Cryptosporidisis in HIV-Infected Individuals: A Global Perspective<br>

Cryptosporidisis in HIV-Infected Individuals: A Global Perspective

Research Article

黑料网

Asma Iqbal^1,2, Mohammad A.K. Mahdy¹, Brent R. Dixon², Johari Surin¹ and Yvonne A.L. Lim^1*

¹Department of Parasitology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia

²Bureau of Microbial Hazards, Food Directorate, Health Canada, Banting Research Centre, 251 Sir Frederick Banting Driveway, P.L.2204E, Ottawa, Ontario, K1A 0K9, Canada

^*Corresponding authors:

Yvonne Lim Ai Lian
Department of Parasitology
Faculty of Medicine
University of Malaya, 50603
Kuala Lumpur, Malaysia
E-mail: limailian@um.edu.my

Received May 14, 2012; Published October 26, 2012

Citation: Iqbal A, Mahdy MAK, Dixon BR, Surin J, Lim YAL (2012) Cryptosporidiosis in HIV-Infected Individuals: A Global Perspective. 1:431. doi:10.4172/scientificreports.431

Copyright: Â© 2012 Iqbal A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

Cryptosporidiosis resulting from infection with the protozoan parasite, Cryptosporidium spp., is a significant opportunistic disease among HIV-infected individuals. It accounts for 10 to 20% of diarrheal cases in HIV-infected individuals living in developed countries, and as much as 50% in developing countries. With the multiple routes of infection of Cryptosporidium due to the recalcitrant nature of its infectious stage in the environment, it is of utmost importance that the formulation of effective and practical control and preventive strategies are based on a firm understanding of its epidemiology. Prevalence data and molecular characterization of Cryptosporidium in HIVinfected individuals is currently available from numerous countries in Africa, Asia, Europe, North America and South America, and it is clear that significant differences exist between developing and developed regions. This review highlights the current global status of Cryptosporidium infections among HIV-infected individuals, and puts forth a contextual framework for the development of integrated surveillance and control programs for cryptosporidiosis in immunocompromised patients. Given that there are few specific chemotherapeutic agents available for cryptosporidiosis, and treatment management is largely based on improving the patientsâ€™ immune status, educating HIV-infected individuals to prevent acquisition of Cryptosporidium infection alone will not be effective in improving the overall health status. Some focus should also be on patient compliance and non-compliance obstacles to the effective delivery of health care.This would require close collaborations amongst public health professionals, governmental officials, epidemiologists, clinicians, molecular biologists, parasitologists, the general public, as well as the targeted HIV-infected individuals themselves.

Keywords

HIV; AIDS; Cryptosporidium; Prevalence; Species; Genotyping; Subgenotyping

Abbreviations

AIDS: Acquired Immune Deficiency Syndrome; HIV: Human Immunodeficiency Virus; CD4: Cluster of Differentiation 4; HAART: Highly Active Antiretroviral Therapy; hsp70: Heat shock protein70; TRAP-C1: Thrombospondin Related Adhesive Protein of Cryptosporidium 1; COWP: Cryptosporidium Oocyst Wall Protein; gp60: Glycoprotein60; SSU: Small Subunit; PCR: Polymerase Chain Reaction; RFLP: Restriction Fragment Length Polymorphism; Cpgp: Cryptosporidium Partial Glycoprotein; MLST: Multilocus Sequence Typing; DHFR: Dihydrofolate Reductase

Introduction and Historical Background

Almost 30 years have lapsed since human immunodeficiency virus type I (HIV-I) was identified as the cause of the acquired immune deficiency disease syndrome (AIDS) [1,2]. Currently, the world is experiencing a global pandemic of HIV infection, and the estimated number of persons living with HIV is 34 million [3]. From its discovery in 1981 until 2010, AIDS has killed more than 25 million people. In 2010 alone, AIDS has claimed an estimated 2.4â€“2.9 million lives, of which more than 390,000 were children. A third of these deaths occurred in sub-Saharan Africa (the hardest-hit region), which is home to approximately 15% of the worldâ€™s population. Among adults, HIV prevalence was 5.2% in Africa which has resulted in an estimated 18 million orphans [3].

Given that HIV progressively weakens the bodyâ€™s immune system, infected people are susceptible to various opportunistic infectious organisms. The enteric protozoan parasite, Cryptosporidium, was first recognized as a human parasite when it was reported as a causative agent of diarrhea in a three-year-old child with self-limiting enterocolitis [4]. Its role as an opportunistic organism was realized when Meisel et al. [5] found Cryptosporidium in a clinical case of immunosuppression. It was not until the emergence of the HIV pandemic in the 1980â€™s that Cryptosporidium became widely recognized as an important human pathogen. The first case of cryptosporidiosis in a homosexual man with AIDS was reported in 1982 [6]. Since then, numerous reports worldwide have identified cryptosporidiosis as a significant pathogen in HIV-AIDS.

There are multiple routes of transmission of cryptosporidiosis in humans, including person-to-person, waterborne, foodborne and zoonotic. For example, Cryptosporidium is currently recognized as one of the most serious and difficult to control waterborne pathogens [7]. In 1993, Cryptosporidium sparked great public health interest after a very large waterborne outbreak in Milwaukee, Wisconsin, which resulted in 403,000 people being affected, with 5,000 confirmed cases of cryptosporidiosis and 100 fatalities, mostly involving immunocompromised individuals [8,9].

Currently, cryptosporidiosis is commonly reported in HIVinfected individuals and is listed as an AIDS-defining illness (Clinical Category C) by the US Centers for Disease Contol and Prevention [10]. The infection in individuals with HIV/AIDS is persistent and lifethreatening and often involves infections of the hepatobiliary and the respiratory tracts in addition to the entire gastrointestinal tract [11]. The clinical profile may be presented as a self-limiting infection, acute or chronic diarrheic with debilitating diarrhea frequently accompanied by weight loss, dehydration, abdominal pain and poor absorption. The chronic nature of these infections contributes to the increase in morbidity and mortality in HIV/AIDS patients [12].

At present, due to limited available molecular epidemiological data, especially amongst immunocompromised patients, the actual global status of the species, genotypes and subgenotypes of Cryptosporidium in HIV-infected individuals is difficult to assess. Moreover, although data on prevalence and molecular characterization are currently available from various countries in regions such as Africa, Asia, Europe, North America and South America, there has been little effort in collating these data and placing information into perspective for future work. Therefore, the aims of the present article were to review the current global knowledge in terms of prevalence and molecular characterization of cryptosporidiosis in HIV-infected individuals and to offer recommendations for an improved understanding of Cryptosporidium infections and its global epidemiology, and control measures in HIV-infected individuals.

Cryptosporidium species and genotypes

Currently, there are 23 species of Cryptosporidium collectively found in humans, mice, cattle, pigs, sheep, horses, goats, cats, dogs, kangaroos, chickens, turkeys, fishes, ferrets, lizards, tortoises, monkeys, and deer [13,14]. Of these, at least seven have been found to infect HIV-infected individuals (Table 1). Due to the lower immunological status of immunocompromized individuals, infections with Cryptosporidium are not only caused by the predominant human species (i.e., C. hominis and C. parvum) but these individuals are also more susceptible to infections by other minor human species, especially C. meleagridis, C. felis, C. muris, C. canis and C. suis.

