3 weeks. The oocysts are immature at the time of shedding and must complete sporulation in the external environment. In this process, two sporocysts, each containing four sporozoites, develop within each oocyst. The time required for sporulation typically takes 2 to 3 days, but may vary depending on the ambient temperature and moisture. Once mature, the resistant oocysts may remain viable and infectious for many months in soil.

Intermediate Hosts

Many animal species, including humans are considered intermediate hosts for this infection. Infection may be acquired via ingestion of oocysts or via carnivorism of tissue containing bradyzoites. After ingestion by a susceptible warm-blooded animal, sporozoites or bradyzoites are released from the disrupted oocyst or tissue and enter macrophages. Within these cells they are transported through the lymphohematogenous system to all organ systems. Survival within macrophages early in infections is because lysosomes are prevented from fusing with phagosomes containing the parasite. Continued intracellular division, termed endodyogeny results in the formation of 8 to 32 tachyzoites, which rupture from the macrophage and may invade any adjacent nucleated host cell to continue the asexual cycle. With the development of host immunity, many of the parasites are destroyed as macrophages become competent killers of the parasite. Within the cells of certain organs, particularly the brain, heart, and skeletal muscle, the trophozoites produce a membrane that surrounds and protects them: Within this tissue cyst, multiplication continues at a more leisurely pace. Eventually, cysts that measure up to 200 μm in diameter are produced and contain more than 1000 bradyzoites. These cysts persist intact for the life of the host or rupture, producing parasitologic relapse. If they are ingested by a carnivore, they survive the digestive enzymes and initiate infection in the new host. The persistence of cysts confers protection against superinfection. This is referred to as premunition.

TOXOPLASMOSIS

EPIDEMIOLOGY

Prevalence and Distribution

Toxoplasmosis occurs in almost all mammals and many birds. Human infections are found in every region of the globe; in general, the incidence is higher in the tropics and lower in cold and/or arid regions. In the United States, the prevalence of positive serologic evidence for the disease increases with age. By adulthood, approximately 50% of individuals worldwide can be shown to have circulating antibodies against T gondii. Seroprevalence in cats may range from about 20% in countries like Japan, where cats are more likely to be kept indoors, to over 70% in some countries where cats are likely to live in rural areas or be feral.

Transmission

Although it is known that humans may acquire toxoplasmosis in a variety of ways, data on their relative frequency are both meager and conflicting. It is likely that the route of transmission varies from population to population, and perhaps from age to age, within any given area. The most important transmission mechanisms of toxoplasmosis are discussed later.

Ingestion of Oocysts

Persons with felinophobia are inclined to the view that the deposition of oocysts in the feces of cats and their subsequent ingestion by the unsuspecting owner is the most common way in which humans acquire this important infection. Disease epidemics of toxoplasmosis associated with exposure to infected cats have been reported. Unfortunately, data from studies relating the frequency of feline exposure to the prevalence of positive serologic tests are conflicting. Acutely infected cats shed oocysts for only a few weeks. It has been shown, however, that chronically infected felines can occasionally reshed oocysts, and prevalence studies have demonstrated that 1% of domestic cats excrete oocysts at any given time. The large number of these structures passed during active shedding and their prolonged survival in the external environment greatly enhance their chance of transmission. Particularly at risk are children at play, who may come in close contact with areas likely to be contaminated with cat feces, and adults responsible for changing a cat’s litter box. It is also possible that insects can mechanically transfer oocysts to human food.

Ingestion of Tissue Cysts

Tissue cysts have been frequently demonstrated in meat produced for human consumption. They are most common in pork (25%) and mutton (10%) and less so in beef and chicken (< 1%). Although such cysts are killed at normal (well-done) cooking temperatures, an impressive array of epidemiologic information links the handling and/or ingestion of raw or undercooked meat with serologic and, occasionally, clinical evidence of disease. Confounding these data is an Indian study that demonstrated no difference between meat eaters and vegetarians in the incidence of positive serologic tests.

Congenital

Approximately 1 of every 500 pregnant women acquires acute toxoplasmosis, and approximately 10% to 20% of the involved women become symptomatic. Regardless of the clinical status of the infected mother, the parasite involves the fetus in 33% to 50% of all acute maternal infections. The risk of transplacental transmission is independent of the clinical severity of the disease in the mother, but does correlate with the stage of gestation at which she is exposed. Fetal involvement occurs in 17% of first-trimester and 65% of third-trimester infections. Conversely, the earlier a fetal infection is acquired, the more severe it is likely to be. Overall, 20% of fetuses experienced severe consequences; a similar proportion develops mild disease. The remainder are asymptomatic.

