Enteric spore-forming opportunistic parasites in HIV / AIDS.

Human Immunodeficiency Virus (HIV) infection causes progressive damage to both limbs of the immune system, which results in a plethora of opportunistic infections. Among the various opportunistic infections, gastrointestinal infections are very common in HIV / Acquired Immunodeficiency Syndrome (AIDS). Opportunistic spore-forming protozoal parasites, namely, Cryptosporidium parvum, Isospora belli, Cyclospora cayetanensis, and Microsporidia, play a major role in causing chronic diarrhea, accompanied with weight loss, in patients with HIV / AIDS. The purpose of this review is to discuss the salient microbiological, clinical, and diagnostic aspects of important enteric spore-forming opportunistic parasites in HIV / AIDS.

INTRODUCTION

Human Immunodeficiency Virus (HIV) infection, causes progressive damage to both limbs of the immune system, which results in a plethora of opportunistic infections. Among the various opportunistic infections, gastrointestinal infections are very common in HIV / Acquired Immunodeficiency Syndrome (AIDS). It is estimated that 30 – 60% and 90% of AIDS patients in developed and developing countries, respectively, suffer from diarrhea.[1]

Opportunistic spore-forming protozoal parasites, namely, Cryptosporidium parvum, Isospora belli, Cyclospora cayetanensis, and Microsporidia play a major role in causing chronic diarrhea, accompanied with weight loss, in patients with HIV / AIDS. Endemicity of a particular enteric parasite in the community is likely to govern the incidence and prevalence of a particular parasitic infection in HIV / AIDS.

It is imperative to detect and treat opportunistic parasitic infections in HIV / AIDS at the earliest, as many of these are associated with severe morbidity and mortality. An array of methods, such as, the direct detection of the parasite or its antigen in the clinical specimen or detection of a specific antibody response, may be used to diagnose parasitic infections. The purpose of this review is to discuss the salient microbiological, clinical, and diagnostic aspects of important, enteric, spore-forming, opportunistic parasites in HIV / AIDS.

CRYPTOSPORIDIUM PARVUM

Ernest Edward Tyzzer first made his observations on the genus Cryptosporidium in 1907, in the gastric crypts of a laboratory mouse. The name Cryptosporidium was given due to the absence of sporocysts within the oocysts, a characteristic of other coccidian parasites. Subsequently, over the next 70 years, Cryptosporidium was observed in the gastrointestinal tract of some 20, mostly healthy animals, in all four classes of vertebrates. For the first time cryptosporidiosis of humans was reported in 1976, however, it was not until the early 1980s that its clinical significance and widespread distribution were recognized.[2]

In the early 1980s, clinical and epidemiological investigations showed that cryptosporidiosis was a common cause of serious and economically significant outbreaks of neonatal diarrhea, particularly in calves and lambs, and also contributed to 4 – 7% of sporadic cases of acute gastroenteritis in humans. Subsequently, Cryptosporidium was found to cause persistent, life-threatening gastrointestinal tract infection in HIV / AIDS, often involving the hepatobiliary and respiratory tracts.[2] The frequency of C. parvum infection in HIV / AIDS patients is reported to range widely between 2.9 and 33% in various studies conducted in India.[13-6]

LIFE CYCLE

C. parvum undergoes both asexual (schizogony) and sexual (gametogony) multiplication in a single host (human or other vertebrates). Infection in humans occurs by ingestion of food and drink contaminated with oocysts of the parasite. Excystation of the mature oocyst in the small intestine releases four sporozoites, which invade the mucosal cells and undergo asexual and sexual multiplication. All the developmental stages are confined to an intracellular, extracytoplasmic location, within parasitophorous vacuoles, in the microvillous region of the host cell. The first intracellular stage is the trophozoite. The trophozoite undergoes three nuclear divisions to form a group of eight merozoites to become the first generation schizont. The merozoites released from the schizont infect other epithelial cells and develop into a second generation schizont, which is composed of four merozoites. The second generation merozoites invade other epithelial cells and form microgametocytes and macrogametocytes. A microgametocyte produces 12 - 16 microgametes, a macrogametocyte transforms into only one macrogamete. After fertilization, a zygote is formed, which later develops into an oocyst. About 20% of the oocysts are thin walled, that is, four sporozoites are covered with a single unit membrane. These oocysts are autoinfective, explaining the overwhelming infection in a susceptible host and development of life-threatening, persistent infection in immunosuppressed patients in the absence of repeated exposure to oocysts.[78] The remaining oocysts are thick walled and are resistant to adverse environmental conditions.