Table 1: Current valid taxonomic nomenclature of Cryptosporidium species and their host ranges.

In recent years, there has been considerable interest in the high prevalence of Cryptosporidium infections in livestock, and the possible role that these animals may play in the zoonotic transmission of this parasite [15]. It may occur through direct contact with oocysts in animal feces, as in the case of animal handlers, veterinarians, farmers and their families, and farm or petting zoo visitors. In the UK, human infection with C. parvum dramatically declined after implementation of several intervention measures that reduce human contact with livestock, which have been previously reported as a risk factor of cryptosporidiosis [16]. Zoonotic transmission, however, may also occur by indirect means through contaminated water or food. For example, produce may become contaminated directly through the use of manure application to crop lands, or indirectly through irrigation or processing with contaminated water. There is also some evidence for the presence of Cryptosporidium oocysts in products of animal origin, such as milk and meat [17]. Livestock have also been implicated as the source of waterborne outbreaks in Canada [18] and England [19].

A large number of prevalence and molecular characterization studies have been done worldwide in recent years on Cryptosporidium infections in dairy calves [20,21]. Among cattle, calves are susceptible to infection shortly after birth and remain so for several months [16]. Infection in dairy calves is most often detected between 8 and 15 days of age, whereas infection in beef calves most often occurs between 1 and 2 months of age [22]. The prevalence of this parasite has generally been reported to be high, with some studies reporting a 100% cumulative prevalence in dairy calves [23,24]. Dairy cattle, and especially dairy calves, may, therefore, pose a greater risk of infection to humans than beef cattle [16]. In addition, cattle living in close proximity to rivers should be considered potential causes of water contamination [21,25]. A recent study showed an increased risk of infection in humans while camping on contaminated land grazed by cattle slurry [26]. This study suggested that an increase in time between grazing and camping was the most important control strategy, but increasing hand-washing frequency and the removal of cattle feces before camping were also beneficial.

Genetic variants of C. parvum isolated from humans that are rarely found in animals have been identified using subgenotyping analysis, suggesting that human infection with C. parvum might have originated from humans themselves [27]. C. meleagridis, a parasite originally described in turkeys [28], has been detected in humans in the UK [19], Thailand [29] and Peru [30,31], implicating domestic pets as a potential source of infection for humans. Dogs and cats seem to be most commonly infected with the predominantly host-adapted C. canis and C. felis [32], and may not represent important zoonotic reservoirs of Cryptosporidium infection. Zoonotic cryptosporidiosis from exposure to pets has not been reported in healthy adults, but transmission of bovine C. parvum from companion animals (cats and dogs) to HIV-infected persons has been reported [33]. In addition, other Cryptosporidium species such as C. felis, C. canis and C. meleagridis have also been shown to infect healthy children and adults [34,35].

Transmission of cryptosporidiosis

Cryptosporidium has different stages which include oocysts, trophozoites, schizonts, merozoites, and sexual stages such as micro- and macrogamonts. Oocysts are the exogenous stage, consisting of four sporozoites within a tough two-layered wall, and range in size from 3.8 by 4.6Â Î¼m to 6.3 by 8.4Â Î¼m, depending on the species [36]. Oocysts are highly infectious, and only 101 to103 oocysts are sufficient to produce infection and disease in susceptible hosts [37]. Cryptosporidium is an obligate parasite, and is therefore capable of completing all stages of its development (asexual and sexual) within a single host [38]. The life cycle starts with the ingestion of the infectious oocyst stage by an appropriate host. Thin-walled oocysts may also develop and can excyst in the intestinal tract causing autoinfection [39] and heavy, persistent infection, with massive shedding of oocysts in the feces of an infected patient [40]. This phenomenon may explain the mechanism of persistent infection in AIDS patients in the absence of successive oocyst exposure [38]. The prepatent period ranges from one to three weeks, whereas the patent period may persist for years, depending on the parasite species and the immune status of the host [41].

Oocysts are most commonly transmitted by the fecal-oral route through direct host-to-host contact, and indirect contamination of food or water [7,18]. Cases of human-to-human transmission have been reported between family members, sexual partners, children in day-care centres, and hospital patients and staff [38]. Zoonotic transmission has also been confirmed by epidemiological studies involving pets, farm animals, and by accidental infection of veterinary workers [9].

Pathophysiology and clinical symptoms

The main site of Cryptosporidium infection is the small intestine, although infection may spread throughout the gastrointestinal tract and extra-intestinal sites. In HIV patients, proximal small intestinal infections generally cause more severe diarrhea and reduced survival rates, compared to heavy infection of the colon which, in the absence of small bowel infection, may result in asymptomatic infection or intermittent diarrhea [42]. This group of high-risk patients frequently experience chronic or intractable disease. The whole gastrointestinal tract, including the gall bladder, pancreatic duct and even the bronchial tree can be affected [43]. Ultrasonic examination of AIDS patients with biliary cryptosporidiosis has revealed a generalized dilation of the bile duct and gall bladder [40]. Esophageal cryptosporidiosis, with parasites attached to the squamous mucosa and the luminal borders of submucosal gland and ducts has been described both in adults and in children with AIDS [44].

While cryptosporidiosis-associated diarrhea almost always resolves within a few days in otherwise healthy individuals, severe diarrhea caused by Cryptosporidium in HIV-infected patients is common and the effect of reduced CD4 T-cell counts on the persistence of diarrhea has been well documented [45]. Cryptosporidium infections in HIV-infected individuals can reduce both their quality of life and life span, especially those who are severely immunosuppressed, with a CD4 T-cell count of less than 200 cells/mm³ [46]. It is evident from previous studies that resistance to and recovery from infection with Cryptosporidium is dependent on T-cell activity [47]. At higher CD4 T-cell levels, spontaneous clearing of the parasite generally takes place [48]. Patients with a CD4 T-cell count of greater than 180 cells/ mm³ usually have a self-limiting infection, whereas most patients with less than 140 cells/mm³ develop severe and persistent infection [49]. Diarrhea is common in HIV-infected patients and is significantly associated with low CD4 T-cell counts [50]. Extra-intestinal cryptosporidiosis, such as biliary cryptosporidiosis is more frequent in patients with CD4 T-cell counts less than 50 cells/mm³ and commonly presents with right upper quadrant pain, nausea, fever, vomiting and often the absence of diarrhea [51].

In developing countries such as Mexico, Haiti and in some countries in Africa, diarrhea has been reported in more than 40% of AIDS patients [52]. Studies from India indicated that diarrhea is the third most common clinical presentation in AIDS patients [53]. The prevalence of cryptosporidiosis in HIV-infected patients with diarrhea has been reported to range from 3 to 16% in developed countries, depending on the population studied, degree of immunosuppression and the use of antiretroviral therapy [44, 54]. A study in Atlanta, USA, found that lower CD4 T-cell counts were predictive of chronic diarrhea [55]. Another study in the USA identified four distinct clinical syndromes of cryptosporidiosis such as transient diarrhea, relapsing illness, chronic diarrhea, and cholera-like illness in AIDS patients with CD4 T-cell counts of less than 200 cells/mm³ [56]. In this patient group, chronic diarrhea and cholera-like illness with severe weight loss predominated, but there was no relationship between intensity of infection and clinical syndrome and intensity of inflammatory response, although there was a trend towards acute inflammation in patients with cholera-like illness. Although reports have indicated that survival rates of HIV-infected patients are influenced by Cryptosporidium infection, there has not been a conclusive link with clinical symptoms such as diarrhea [56].