Miscellaneous

In addition to causing congenital infection, tachyzoites have been responsible for disease transmission in several other situations, including laboratory accidents, transfusions of whole blood and leukocytes, and organ transplantation. Because tachyzoites may survive for several hours in body fluids or exudates of acutely infected humans, it is possible for infection to occur after contact with such materials.

PATHOGENESIS AND IMMUNITY

In primary infection, the proliferation of tachyzoites results in the death of involved host cells, stimulation of a mononuclear inflammatory reaction, and parasite-specific antibody and cellular responses. In immunodeficient hosts, such as those with human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS), latent infections reactivate and rapid organism proliferation ensues, producing numerous widespread foci of tissue necrosis. The consequences are most serious in organs such as the brain, where the potential for cell regeneration is limited.

In normal hosts, acute infection is rapidly controlled with the development of humoral and cellular immunity. Extracellular parasites are destroyed, intracellular multiplication is hindered, and tissue cysts are formed. Except for lysis of extracellular parasites by antibody and complement, cell-mediated immunity appears to play the principal role in this process, mediated in part by IL-2, interferon-α, and cytotoxic T cells. Immunity appears to be lifelong, most likely due to the persistence of the parasite in the tissue cysts. The cysts, which are found most frequently in the brain, retina, heart, and skeletal muscle, normally produce little or no tissue reaction. The suppression of cell-mediated immunity that accompanies serious illness, or the administration of immunosuppressive agents, may lead to the rupture of a cyst and the release of trophozoites. Their subsequent proliferation and the intense antibody reaction to their presence result in an acute exacerbation of the disease.

TOXOPLASMOSIS: CLINICAL ASPECTS

MANIFESTATIONS

In most patients, infection with T gondii is completely asymptomatic. Clinical manifestations, when they do appear, vary with the type of host involved. In general, they may be grouped into one of the three syndromes listed below.

Congenital Toxoplasmosis

Immune mechanisms are poorly developed in utero. Thus, a large proportion of fetal infections results in clinical illness. If the infection spreads to the central nervous system, the outcome is often catastrophic. Abortion and stillbirth are the most serious consequences. Liveborn children may demonstrate microcephaly, hydrocephaly, cerebral calcifications, convulsions, and psychomotor retardation. Disease of this severity is usually accompanied by evidence of visceral involvement, including fever, hepatitis, pneumonia, and skin rash. Infants infected with toxoplasmosis later in prenatal development demonstrate milder disease. Many appear healthy at birth but develop epilepsy, retardation, or strabismus months or years later. Probably the most common delayed manifestation of congenital toxoplasmosis is chorioretinitis. This condition, which is thought to result from the reactivation of latent tissue cysts, typically presents during the second or third decade of life as recurrent bouts of eye pain and loss of visual acuity. The lesions are usually bilateral but focal. If the retinal macula is not involved, vision improves as the inflammation subsides. Toxoplasma gondii accounts for 25% of all cases of granulomatous uveitis seen in the United States.

Normal Host

The most common clinical manifestation of toxoplasmosis acquired after birth is asymptomatic localized lymphadenopathy. The cervical nodes are most frequently involved, but nontender enlargement of other regional groups, including the retroperitoneal nodes, also occurs. At times, adenopathy is accompanied by fever, sore throat, rash, hepatosplenomegaly, and atypical lymphocytosis, thus mimicking the clinical and laboratory manifestations of infectious mononucleosis. Occasionally, the normal host develops severe visceral involvement, which may be manifested as meningoencephalitis, pneumonitis, myocarditis, or hepatitis. Chorioretinitis after postnatally acquired infection, though documented, is uncommon. Unlike congenitally acquired ocular disease, it occurs during midlife and is generally unilateral.

Immunocompromised Host

In the immunocompromised host, toxoplasmosis is a serious, often fatal disease. If primary infection is acquired while a patient is undergoing immunosuppressive therapy for malignancy or organ transplantation, widespread dissemination of the infection with necrotizing pneumonitis, myocarditis, and encephalitis may occur. More commonly, acute disease in this population results from the activation of chronic, latent infection by immunosuppressive therapy, or from the acquisition of a concurrent immunosuppressive infection, particularly AIDS. Encephalitis occurs in 50% of such cases and in more than 90% of fatal cases. Toxoplasmic encephalitis is particularly common in AIDS patients; it is seen in approximately 10% of those with circulating toxoplasma antibodies. As such, it is a major cause of morbidity and mortality in this patient population. Clinically, encephalitis may present as a meningoencephalitis, diffuse encephalopathy, or mass lesion. Acute toxoplasmosis has also been seen as a result of organ transplantation in which immunosuppressive drugs were given to prevent organ rejection but resulted in a reactivation of latent cyst forms.