CLINICAL MANIFESTATION

In immunocompetent individuals, C. parvum causes a self-limiting infection, manifesting as nausea, low-grade fever, abdominal cramps, anorexia, and 5 – 10 watery stools per day. However, in AIDS patients, it causes profuse, watery diarrhea with fluid loss of over 10 L/day. In studies of human enteric cryptosporidiosis on human colon adenocarcinoma cells (Caco-2 cell monolayers), secretory diarrhea due to enterotoxic activity was demonstrated in most patients. In general, the duration and severity of diarrheal illness is directly related to the degree of the immunocompromised state. Cryptosporidiosis is not always confined to the gastrointestinal tract. Additional symptoms (respiratory infection, cholecystitis, hepatitis, and pancreatitis) have been associated with extraintestinal infections.[7]

DIAGNOSIS

For diagnosis, oocysts can be demonstrated in the fresh as well as preserved stool samples by modified acid-fast stains and immunoflourescent staining. Oocysts appear round, 4 – 6 μm in size, with the mature oocysts containing sporozoites. The number of oocysts is directly correlated with the consistency of the stool; the more diarrheic the stool, the more the oocysts present. It is also possible to detect the cryptosporidial antigen in fresh, frozen or fixed fecal material by immunoassays. When using an immunoassay, it must be ensured that the stool specimen fixative is compatible with the immunoassay reagents. Stool concentration techniques such as Sheather's sugar floatation can be employed to increase the yield of modified acid-fast stains and immunoflourescent staining, as these tests detect oocysts. However, as immunoassays detect Cryptosporidium antigen, the specimen need not be concentrated. Polymerase chain reaction (PCR) is more sensitive, specific, and allows identification of Cryptosporidium genotypes, but requires more ‘hands-on’ time and expertise and is more expensive. Various stages of the parasite can also be detected in histology preparations from all levels of the intestinal tract, with the jejunum being the most heavily infected site.[7]

TREATMENT AND PREVENTION

No effective therapy for cryptosporidiosis has been identified since the emergence of cryptosporidiosis as a significant disease in humans, therefore, detection of this parasite in immunocompromised hosts, especially those with AIDS, usually carries a poor prognosis. Various drug regimens have been evaluated for treating Cryptosporidiosis; one of the successful regimens has been a combination therapy with paromomycin 1 g BD with azithromycin 600 mg, for four weeks, followed by paromomycin monotherapy for eight weeks. Nitazoxanide has currently been evaluated for chronic cryptosporidiosis. HAART has shown a dramatic response to cryptosporidiosis. The rise in CD4 count correlates with the resolution of diarrhea. The reduction or elimination of oocysts from the environment forms the mainstay of the control of cryptosporidiosis, but it is difficult due to environmentally resistant oocysts, numerous potential reservoir hosts for zoonotic transmission, widespread environmental distribution, and occurrence of asymptomatic infections.[7]

ISOSPORA BELLI

Virchow discovered I. belli in 1860. I. belli is considered to be the only species of Isospora that infects humans, and no other reservoir hosts are recognized for this infection. The frequency of I. belli infection in HIV / AIDS patients is reported to range widely between 2.5 to 26.1%, in various studies conducted in India.[13-6] Transmission of infection occurs through ingestion of water or food contaminated with mature, sporulated oocysts

Schizogonic and gametogonic stages occur within the epithelial cells of the distal duodenum and proximal jejunum. The sporulated oocyst is the infective stage, which on excystation, releases eight sporozoites from two sporocysts, which penetrate the mucosal cells and initiate the life cycle. In the epithelium, the sporozoites develop into trophozoites, which undergo asexual multiplication (schizogony). The schizonts contain various numbers of merozoites, which are liberated after maturation and invade the adjacent epithelial cells. The merozoites may develop through the schizogony cycle again or enter gametogony and transform into macrogametocytes and microgametocytes. After fertilization, a zygote is formed, and it matures into an oocyst. Oocysts are passed in the stool, and are long, oval, and measure 20 – 30 μm by 10 – 19 μm. Further development occurs outside the body, with the development of two mature sporocyts, each containing four sporozoites, which can also be recovered from the fecal specimen.[78]