Treatment of cryptosporidiosis

Despite the magnitude and severity of Cryptosporidium infection in HIV-infected individuals, its pathogenesis is poorly understood, and there is currently no drug of choice. However, with the introduction of Highly Active Antiretroviral Therapy (HAART), the incidence of cryptosporidiosis has declined [57], and it is clear that chronic diarrhea and cryptosporidial infection often resolves with the increase in CD4 T-cell numbers [58]. In the severely affected immunocompromised patient, drug treatment is of uncertain, and probably limited, efficacy, and the infection responds best to an improved hostâ€™s immune status, for example, by means of HAART. Conversely, relapses can occur following deterioration in immune function, if infection has been suppressed and not completely cleared [43]. Usually, the aim of treatment is to reduce the duration of diarrhea, prevent complications, eliminate the organism from the host, and reduce mortality [59].

As diarrhea is self-limiting in people with a good immune status, efforts should be made to improve the immune status in immunocompromised patients using HAART, as this often leads to resolution [59]. To ensure absorption of antiretroviral drugs, symptomatic treatment with loperamide and/or opium tincture, as controlled prescriptions given at the maximum doses is advised. If this is unsuccessful, treatment with other anti-diarrheal medications can be attempted. Sufficient hydration of the patient is also important, and may involve infusions in the case of severe dehydration.

The emergence of cryptosporidiosis has triggered the screening of many compounds for potential anti-cryptosporidial activity, but unfortunately the majority has been ineffective. Currently, the drugs most commonly used to treat cryptosporidiosis include paromomycin, spiramycin, azithromycin and rifaximin. In 2001, Rossignol reported good results in individual cases of cryptosporidiosis when treated with the antihelminthic agent, nitazoxanide (brand names Alinia and Cryptaz). Nitazoxanide has been found to be effective in a small, randomized study and is possibly the first drug with real efficacy for treating cryptosporidiosis [60]. In the American Expanded-Access Program (EAP), almost two-thirds of the patients responded to treatment [61]. In June 2005, nitazoxanide was licensed for Cryptosporidium-associated diarrhea in the US. However, there is no approval yet for AIDS patients, either in the US or in Europe.

Immune reconstitution using HAART is the treatment of choice for those with HIV-related immunodeficiency, as it drastically diminishes the occurrence of life-threatening diarrhea [62]. Some studies using protease inhibitors, especially serine and cysteine protease, as a part of HAART in the treatment of HIV infection, have shown an improvement in the health status of AIDS patients suffering from cryptosporidiosis [63]. Thus, combination therapy, restoring immunity in AIDS patients along with antimicrobial treatment of Cryptosporidium infections, is necessary. Although HAART has been found to be effective, this treatment regime is not widely available, and is too expensive in many developing countries where cryptosporidiosis is particularly prevalent in HIV-infected patients [59,64]. This is true especially in developing countries such as in the African and Asian regions. Hence, effective control and prevention of cryptosporidiosis is of utmost importance.

HIV-cryptosporidiosis in Africa

HIV/AIDS is a major public health concern in Africa, where there are 22.9 million HIV cases and the largest burden of AIDS cases in the world [3]. Although Africa is inhabited by an estimated 14.7% of the global population, it contributed 66.6% of the global AIDS deaths in 2010 [3]. The potent combination of HIV and extreme poverty also results in intestinal parasitic infections being widespread in Africa [65]. Among the intestinal parasitic infections, cryptosporidiosis is one of the most common, and is of particular concern among HIV-infected individuals. Currently, 16 reports on cryptosporidiosis among HIV-infected individuals are available from 11 countries in Africa, namely Cameroon, Ethiopia, Equatorial Guinea, Guinea-Bissau, Kenya, Nigeria, South Africa, Tanzania, Tunisia, Uganda, Zambia and Zimbabwe, with prevalences ranging from 3.8 % to 73.6% [66-80] (Table 2). Uganda has recorded the highest rate (73.6% of 91 cases) of cryptosporidiosis in HIV-infected individuals [72], whilst Cameroon has the lowest (3.8% of 154 cases). Rates of cryptosporidiosis do not, however, correlate with the number of HIV cases each country has. For example, Uganda contributes 5.4% of HIV people on the African continent, whilst Cameroon contributes 6.1%. The African country with the highest HIV rate is South Africa (18%). Prevalence and species distribution of Cryptosporidium spp. vary greatly with the regions or countries studied, and even within specific demographic groups (i.e., children, adults, and women). This creates a complex picture of the epidemiology of cryptosporidiosis. Studies on cryptosporidiosis in African HIV/AIDS patients focused mainly on hospitalized patients taking into consideration not only symptomatic adults, as is usually the case, but also paediatric cases [72,77].

Table 2: Available information on the prevalence of cryptosporidiosis in HIV-infected individuals by region.

Studies in Ethiopia, which contributes 2.1% of the African HIV/AIDS patients [3], have reported high prevalences of cryptosporidiosis (11% to 25%) [65,74,80]. Diarrhea and low CD4 T-cell counts have been found to be significant predictors of cryptosporidiosis in this country [65,66,68,71,73,74,76,77] This was evident in a study of 52 Ethiopian HIV patients with chronic diarrhea, whereby 11% had Cryptosporidium infections [74]. Similar findings were also obtained when 20.1% of 214 HIV/AIDS patients with CD4 T-cell counts of less than 200 cells/mm³ had cryptosporidiosis, with diarrhea as the most frequent symptom [65]. A prospective cross-sectional study of HIV-seropositive adult patients admitted to a government hospital in Nairobi, Kenya, revealed Cryptosporidium as the most common pathogen (17% of 75 cases). Thirty-one patients died, and detection of Cryptosporidium oocysts was the single most significant predictor of death (P<0.05) [67]. Among Nigerian HIV-infected adult patients with chronic diarrhea, C. parvum was found to be the most common parasitic infection (16.8% of 101 cases) [76], whilst another study indicated a very high percentage of cryptosporidiosis (52.7%) amongst 100 HIV-infected individuals presenting with diarrhea [77]. However, in Zimbabwe, only 9% of 82 of HIV-infected patients with diarrhea were found to have C. parvum [68]. In hospitalized children with diarrhea between the ages of 15 months and 5 years in Zambia, Cryptosporidium was again the most common parasite identified, being isolated from 14% of 44 HIV-seropositive children. Those with chronic diarrhea had significantly higher cryptosporidiosis rates compared to those with acute diarrhea [66]. Although many studies concurred that detection of Cryptosporidium is more significant among those with diarrhea, Houpt et al. [71] reported otherwise in Tanzania. In this study, the percentage of Cryptosporidium-positive patients with diarrhea (18% of 61 cases) was not significantly different from the number of Cryptosporidium-positive patients without diarrhea (16.6% of 66 cases) in HIV infected individuals (p > 0.05) [71].