DIAGNOSIS

The diagnosis of toxoplasmosis may be established by a variety of methods. In acute toxoplasmic lymphadenitis, the histologic appearance of the involved nodes is often pathognomonic. The trophozoite may be demonstrated in tissue with Wright or Giemsa stain. Electron microscopy and indirect fluorescent antibody techniques have also been used successfully on heart transplant or brain tissue obtained by biopsy. Although tissue cysts are selectively stained by periodic acid–Schiff, their presence is not indicative of acute disease. Isolation of the organism can be accomplished by inoculating blood or other body fluids into mice or tissue cultures. Inoculation of other tissues is not usually helpful because a positive result may only reflect the presence of latent tissue cysts.

Serologic procedures are the primary method of diagnosis. To establish the presence of acute infection, it is usual to demonstrate a fourfold rise in the IgG antibody titer between acute and convalescent serum specimens. Peak titers are often reached within 4 to 8 weeks, so the acute serum must be collected early during illness. Of the many tests developed for the detection of IgG antibodies, indirect hemagglutination, indirect fluorescent antibody, or enzyme immunoassay (EIA) tests are the tests most frequently used. Titers may remain high for many years.

The detection of IgM antibodies provides a more rapid confirmation of acute infection. These antibodies appear within the first week of infection, peak in 2 to 4 weeks, and may slowly revert to negative. It also appears that immunoglobulin-M (IgM) antibodies are produced after reactivation of latent disease. EIA for IgM antibody is now commonly used. Examination of tissues, urine, and other body fluids for the presence of toxoplasma antigen, or DNA by the PCR, have been shown to be useful adjunctive tests in immunocompromised individuals and in the diagnosis of congenital infections.

TREATMENT AND PREVENTION

Usually, patients infected with toxoplasmosis do not require therapy unless symptoms are particularly severe and persistent or unless vital organs, such as the eye, are involved. Immunocompromised and pregnant women, however, should be treated if acute infection (or reactivation) is documented (Table 51–3). Routine serial serologic testing of such individuals would allow early detection of infected persons and enhance the prospects of a successful outcome. It is now clear that early treatment of acutely infected pregnant women significantly reduces the incidence of severe congenital infections and reduces the ratio of benign to subclinical forms in infants. At present, the most commonly used therapeutic regimen in the United States for toxoplasmosis is the combination of pyrimethamine and sulfonamides. Unfortunately, the former drug is teratogenic and should not be used in the first trimester of pregnancy; spiramycin, a cytostatic macrolide, is often substituted in this setting.

Although the pyrimethamine–sulfonamide combination is very effective against trophozoites, it is inactive against the cyst forms. Because both parasitic forms are present in patients with toxoplasmic encephalitis, recrudescence of illness generally follows completion of standard therapy in patients with AIDS. This may be prevented by initiating chronic, low-dose suppressive therapy after completion of the standard regimen. Atovaquone, a recently introduced hydroxynaphthoquinone, possesses activity against both trophozoites and cysts. Its use, therefore, may result in radical cure of toxoplasma encephalitis, eliminating the need for chronic suppression.

Prevention of toxoplasmosis should be directed primarily at pregnant women and immunologically compromised hosts. Hands should be carefully washed after handling uncooked meat. Cysts in meat can be destroyed by proper cooking (56°C for 15 minutes) or by freezing to –20°C. Cat feces should be avoided, particularly the changing of litter boxes.

Cryptosporidia (“hidden-spore”) are small parasites that can infect the intestinal tract of a wide range of mammals, including humans. Like many other apicomplexan parasites, they are obligate intracellular organisms that exhibit alternating cycles of sexual and asexual reproduction within the gastrointestinal tract of the same host. Long recognized as an important cause of diarrhea in animals, cryptosporidia were not identified as causes of human enteritis until 1976, when first observed in a patient with a congenital IgA immunodeficiency. The advent of the AIDS epidemic sparked an intense interest in this parasite as a problem in humans. There are at least 26 different species of Cryptosporidium currently recognized. Of 20 species and genotypes that infect humans, the most common are zoonotic species, Cryptosporidium parvum and a species, Cryptosporidium hominis, that only infects humans. The former is more likely to be encountered in rural populations, whereas the latter dominates in urban settings.