I. belli infection in immunocompetent people generally presents as transient diarrhea and is also implicated in traveler's diarrhea. However, in the immunosuppressed, particularly those with AIDS, it often presents with profuse diarrhea, associated with weakness, anorexia, and weight loss. Extraintestinal infection in AIDS patients has also been reported with the involvement of mesenteric and mediastinal lymph nodes, liver, and spleen, with the most probable mode of spread to the extraintestinal sites being through the lymphatic channels.[7]

Oocysts can be easily identified in wet preparations either directly or after concentration. However, preservation of stool in polyvinyl alcohol and subsequent microscopic examination may not be appropriate, as it becomes difficult to observe the oocyst wall. Oocysts can also be stained with auramine-rhodamine stains, but should be confirmed with wet smears or acid fast stains, particularly if the stool contains other cells or excess artifact material. Intestinal biopsy may demonstrate the organisms, while stool examination may be negative, due to the small number of organisms. Oocysts appear ellipsoidal, 20 – 30 μm long and 10 – 19 μm wide. Mature oocysts contain two sporocysts with four sporozoites each. However, the usual diagnostic stage is the immature oocyst containing a spherical mass of protoplasm.[7]

The drug of choice for treatment is trimethoprime-sulfamethoxazole. In patients allergic to sulfonamides, pyrimethamine alone is given as treatment. In HIV / AIDS patients with recurrent-to-persistent infection, the therapy must be continued indefinitely. Prevention of isosporiasis is possible with improved personal hygiene measures and sanitary conditions, to eliminate possible fecal-oral transmission from contaminated food, water, and possible environmental surfaces.[7]

Cyclospora Cayetanensis

Human infections with C. cayetanensis, first discovered in the late 1880s, have recently emerged as an infectious disease with worldwide distribution (United States, Caribbean, Central and South America, Southeast Asia, Eastern Europe, Australia, and Nepal). A number of outbreaks have been reported in the United States and Canada, involving more than 1,400 cases. The organism has been demonstrated in the feces of immunocompetent travelers to developing countries, immunocompetent subjects with no travel history, and in patients with AIDS. Numerous food and water-borne outbreaks have been reported in the recent past.[7] In a recent study conducted at Pune, Cyclospora was reported in 0.7% of the HIV-infected individuals presenting with diarrhea.[1] However, in a study conducted in North India, cyclosporiasis was reported in 3.3% of the 120 HIV-seropositive subjects.[1]

0Infection occurs by ingestion of food contaminated with feces containing mature oocysts of C. cayetanensis. Freshly passed oocysts are immature and it takes approximately five days for maturation to occur, therefore, these are not visible in stool examination. Mature oocysts contain two sporocysts, each containing two sporozoites. The life cycle, although not completely understood, is thought to be similar to that of I. belli.[7]

In an immunocompetent host, a transient diarrhea lasting for three to four days, associated with malaise, low-grade fever, fatigue, anorexia, vomiting, myalgia, and weight loss is seen. In an AIDS patient, the diarrhea may persist for 12 weeks or more. Biliary disease has also been reported.[7]

Oocysts can be demonstrated on direct examination of wet smear or after modified acid-fast and modified safranin staining, decolorizing with 1% sulfuric acid is recommended, as 3 – 5% sulfuric acid may be too strong for Cyclospora. Even with 1% sulfuric acid as a decolorizer, some oocysts may appear pale in color. Concentration of fecal specimens is recommended prior to staining. Cylospora oocysts are spherical and measure 8 – 10 μm in diameter. On a wet smear, oocysts appear non-refractile. Oocysts autofluoresce strong green (450 – 490 DM excitation filter) or intense blue (365 DM excitation filter) under UV epifuorescence. On modified acid-fast staining, using 1% sulfuric acid, the organisms appear acid-fast variable and those that do not stain may have a wrinkled appearance. On modified safranin staining, oocysts appear brilliant reddish orange.[7]

The drug of choice for treatment is trimethoprime-sulfamethoxazole. HIV / AIDS patients may require higher doses and long-term maintenance treatment in comparison to treatment in immunocompetent hosts. As the parasite is not killed with routine chlorination and drinking water treated by halogination may not be safe, boiling is recommended. Fresh fruits and vegetables should be thoroughly washed or peeled prior to eating.[7]

MICROSPORIDIA

Microsporidia comprises of more than 1,200 species, which parasitize a wide variety of invertebrate and vertebrate hosts. These organisms have long been known to be causative agents of economically important diseases in insects, fish, and mammals. However, with the advent of HIV / AIDS, they have emerged as important opportunistic pathogens.[9]