Studies associating cryptosporidiosis with diarrhea have been generally evaluated amongst those infected with HIV-1 type virus. However, for the relatively uncommon HIV-2 type which is concentrated in West Africa, and rarely found elsewhere, information about the occurrence of Cryptosporidium in cases with chronic diarrhea is limited. In 2001, patients from Guinea-Bissau, the country with the highest prevalence of HIV-2 in the world [3] were evaluated and results showed that 25% of 37 HIV-2-positive patients were infected with C. parvum [69].

Recently, advances in molecular techniques have increased the sensitivity of Cryptosporidium detection, as well as allowing for a simultaneous genetic characterization of the isolates obtained from African HIV/AIDS patients. The most common PCR target gene utilised in molecular studies on Cryptosporidium in Africa is 18s rRNA [70,72,75,78,81,82], although some studies have also targeted hsp70, acetyl-CoA, TRAP, COWP and gp60 genes [70,81] (Table 3). Currently, molecular data are available from Equatorial Guinea, Kenya, Malawi, South Africa, Tunisia and Uganda. C. hominis was found to be the most common species reported in HIV-infected individuals in most of these countries, followed by C. parvum [70,72,75,78,81,82]. One exception was in Equatorial Guinea, where 36.7% of 185 HIV-infected patients were identified as positive for Cryptosporidium, and PCR-restriction fragment length polymorphism (RFLP) analysis revealed that C. parvum was the most common species found at 52.9%, while C. hominis was reported at 44.1% [79]. This study also provided some evidence for zoonotic transmission as C. meleagridis was reported in 2.9% of these samples. In addition to Equatorial Guinea, C. meleagridis has also been reported in Kenya [81,82], Tunisia [78] and Uganda [72], whereas C. muris has been reported only in Kenya [82,83]. However, C. felis, a species which has been reported in humans in other areas of the world, has not been reported in Africa.

Table 3: Molecular characterisation of Cryptosporidium detected in HIV-infected individuals by region.

South Africa has the highest rate of reported HIV/AIDS cases in Africa [3]. Currently, the only available subgenotyping information is based on 24.8% of 101 HIV-infected South African children found positive for Cryptosporidium. The study analyzed nucleic acid and amino acid sequence polymorphisms at the Cpgp40/15 locus of 20 C. parvum isolates from these children. Fifteen isolates exhibited one of four previously identified genotype I (now known as C. hominis) alleles at the Cpgp40/15 locus (Ia, Ib, Ic, and Id), while five isolates displayed a novel set of polymorphisms that defined a new Cpgp40/15 genotype I alleles, designated Ie. Only 15 of these isolates exhibited concordant C. hominis at the TRAP and COWP loci, while five isolates (all of which displayed Cpgp40/15 genotype Ic alleles) displayed C. parvum at these loci. Children infected with isolates having genotype Ic alleles were significantly older than those infected with isolates displaying other C. hominis genotypes [70].

The occurrence of these species and genotypes has revealed substantial variation amongst the different geographical locations studied, and amongst the populations selected for these studies. The generally high prevalence of C. hominis infections indicates that humans are a major source of infection and that person-to-person transmission has played a major role in the spread of Cryptosporidium infection in Africa, especially among HIV-infected patients. In addition, with the presence of zoonotic species such as C. parvum, C. meleagridis and C. muris, the role of infected animals in transmitting Cryptosporidium to humans must not be underestimated. This was evident in Tunisia, where C. meleagridis was mainly identified in children living on farms and having close contact with animals [78].

HIV-cryptosporidiosis in Asia

Asia is the largest continent in the world and is home to 60% of the global population. In terms of the number of people living with HIV, Asia has the second highest number of cases of HIV and AIDS (~ 4.7 million HIV cases) in the world after Africa [3]. Regionally, however, the number of cases has decreased since 2000 due to the advent of HAART. While the annual number of AIDS-related deaths in South and South-East Asia in 2008 was approximately 12% lower than the mortality peak in 2004, the rate of HIV-related mortalities in East Asia (China, Taiwan, Japan, Korea, and Vietnam) continues to increase, with the number of deaths in 2008 more than three times higher than in 2000 [84].

Currently, Asia has the greatest number of published studies (36) on cryptosporidiosis in HIV-infected individuals, with data available from nine countries including: Cambodia, Indonesia, India, Iran, Korea, Malaysia, Nepal, Taiwan, and Thailand. These studies have shown a wide range of Cryptosporidium infection rates (i.e., 0.5% to 56.5%) in HIV-infected individuals with or without diarrhea [48,50,57,85-118] (Table 2). The highest prevalence was recorded in a study from India, where 56.5% of 200 HIV-infected individuals had cryptosporidiosis [50], whereas a study in Taiwan reported only 0.9% of 1,044 HIV-infected patients harbouring Cryptosporidium [57].

Among the southeastern Asian countries, Thailand has a relatively high number (1.4% of global prevalence) of HIV cases [3]. Research on cryptosporidiosis in this country has been substantial, with 10 reports on HIV-infected individuals documenting prevalences ranging from 5.7% to 28.7% [109-118]. Similar to Africa, studies in Thailand also indicated higher rates of cryptosporidiosis amongst HIV-infected individuals with CD4 T-cell counts of less than 100 cells/mm³ [110,112-114,116]. Another country with high rates of HIV infection is Cambodia, with 0.8% of the global numbers [3]. However, except for one case-control study, very little is known about the epidemiology of HIV infection, or its co-infections in this country. This case-control study involving 80 HIV-infected individuals found that 45% of these patients harboured Cryptosporidium. Using PCR-RFLP, this study showed the presence of C. hominis in chronic diarrheic patients, and both C. hominis and C. parvum in asymptomatic patients [85]. In Malaysia, the first cryptosporidiosis case was reported in 1984 [119]. Subsequently, there were 5 reports with prevalence ranging from 3% to 23% [103-107]. Given that 70% of HIV-infected individuals in Malaysia are intravenous drug users, a study was carried out to determine the prevalence of Cryptosporidium amongst asymptomatic intravenous drug users, and it was found that 23% of 168 patients were positive [103]. Among hospitalized HIV patients, 3% to 16% were positive with cryptosporidiosis [104-107]. Two studies indicated that HIV patients with CD4 T-cell counts of less than 200 cells/mm³ had cryptosporidiosis [105,107], and these patients were more likely to have diarrheal symptoms. A recent study in Indonesia associated the occurrence of cryptosporidiosis (4.9% of 318 cases) in HIV-infected individuals with chronic diarrhea and CD4 T-cell counts less that 50 cells/mm³ [86].

Although limited, information on cryptosporidiosis in HIV-infected individuals is available from eastern Asia, namely Korea and Taiwan, both of which have HIV/AIDS cases of less than 0.1% [3]. Guk et al. [102] reported C. parvum infections in 10.5% of 65 HIV-infected individuals who visited Seoul National University Hospital in Korea. Interestingly, another study in Korea has suggested that naturally infected pigs may be a significant reservoir for C. parvum in human, and that pigs should be viewed as being more important than calves, because the number of pigs overwhelms the number of calves in Korea [120].