PARASITOLOGY

MORPHOLOGY

Regardless of animal host, all species of this tiny (2-6 μm) parasite appear morphologically identical. The organisms appear as small spherical structures arranged in rows along the microvilli of the epithelial cells. They are readily stained with Giemsa and hematoxylin–eosin. Although they remain external to the cytoplasm of the intestinal epithelial cell, they are clearly enveloped by a membrane of host cell origin. They are thus said to be intracellular but extracytoplasmic. The parasite replicates at this site giving rise to oocysts. Oocysts shed into the intestinal lumen contain four sporozoites that are not contained within sporocyst structures like their relative, Toxoplasma. Their cell wall provides the unusual property of acid-fastness, allowing them to be visualized with stains generally employed for mycobacteria. Oocysts are typically 5 to 6 μm in diameter.

LIFE CYCLE

Infective oocysts are excreted in the stool of the parasitized animal. Unlike those of Toxoplasma, cryptosporidia oocysts are fully mature and immediately infective to the next host on passage in the feces. After ingestion by another animal, sporozoites are released from the oocyst and attach to the microvilli of the small intestinal epithelial cells, where they are transformed into trophozoites. These divide asexually by multiple fission (schizogony) to form schizonts containing eight daughter cells known as type 1 merozoites. On release from the schizont, each daughter cell attaches itself to another epithelial cell, where it repeats the schizogony cycle, producing another generation of type 1 merozoites. In the absence of effective immunity, this phase may constitute an autoinfective portion of the life cycle allowing perpetuation of the infection.

A second generation of schizonts follows with the formation of four merozoites. These merozoites are destined to invade intestinal cells and give rise to male (microgametocyte) and female (macrogametocyte) sexual forms. Gamete development ensues and after fertilization, the resulting zygote develops into an oocyst that is shed into the lumen of the bowel. The majority, approximately 80%, possesses a thick protective cell wall that ensures their intact passage in the feces and survival in the external environment.

Approximately 20% fail to develop the thick protective wall. The cell membrane ruptures, releasing infective sporozoites directly into the intestinal lumen and initiating a new “autoinfective” cycle within the original host. In the normal host, the presence of innate or acquired immunity dampens both the cyclic production of type 1 merozoites and the formation of thin-walled oocysts, halting further parasite multiplication and terminating the acute infection. In the immunocompromised, both presumably continue, explaining why such individuals develop severe, persistent infections in the absence of external reinfection.

CRYPTOSPORIDIOSIS

EPIDEMIOLOGY

Cryptosporidiosis appears to involve most vertebrate groups. In all species, infection rates are highest among the young and immature. Experimental and epidemiologic data suggest that domestic animals constitute an important reservoir of disease in humans. Transmission from young animals at petting zoos to children has been documented. Outbreaks of human disease in day care centers, swimming pools, hospitals, and urban family groups indicate that most human infections result from person-to-person transmission. In Western countries, between 1% and 4% of small children presenting to medical centers with gastroenteritis have been shown to harbor cryptosporidia oocysts. In Third World countries, the rates have varied from 4% to 11%. In some outbreaks of diarrhea in day care centers, most attendees were found to have oocysts in their stool.

Infection rates of cryptosporidiosis in adults suffering from gastroenteritis is approximately one-third of that reported in children; it has been highest in family members of infected children, medical personnel caring for patients with cryptosporidiosis, male homosexuals, and travelers to foreign countries. In the United States, the parasite was identified in 15% of patients with AIDS and diarrhea at the onset of the epidemic. Because of the advent of antiretroviral therapies, this has been reduced to 1% to 2%; in Haiti and Africa, high percentages of such individuals who do not have access to antiretroviral therapies may be involved. Asymptomatic carriage is uncommon in these populations. In many developing countries, most children may acquire multiple infections with Cryptosporidium before the age of 5 years. After that infections can still be detected, but symptoms may be absent suggesting that constant exposure may help maintain a measure of immunity.