Microsporidia are unicellular obligate intracellular protozoan parasites with spores of unicellular origin. The spore is the infective stage, the only stage able to survive outside of the host cell. The spores contain a polar tubule, which is an extrusion mechanism for injecting the infective spore contents into host cells. Extensive multiplication of the parasite occurs in the host cell cytoplasm, which includes repeated divisions by binary fission (merogony) or multiple fission (schizogony) and spore production (sporogony). Both merogony and sporogony can occur concurrently in the same cell. Sporogony results in the formation of thick walled oval to cylindrical spores ranging from 1.5 × 2.5 μm to 2.5 × 4.0 μm. Thick walls renders them environmentally resistant and difficult to stain.[7]

It was not until the AIDS pandemic began in the mid 1980s that microsporidia were recognized as agents of human disease. Currently, 15 microsporidian species have been implicated in human infection, out of which most, if not all, are associated with HIV infection. Infection most probably occurs by ingestion or inhalation of spores from environmental sources or by zoonotic transmission.[79] In humans, the most common microsporidial infections are due to Entercytozoon bieneusi and Encephalitozoon intestinalis. Both species have been found worldwide, mainly in HIV-infected patients with chronic diarrhea, and also in immunocompetent persons with acute, self-limited diarrhea.[79]

E. bieneusi causes intractable diarrhea accompanied with fever, malasie, and weight loss in AIDS patients, particularly with a CD4 -T Lymphocyte count of less than 200/μl. E. bieneusi is also implicated in AIDS-related sclerosing cholangitis. In HIV-negative, immunocompetent, and otherwise healthy persons, E. bieneusi infections are associated with self-limited diarrhea. E. bieneusi might also persist as an asymptomatic infection in immunocompetent humans.[79] In a study conducted in North India, E. bieneusi was reported in 2.5% of the HIV-infected individuals.[6]

E. intestinalis is the second most prevalent microsporidial species infecting humans. Prevalence of E. intestinalis infections in HIV / AIDS is reported to range from 0 to 7.3% in various studies.[10-12] Dissemination to the kidneys, lower respiratory tract, and biliary tract can also occur.

Enteric infections can be diagnosed by light microscopy of stool specimens, tissue sections, and touch preparations from small intestinal biopsy specimens; electron microscopy of tissue sections; antigen detection; PCR; culture; and antibody detection. The spores stain poorly with hematoxylin and eosin, but can be better visualized by using Modified Trichrome, Modified Gram stains (Brown-Brenn, Brown-Hopps) and Giemsa-stained touch preparations. Tissue examination by electron microscopy is considered the gold standard for confirmation and is used for identification of the species. However, this option is not available at all laboratories and sensitivity is lower than other methods for the detection of spores in the stool or urine. Use of chemifluorescent agents (optical brightening agents) such as calcofluor, Fungi-Fluor or Uvitex 2B is now recommended as a simple and sensitive screening method for the detection of microsporidial spores in stool specimens. However, these methods are nonspecific as objects other than microsporidial spores will also fluoresce, especially when examining the stool specimen. Microsporidial spores can also be demonstrated in the clinical specimens by indirect immunofluorescence, which is considered to be more sensitive than other staining methods. PCR testing of the intestinal biopsy specimens may be helpful for diagnosis of microsporidiosis in HIV-positive patients having negative stool and biopsy results. Molecular methods would also prove immensely useful for the identification of isolates to the species level. However, commercial PCR-based methods are not yet available. In vitro culture of E. intestinalis, but not E. bieneusi is possible using various cell lines. In vitro culture can be used to assess the efficacy of antimicrobial agents on microsporidian species, apart from enabling the development of immunological reagents for diagnosis and species differentiation. However, this technique is not practical for routine clinical diagnosis. Although a variety of serologic tests for antibody detection are available, preliminary results are controversial and may not be relevant for immunocompromised patients, and have therefore not been found to be useful tools for diagnosis of microsporidiosis.[713]

Evidence indicates that a complete parasitological cure is possible with albendazole. Albendazole is more effective in treating infections with E. intestinalis as compared to E. bieneusi. The sources of human infections have not been fully defined. Based on the existing knowledge, the possibility of human-to-human, animal-to-human, and environment-to-human transmission exists. As freshly formed spores are infectious, precautions while handling body fluids and frequent hand washing may be important in preventing primary infections in health care settings.[7]