India accounts for approximately 50% of the HIV infections in Asia [3]. There are 13 reports available on the prevalence of Cryptosporidium infections from different parts of India, with prevalences ranging from 4.7% to 56.5% in HIV-infected individuals [48,50,87-96]. As observed in other parts of Asia, there is a paucity of data on the correlation of CD4 T-cell counts and the etiology of diarrhea among HIV-infected individuals. However, in one study, diarrhea was determined to be the most common symptom in HIV/AIDS patients with CD4 T-cell counts of less than 200 cells/ mm³, and Cryptosporidium infection was the highest (56.5% of 200 cases) and statically significant as compared with the other intestinal parasites [50]. In contrast, another study did not find any significant correlation between diarrhea and Cryptosporidium infection [92,95]. A study in India analysed the correlation between CD4 T-cell counts of HIV patients and the occurrence of intestinal protozoal infections in different seasons, and the results showed that the highest incidence of Cryptosporidium infection (39.8% of 366 cases) occurred during the rainy season [96]. In Nepal, cryptosporidiosis was reported in 10.7% of 75 HIV-infected individuals, associated with chronic watery diarrhea of more than one monthâ€™s duration [108].

In western Asia (i.e., Iran), there have been four reports on cryptosporidiosis in HIV-positive individuals. Using Ziehl-Neelsen staining and conventional microscopy method, the prevalence was determined to be between 0.9% and 26.7% [98-101]. Zali et al. [98] highlighted the significance of intestinal parasites among HIV-infected individuals, especially those with low immunity presenting with diarrhea. In another study [100], it was found that 26.7% of 75 HIV-infected individuals were infected with Cryptosporidium, and the probability of infection with opportunistic parasites increased as the number of CD4 T-cells decreased in the patients.

Determining the true prevalence of cryptosporidiosis in any region is important for epidemiological data; hence, a more sensitive and reliable technique is required to improve the detection rate [106]. Although most of the prevalence studies conducted in Asia have relied upon microscopy, from 2000 onwards, molecular techniques have also been employed [29,57,82,85,91,93,106,118,121-124] (Table 3). The use of microscopic diagnostic techniques (Niehl-Neelsen staining) in combination with a nested-PCR, offers one of the most sensitive methods for detecting Cryptosporidium. Based on genotyping studies, C. hominis was more prevalent than C. parvum in Cambodia [85], India [91,93], Taiwan [57], Thailand [121] and Vietnam [83]. However, in Iran [122,123] and Malaysia [105,106] C. parvum was more common.

Lim et al. [105] published the first report of C. hominis, C. meleagridis and C. felis from Malaysian HIV patients. The sequencing of amplicons derived from SSU revealed that C. parvum was the most commonly detected species at 64% of 25 cases (16 isolates), followed by C. hominis (six isolates), C. meleagridis (two isolates) and C. felis (one isolate). Sequencing of the gp60 gene identified C. parvum subgenotype IId and C. hominis subgenotypes Ia, Ib, Id, Ie and If in HIV patients [105]. Further subgenotyping analysis demonstrated C. parvum subgenotype IIdA15G2R1 and C. hominis subgenotypes IaA14R1, IbA10G2R2, IdA15R2, IeA11G2T3R1 and IfA11G1R2. Another study in Malaysia reported that of 346 fecal samples from HIV-positive patients, microscopically Cryptosporidium positive samples (i.e., 43 samples) were also tested for Cryptosporidium spp. by PCR targeting the small subunit of nuclear ribosomal RNA (SSU rRNA), and 32 (74.4% of 43) were found positive. Subsequent sequence analysis identified four distinct Cryptosporidium species which included C. parvum (84.3%) as the most frequently detected species, followed by C. hominis (6.3%), C. meleagridis (6.3%) and C. felis (3.2%) [125]. Subgenotype analysis targeting partial 60kDa glycoprotein (pgp60) gene identified 18 (5.2% of 346 cases) isolates as Cryptosporidium-positive, with 72.2% of the 18 identified as C. parvum and 27.7% as C. hominis. Gp60 analysis revealed that the C. parvum isolates included subgenotypes IIaA13G1R1 (two isolates), IIaA13G2R1 (two isolates), IIaA14G2R1 (three isolates), IIaA15G2R1 (five isolates) and IIdA15G1R1 (one isolate). C. hominis was represented by subgenotypes IaA14R1 (two isolates), IaA18R1 (one isolate) and IbA10G2R2 (two isolates) [124]. These data demonstrated the potential significance of zoonotic transmission of C. parvum as it was the predominant species in HIV individuals in Malaysia. The advantage of using subgenotyping is in clarifying the epidemiology of Cryptosporidium and raising interesting questions with regards to its population genetics. Besides the two most common human species (C. parvum and C. hominis), C. meleagridis and C. felis are also relatively common in India [91,93], Taiwan [57] and Thailand [29]. Other less common species included C. canis, which has only been reported in Thailand [29], and C. muris in India and Thailand [91,93,121].

Given that in some countries C. parvum was more prevalent, the role of zoonotic transmission was further evaluated. In a study carried out in India, 48 isolates of Cryptosporidium were identified, with 33 being identified as C. hominis. Of those patients who have potentially acquired infection via zoonotic transmission, molecular characterization identified eight as C. parvum, five as C. felis, one as C. meleagridis, one as C. muris and one as C. parvum (mouse genotype). Twenty one of these patients confirmed contact with animals such as cows, goats and dogs in both urban and rural households [91]. Based on analysis of the Cpgp40/15 locus, the subgenotypes found among 31 C. hominis isolates from Indian HIV patients included Ia, Ib, Ic, Id and If, with one mixed infection with subgenotypes Ic and Id, whereas among five C. parvum isolates, four were IIa or IIb, which have similar-sized fragments, and one was IIc [91].

In Thailand, molecular characterisation studies showed that only C. meleagridis and C. muris were present in HIV-infected children, whereas C. hominis predominated in HIV-infected adults [121]. The epidemiology of chronic diarrhea in adults with late-stage HIV infection was investigated [29]. During this investigation, [34] Cryptosporidium isolates were obtained from the patients who had symptomatic cryptosporidiosis. Genotyping of these isolates, by RFLP analysis and DNA sequencing of the 18S rRNA gene, indicated that 17 were C. hominis, with the rest being C. meleagridis (seven isolates), C. parvum (five isolates), C. felis (three isolates) and C. canis (two isolates). This was the first report of C. canis and C. parvum in HIV-infected Thai patients [29]. The Asian and African regions show an obvious difference in genetic characterization of Cryptosporidium species. As with the African regions, direct or indirect transmission of oocysts through human-to-human or waterborne routes appears to be most common in Asia, where infections with C. parvum and C. hominis predominate.Â However, the presence of zoonotic species, including C. canis, C. felis, C. muris, and C. meleagridis, in humans indicates that animal reservoirs may also be important. Â Therefore, continued molecular characterization of Cryptosporidium species and genotypes, combined with field epidemiology, is likely to lead to more rational approaches to disease control in this region and elsewhere.