Because oocysts are found almost exclusively in stool, the principal transmission route of cryptosporidiosis is undoubtedly by direct fecal–oral spread. Transmission via contaminated water has been documented. Most noteworthy was the outbreak involving municipal water in the city of Milwaukee in 1993. An estimated 403 000 people were infected via primary and then secondary spread of the organism. The hardy nature of the oocysts, which do not respond to conventional chlorine treatment of water and many other commonly used disinfectants, makes it likely that there is also indirect transmission via contaminated food and fomites. Flies and shellfish have been incriminated as transport hosts for this parasite.

PATHOGENESIS AND IMMUNITY

Although the jejunum is most heavily involved, cryptosporidia have been found throughout the gastrointestinal, and even in the respiratory, tract, particularly in immunocompromised patients. Cryptosporidial cholecystitis is seen with some frequency in AIDS patients with enteritis. By light microscopy, bowel changes appear minimal, consisting of mild-to-moderate villous atrophy, crypt enlargement, and a mononuclear infiltrate of the lamina propria. The pathophysiology of the diarrhea is unknown. The vital role played by the host’s immune status in the pathogenesis of the disease is indicated by both the enhanced susceptibility of the young to infection and the prolonged severe clinical disease seen in immunocompromised patients. Indirect evidence suggests that antibodies in the intestinal lumen exert a protective effect against initial C parvum infection. Experimental animal studies indicate that CD4+ T lymphocytes and interferon play independent roles in the immunologic clearance of the parasite.

CRYPTOSPORIDIOSIS: CLINICAL ASPECTS

MANIFESTATIONS

Immunocompetent patients usually note the onset of explosive, profuse, watery diarrhea 1 to 2 weeks after exposure. Typically, cryptosporidiosis persists for 5 to 11 days and then rapidly abates. Occasionally, purging, accompanied by a mild malabsorption and weight loss, continues for up to 1 month. A few patients complain of nausea, anorexia, vomiting, and low-grade fever. Except for its shorter duration, more prominent abdominal pain, and relative lack of flatulence, the clinical manifestations of cryptosporidiosis closely resemble those produced by Giardia lamblia. Radiographic and endoscopic examinations of the gut are either normal or demonstrate mild, nonspecific abnormalities. Recovery is complete.

Cryptosporidiosis has been described in patients with a broad range of immunodeficiencies, including childhood malnutrition in Third World countries, AIDS, congenital hypogammaglobulinemia, and in those resulting from cancer chemotherapy and immunosuppressive management of organ transplantations. In such patients, cryptosporidiosis is usually indolent at onset, and manifestations are like those seen in normal hosts, but the diarrhea is more severe. Fluid losses of up to 17 L/day have been described. Patients with biliary cryptosporidiosis present with typical manifestations of cholecystitis and cholangitis. Unless the immunologic defect is reversed, the disease usually persists for the duration of the patient’s life. Weight loss is often prominent. The prognosis depends on the nature of the underlying immunologic abnormality; 50% of the patients with AIDS die within 6 months. Although other intercurrent infections are usually the direct cause of death, malnutrition and complications of parenteral nutrition contribute.

DIAGNOSIS

The diagnosis of cryptosporidiosis is established by the recovery and identification of Cryptosporidium oocysts in a recently passed or preserved diarrheal stool. Oocyst excretion is most intense during the first week of illness, tapers during the second week, and generally stops with the cessation of diarrhea. Because cryptosporidia oocysts are acid-fast, a presumptive identification can be established with any one of the acid-fast staining procedures developed for mycobacteria (Figure 51–10). This is best used in the hands of a competent diagnostician with experience. A direct immunofluorescence antibody stain using a monoclonal antibody to oocyst wall has been introduced, and is superior to acid-fast stains, but more time consuming and expensive. When direct examinations are negative, concentration procedures are used and the concentrate restained. Immunofluorescence and EIAs for the detection of anticryptosporidial antibodies are available as are EIAs and PCR methods for application to stool samples.

TREATMENT AND PREVENTION

In the immunocompetent patient, the disease is self-limited and attempts at specific antiparasitic therapy are not warranted; rehydration may be required in small children. In the immunocompromised host, the severity and chronicity of the diarrhea warrant therapeutic intervention. Unfortunately, there is no uniformly effective anticryptosporidial agent available now. Paromomycin, a luminal antimicrobial, has been shown to reduce the intensity of diarrhea in some patients, and parenteral octreotide acetate, a somatostatin analog, has been useful in decreasing stool volumes. Macrolide antimicrobials have also been suggested in difficult cases. Nitrazoxanide, a synthetic drug, has been approved for use in all patients over 1 year of age in the United States and is reported to have a cure rate of 72% to 88% by the CDC. Parasitologic cure with this drug approaches 80%. The only uniformly successful approach has been the reversal of underlying immunologic abnormalities. When appropriate, withdrawal of cancer chemotherapy agents or immunosuppressive drugs may result in a cure.

The stools of patients with cryptosporidiosis are infectious. Stool precautions should be instituted at the time the diagnosis is first suspected; for the immunosuppressed patient, this should be whenever diarrhea, regardless of the presumed cause, is first noted. This is particularly important in cancer chemotherapy and transplantation units, where spread of the disease from a symptomatic patient to other immunosuppressed patients can have life-threatening consequences. Oocysts can survive for many months in the external environment and have been found in most water sources across the United States. The infectious dose for this parasite is acknowledged to be very low.

CYCLOSPORA AND ISOSPORA

This parasite was first recognized in the 1980s, but it was not until 1993 that it was shown to be closely related to both Cryptosporidium and Toxoplasma. The species that infects humans is Cyclospora cayetanensis. The species name has been derived from the University in Lima, Peru, where much initial work was done on this parasite. This parasite gained notoriety because of foodborne outbreaks of illness that were ultimately linked to raspberries imported into the United States. Similar outbreaks have now been documented in many countries. Humans appear to be the only host for this parasite and its normal endemicity is usually linked to underdeveloped countries.

The parasite has an oocyst that measures 8 to 10 μm in diameter and stains acid-fast variable. It is remarkably like Cryptosporidium in its appearance, but is larger (Figure 51–11). A big difference between the two is that it takes a week or longer for the oocyst to complete sporulation outside the host. Because of this, direct person-to-person transmission is unlikely. Complete sporulation results in an oocyst with two sporocysts each containing two sporozoites. Where both parasites are present in populations, Cryptosporidium has both a greater incidence and prevalence.

Symptoms of cyclosporiasis mimic those of Cryptosporidium. Cyclospora is treatable with trimethoprim–sulfamethoxazole and for this reason it is important to correctly identify this parasite in stool sample as Cryptosporidium does not respond to this drug.

Isospora belli, now named Cystoisospora belli, is another protozoan closely related to Toxoplasma. Like Toxoplasma, oocysts contain two sporocysts, each with four sporozoites. Oocyst, however, are much larger in size (25-30 μm) and almost football shaped (Figure 51–13). They can be stained with modified acid-fast and trichrome procedures. Clinical disease resembles that of cryptosporidiosis and this parasite responds to treatment with trimethoprim–sulfamethoxazole. This parasite has a worldwide distribution in subtropical areas, but is not that prevalent. It is recognized as a problem in the immunocompromised.

The inclusion of this parasite group in this chapter is because at one time they were placed in the same taxonomic grouping (Sporozoa) as were the other organisms discussed in this chapter. Once classified among the Protozoa they are now known to be fungi and are placed in their own phylum, the Microspora. As the name implies, this group of organisms is characterized by producing small spores. There are over 1200 known species parasitizing a very wide variety of eukaryotic hosts.

These parasites came to our attention because of the advent of the HIV/AIDS pandemic and they are still recognized largely as parasites of the immunocompromised. There have, however, been reports of infections caused by these organisms in children of certain African countries.

At least 14 different species have been recorded from humans. The principal ones of concern are Enterocytozoon bieneusi and three different species of Encephalitozoon. Enterocytozoon bieneusi inhabits the intestinal tract and causes diarrhea. Encephalitozoon spp. can disseminate to a wide variety of organ sites within the body. Infections begin by the ingestion of spores that discharge a polar filament in the environment of the small intestine. Sporoplasm containing a nucleus travels through this polar filament into host cells that have been punctured. Because of this rapid discharge process, the microsporidia have been referred to as nature’s perfect syringe. The sporoplasm and nuclei divide within infected cells ultimately resulting in the formation of more spores which can continue the life cycle (Figure 51–12).

The easiest way to diagnose these infections is from stained clinical smears, especially of fecal samples. A quick hot chromotrope technique or acid-fast trichrome is often used to stain the spores (Figure 51–13). Chemofluorescent stains such as calcofluor white are also useful.

Immune resolution using antiretrovirals resolves enteric microsporidiosis. Fumagillin has proven efficacious against E bieneusi. Albendazole has proven useful against disseminated microsporidiosis and a combination of fumagillin drops and albendazole is recommended for ocular infections.