HIV-cryptosporidiosis in Europe

The number of AIDS cases has declined slightly in western and central Europe due to widespread availability of antiretroviral drugs, especially HAART, in this region [84]. The prevalence of Cryptosporidium infection among HIV-infected individuals in Europe, as reported from Denmark, Italy, France, Portugal, Spain, Turkey, and the UK, ranges from 8% to 39% [126-136] (Table 2). In the mid 1990â€™s, the epidemiology of cryptosporidiosis among European patients with AIDS was described based on a cohort of 6,548 AIDS patients seen at 52 centres across 17 European countries. Countries were classified into three regions; north Europe (Denmark, Sweden, Finland, Netherlands, UK, Ireland, northern Germany), central Europe (Belgium, France, Luxembourg, Switzerland, southern Germany), and south Europe (Greece, Israel, Italy, Portugal and Spain). Overall, cryptosporidiosis was diagnosed in 6.6% of these patients. Cryptosporidiosis was reported in a significantly higher proportion of central Europeans compared to southern Europeans [137]. Waterborne transmission associated with drinking water and swimming pool contact is now one of the most common causes of human cryptosporidiosis in Europe and the UK [138].

In addition to the routes of transmission mentioned above, a nosocomial outbreak of cryptosporidiosis in AIDS patients occurred at a hospital in Copenhagen, Denmark. Thirty percent of 60 HIV-infected patients were affected during this outbreak. The source of the outbreak was identified as ice from an ice machine in the ward, which was contaminated by an incontinent, psychotic patient with cryptosporidiosis [128]. In Lisbon, Portugal, a study assessed the prevalence and factors associated with Cryptosporidium infection among 465 HIV-infected individuals. Cryptosporidiosis was reported in 36 (8% of 465) patients. Prevalence of cryptosporidial infection was higher among HIV-infected individuals whose exposure category was through sexual contact than among patients in other HIV exposure categories (, with few cases occurring in intravenous drug users [133]. The first Italian case of human C. felis infection occurred in a homosexual HIV-positive man who, in spite of a very low CD4 T-cell count, successfully recovered with paromomycin treatment. While this individual did not have a cat at home, the city where he lives (Rome) is home to a plethora of stray and domestic cats (approximately 0.1 cats per inhabitant). Infection may have occurred upon accidental contact with oocysts in the environment [139]. Also in Italy, a study reported nine cases of cryptosporidiosis in AIDS patients. Formalin-fixed faecal specimens were treated to obtain high quality genomic DNA for amplification and sequencing of the 60-kDa glycoprotein (gp60) gene. Sequence analysis revealed that one of these patients was infected with C. hominis whereas the remaining eight patients were infected with C. parvum. Interestingly, the patients showing severe cryptosporidiosis harboured two subgenotypes within the C. parvum family IIc (IIcA5G3 and IIcA5G3R2), whereas patients with moderate or mild infections showed various subgenotypes of the C. parvum family IIa (IIaA14G2R1, IIaA15G2R1, IIaA17G3R1 and IIaA18G3R1). DNA extraction and genotyping of Cryptosporidium species is a challenging task on formalin-fixed stool samples, whose diagnostic outcome is dependent on the age of the sample. This method represented a step forward in routine diagnosis, and improved the epidemiology of HIV-related clinical cases. Due to the need to elucidate genetic richness of Cryptosporidium human isolates, this approach represents a useful tool to correlate individual differences in symptoms to subgenotyping lineages [140].

In Europe, studies on cryptosporidiosis have employed molecular techniques much earlier than other areas of the world. Generally, C. parvum is more common in Europe, followed by C. hominis, C. meleagridis, C. felis, and C. muris [45,81,136,139,141-147] (Table 3). No reports of C. canis nor C. suis have been documented thus far. In the UK, C. parvum was detected in both animals and humans, and C. parvum bovine genotype was found to be dominant. With regards to cryptosporidiosis outbreaks, it was speculated that C. hominis is more infectious to humans and is therefore better adapted to this host species [19,27,143,148,149].

In France, 13 C. parvum isolates typed using PCR-RFLP at the 18S rRNA gene locus indicated possible zoonotic transmission, while six C. hominis isolates were also reported [141]. The prominence of zoonotic species was also reported by Guyot et al. [143] who found that the majority of HIV-infected patients were infected with cattle (22 out of 46 cases) or human (14 out of 46 cases) genotypes of C. parvum. In addition, three patients were infected with C. meleagridis, six with C. felis, and one with C. muris. This was the first report of these three zoonotic species in humans reported in France, and the findings indicated that immunocompromised individuals are susceptible to a wider range of Cryptosporidium species and genotypes [143]. The same observation was made in a study in Switzerland [81].

Similarly, in Portugal, molecular analysis showed that 22 HIV-infected patients were infected with C. parvum, 11 with C. hominis, four with C. felis, and three with C. meleagridis [146]. Of those infected with C. meleagridis and C. felis, three were heterosexual, two were homosexual persons, one was an intravenous drug user and one acquired HIV infection through vertical transmission [146]. Another study in Portugal characterized 16 C. parvum, seven C. hominis, three C. felis and three C. meleagridis isolates using PCR-RFLP, and subsequent DNA sequencing analysis of 18s rRNA and 60-kDa glycoprotein genes showed extensive genetic diversity in both C. parvum and C. hominis isolates. Seven alleles were identified, three corresponding to C. hominis and four corresponding to C. parvum [144]. A further subgenotyping study characterized Cryptosporidium spp. from HIV-seropositive patients, as well as cattle, sheep and wild ruminants, from different regions of Portugal using a gp60-based PCR sequencing protocol. Results showed 14 C. hominis and nine C. parvum isolates, with five different subgenotypes belonging to five subgenotype families in C. hominis, and eight subgenotypes belonging to four subgenotype families in C. parvum. Seven of the eight C. parvum subgenotypes were observed in human parasites, while only four subgenotypes were observed in animals. All animal parasites in family IIa were of the IIaA15G2R1 subgenotype, with the exception of those from seven calves that exhibited the subgenotype IIaA16G2R1. Human parasites in the IIa family were identified as subgenotype IIaA15G2R1, but not IIaA16G2R1. In humans, four subgenotypes within the subgenotype family IId were identified (IIdA17G1, IIdA19G1, IIdA21G1, and IIdA22G1), two of which, IIdA17G1 and IIdA21G1, were also found in calves and sheep, respectively. All wild ruminants had the same subgenotype (IIa), which was also the predominant subgenotype in cattle all over Portugal, and was found in nine HIV-infected individuals [145]. These findings highlight the significance of zoonotic transmission. In Switzerland, HIV-infected individuals were found to harbour C. hominis Ib and Id subgenotypes and C. parvum IIa and IId subgenotypes [147].

HIV-cryptosporidiosis in North America

As in Europe, the incidence of AIDS-opportunistic illness in the US has also decreased following the introduction of HAART [150]. However, there are some reports of Cryptosporidium infection among HIV-positive children, with acute diarrheal disease, as well as among asymptomatic HIV-positive individuals in the US with prevalence rates ranging from 3.5% to 10.8% [55,150-152] (Table 2).

Data from the AIDS surveillance registry for the 10-year period 1983â€“1992 found an overall rate of 3.8% of individuals to be positive for cryptosporidiosis during the study period. Significant risk factors for cryptosporidiosis included sexual contact (3.9%), immigrants from Mexico (5.2%) and Latinos (4.2%) [153]. Seasonal variation was also reported, in New Orleans, Louisiana, where a greater number of cryptosporidiosis cases were observed among HIV-infected individuals in the spring compared to other seasons [150]. A prospective cohort study in Atlanta, Georgia, determined the role of enteric parasites in acute and chronic diarrhea in patients infected with HIV. This report described the incidence of diarrhea and its association with CD4+ T-lymphocyte counts, and cryptosporidiosis was found in 10.8% of 602 HIV-infected individuals [55]. Other than the US, the only report of cryptosporidiosis in HIV-infected individuals in North America was from Cuba, where 11.9% of 67 AIDS patients with cryptosporidiosis had continuous diarrhea [154].

Regarding the species of Cryptosporidium reported in HIV-infected individuals in the US, the most prevalent was C. hominis, followed by C. parvum, C. canis, and finally C. muris (Table 3). A study in the US characterized 13 Cryptosporidium isolates from people with AIDS and found that 10 were C. hominis and three were C. parvum [155]. A subsequent study in the US found that five of 10 AIDS patients with cryptosporidiosis were infected with C. hominis, one had C. parvum, three had C. felis and one had C. canis [156].

Characterization of isolates from outbreaks and sporadic cases of human cryptosporidiosis using PCR-RFLP and DNA sequencing confirmed that C. parvum is highly conserved at the TRAP-C2 locus. Of 17 Cryptosporidium isolates from HIV-infected patients in New Orleans, Louisiana, two demonstrated the bovine-genotype pattern now known as C. parvum, while the rest were similar to C. hominis [157]. More recently genotyping and multilocus sequence typing techniques employed to determine the transmission patterns of cryptosporidiosis among HIV-infected individuals in Jamaica, demonstated that 25 individuals had C. hominis, seven had C. parvum, one had C. canis, one had C. felis, and one had both C. hominis and C. felis. Subgenotyping of C. hominis and C. parvum using sequence analysis of the gp60 gene demonstrated that 22 C. hominis isolates were of the subgenotype IbA10G2, and three were subgenotype IeA12G3T3. All seven C. parvum specimens belonged to subgenotype IIcA5G3d. This finding suggested that the anthroponotic route of transmission predominated [158].

HIV-cryptosporidiosis in South America

A significant number of cases of cryptosporidiosis have been reported in HIV-infected individuals from various countries in South America. Data is available from Brazil, Chile, Colombia, Peru and Venezuela with prevalences ranging from 4% to 22.8% [12,30,31,159-169] (Table 2). In addition to geographic variability, there also appears to be seasonal differences in Cryptosporidium infection, with more infections occurring in warmer or more humid months [12].

The greatest numbers of studies on the prevalence of Cryptosporidium infection were reported from Brazil, with seven studies conducted in different regions on both symptomatic and asymptomatic HIV-infected adults and children. Prevalences in these studies ranged from 4% to 19.1% [159,161,164,165,168,169]. Using molecular tools, six species of Cryptosporidium have been identified among HIV-infected individuals in South America. These include C. hominis, C. parvum, C. meleagridis, C. muris, C. canis and C. suis [30,31,34,167,170-173] (Table 3). Currently, the occurrence of C. suis in HIV-infected individuals is only reported in Peru [30]. Recently, the predominance of C. hominis over C. parvum was reported [173]. Seventeen isolates were identified as C. hominis, five as C. felis, four as C. parvum, and one as C. canis. These findings suggested that while human-to-human transmission predominates in urban environments of Brazil, the cat species C. felis may play a potential role in the zoonotic transmission of cryptosporidiosis [173].

Similar findings on the diversity of Cryptosporidium spp. were also reported in Peru. Cryptosporidium oocysts were detected in 13.3% of 2,672 HIV-infected patients. Using PCR-RFLP, these authors identified six species; C. hominis (204 isolates) was the most frequently detected, followed by 38 isolates of C. meleagridis, 34 of C. parvum, 12 of C. canis, 10 of C. felis, and one of C. suis. These findings indicate that C. hominis is the predominant species in Peruvian HIV-positive persons, and that zoonotic Cryptosporidium spp. account for about 30% of cryptosporidiosis in these patients [30]. In addition, mixed Cryptosporidium infection was found to be more frequent and persist longer in HIV-infected individuals than in the general population [171]. Distribution of the five most common Cryptosporidium spp., namely C. hominis, C. parvum, C. meleagridis, C. felis, and C. canis, were found to be the same in HIV-infected adults and children [34].

Another genotyping and subgenotyping study in Peru, determined that 230 (9.2% of 2,490) HIV-infected individuals were infected with Cryptosporidium. Of these, there were 141 C. hominis isolates, 22 C. parvum, 17 C. meleagridis, six each of C. canis and C. felis, and one C. suis. All C. parvum isolates belonged to subgenotype family IIc, which is considered anthroponotic in origin. Although Cryptosporidium infections were associated with diarrhea, only those infections with C. canis, C. felis, and subgenotype family Id of C. hominis were associated with diarrhea without vomiting, while infections with C. parvum were associated with both chronic diarrhea and vomiting. These results demonstrated that different Cryptosporidium genotypes and subgenotype families are linked to different clinical manifestations [31].

Conclusion

This review confirms that HIV-positive individuals worldwide are at high risk of acquiring Cryptosporidium infection. The prevalence of cryptosporidiosis in these individuals, however, varies considerably amongst studies, depending on where the study was conducted, the season, the age of the populations studied, the stage of the disease, and the laboratory methods used. Cryptosporidiosis in HIV-infected people may result in severe and prolonged diarrhea, especially in patients with low CD4 T-cell counts, while at higher CD4 T-cell levels, spontaneous clearance of the parasite generally occurs. Due to the availability of HAART in developed nations (i.e., countries in Europe and North America), there has been a reduction in the prevalence of Cryptosporidium infection in HIV/AIDS patients. However, in the resource poor settings of developing countries (i.e., countries in Africa, Asia and South America), patients usually go undiagnosed for long periods and present late in the course of the disease [49]. With lack of access to HAART in some of these countries, infections with Cryptosporidium are usually prolonged and, in some instances, fatal. With limited effective treatment available for cryptosporidiosis, an intact immune system is crucial in resolving the infection [166].

In the last decade, numerous molecular tools have been employed to enhance the molecular epidemiology of Cryptosporidium by detecting and differentiating Cryptosporidium spp. at the species, genotype and subgenotype levels, which significantly improves our understanding of the transmission of cryptosporidiosis in humans and animals, and provides a better understanding of population genetics of Cryptosporidium transmission in humans [27]. The high prevalence of C. hominis reported in many of these studies indicates that humans are a major source of infection, and that person-to-person transmission probably plays a major role in the spread of Cryptosporidium infection in HIV-infected patients. However, the presence of C. parvum, C. meleagridis, C. muris, C. canis and C. suis, especially in African, Asian, European and South American countries, is suggestive of zoonotic transmission, with infected individuals having direct or indirect (e.g., contaminated water or foods) contact with animals. These findings also demonstrate that immunocompromised individuals are susceptible to a wide range of Cryptosporidium species and genotypes.

Future Priorities

Data is currently limited in a few high-priority areas with respect to cryptosporidiosis in HIV-infected people. For example, further information is required on the relative risks of acquiring cryptosporidiosis from drinking water, poor hygiene, unsafe sexual practices, direct or indirect contact with animals, household or nosocomial infections, oocyst-contaminated foods, and other sources. Another area that requires further study is in the association amongst clinical manifestations, CD4 T-cell counts, and the molecular type of Cryptosporidium in HIV/AIDS patients. Clinical studies will be needed to clearly define the asymptomatic carrier rate for different species, genotypes and subgenotypes of Cryptosporidium in HIV-infected patients who recover from a clinical episode of cryptosporidiosis and who have CD4 T-cell counts of greater than 200 cells/mm³. It will also be important to determine if such carriers are likely to develop severe cryptosporidiosis if their CD4 T-cell count drops below this level. Finally, as there are currently few viable options for drug treatment, especially in d eveloping countries, continued screening of compounds for their efficacy in treating cryptosporidiosis is imperative. In particular, accessible, affordable, and efficacious drugs for the treatment of diarrhea caused by infection with Cryptosporidium spp., or for the outright clearance of infection, will be of considerable importance in minimizing the impact of this global opportunistic pathogen on HIV/AIDS patients.

A number of laboratory methodologies will need to be addressed in order to fill some of these research gaps. For example, sensitive, specific, and validated standardized methods for the routine detection and molecular characterization of human Cryptosporidium isolates identified in surveillance studies worldwide, as well as isolates from animals and environmental sources (e.g., water and foods), are required to facilitate epidemiologic studies of cryptosporidiosis, and more firmly identify the possible sources of contamination. The use of second generation molecular diagnostic tools in conjunction with traditional epidemiological methods such as gp60-based subgenotyping, and more recently multilocus subgenotyping, can be used to determine the species, genotypes and subgenotypes present and will be very useful in identifying the source(s) of infections, whether anthroponotic or zoonotic. Development and incorporation of these methods, as well as those for viability determination of oocysts, will improve the efficiency and accuracy of surveillance studies and outbreak investigations, and could also facilitate screening of potential therapeutic agents for infections due to Cryptosporidium species.

Continued efforts in this area will provide the scientific data needed to better advise public health professionals, and the general public, on the sources of infection with Cryptosporidium and the means to reduce the likelihood of transmission. Regional and national reporting systems for collecting data on the numbers of cases of cryptosporidiosis are also needed to better quantify the public health impact of this disease, and to identify illness outbreaks. In light of the evidence supporting zoonotic transmission of cryptosporidiosis in HIV-infected individuals worldwide, prevention and control measures against cryptosporidiosis also need to be adopted and regulated at the farm level. The role of the veterinarian in diagnoses, treatment and guidance concerning cryptosporidiosis is significant in the management and prevention of this disease in farm animals as well as in companion animals. Since these animals have been implicated as a major source of Cryptosporidium transmission to humans, management of the disease in animals will likely translate into fewer human infections.

Finally, in order to develop effective disease reduction strategies related to Cryptosporidium, smart partnerships need to be built amongst governments, agencies, and health care workers responsible for the care of HIV-infected patients worldwide, as well as the targeted HIV-infected population itself. By addressing the data gaps mentioned above, the prevention and management of clinical disease in HIV-infected patients, and other immunocompromised individuals, could be significantly improved. Communication of the findings of such studies would also serve to focus the immunocompromised patientsâ€™ attention on avoidance of exposures that would put them at greater risk.

References

BarrÃ©-Sinoussi F, Chermann JC, Rey F, Nugeyre MT, Chamaret S, et al. (1983)

Broder S, Gallo RC (1984)

Global Report (2011)

Nime FA, Burek JD, Page DL, Holscher MA, Yardley JH (1976)

Meisel JL, Perera DR, Meligro C, Rubin CE (1976)

Ma P, Soave R (1983)

Ramirez NE, Ward LA, Sreevatsan S (2004)

MacKenzie WR, Schell WL, Blair KA, Addiss DG, Peterson DE, et al. (1995)

Mosier DA, Oberst RD (2000)

(1992)

Tzipori S, Widmer G (2008)

De Oliveira-Silva MB, de Oliveira LR, Resende JC, Peghini BC, Ramirez LE, et al. (2007)

Fayer R (2010)

Traversa D (2010)

Dixon BR (2009)

Hunter PR, Thompson RC (2005)

Dixon B, Parrington L, Cook A, Pintar K, Pollari F, et al. (2011)

Hunter PR, Thompson RC (2005)

Fayer R, Morgan U, Upton SJ (2000)

McLauchlin J, Amar C, Pedraza-DÃaz S, Nichols GL (2000)

Starkey SR, White ME, Mohammed HO (2007)

Ziegler PE, Wade SE, Schaaf SL, Stern DA, Nadareski CA, et al. (2007)

Atwill ER, Johnson E, Klingborg DJ, Veserat GM, Markegard G, et al. (1999)

O'Handley RM (2007)

Coklin T, Farber JM, Parrington LJ, Coklin Z, Ross WH, et al. (2010)

Farizawati S, Lim YA, Ahmad RA, Fatimah CT, Siti-Nor Y (2005)

Hill A, Nally P, Chalmers RM, Pritchard GC, Giles M (2011)

Xiao L, Ryan UM (2004)

Xiao L, Fayer R, Ryan U, Upton SJ (2004)

Gatei W, Suputtamongkol Y, Waywa D, Ashford RW, Bailey JW, et al. (2002)

Cama VA, Bern C, Sulaiman IM, Gilman RH, Ticona E, et al. (2003)

Cama VA, Ross JM, Crawford S, Kawai V, Chavez-Valdez R, et al. (2007)

Abe N, Sawano Y, Yamada K, Kimata I, Iseki M (2002)

Fayer R, Trout JM, Xiao L, Morgan UM, Lai AA, et al. (2001)

Xiao L, Bern C, Limor J, Sulaiman I, Roberts J, et al. (2001)

Pedraza-DÃaz S, Amar C, Iversen AM, Stanley PJ, McLauchlin J (2001)

Fayer R (1997) Cryptosporidium and Cryptosporidiosis. CRC Press LLC 2000 New York, USA.

DuPont HL, Chappell CL, Sterling CR, Okhuysen PC, Rose JB, et al. (1995)

Current WL, Garcia LS (1991)

Putignani L, Menichella D (2010)

Chen XM, LaRusso NF (2002)

Ayuo PO (2009)

Clayton F, Heller T, Kotler DP (1994)

Chalmers RM, Davies AP (2010)

Rotterdam H, Tsang P (1994)

Hunter PR, Nichols G (2002)

Lee JK, Song HJ, Yu JR (2005)

Riggs MW (1997) Immunology: host response and development of passive immunotherapy and vaccines. In: Fayer R (Ed.), Cryptosporidium and Cryptosporidiosis. CRC Press.

Gupta S, Narang S, Nunavath V, Singh S (2008)

Current WL, Bick PH (1989)

Sadraei J, Rizvi MA, Baveja UK (2005)

Montero JA, Sinnott JT, Holt DA, Lloyd C (2001) Biliary cryptosporidiosis: current concepts. Infect Med 18: 312-316.

SÃ¡nchez-Mejorada G, Ponce-de-LeÃ³n S (1994)

Ballal M, Prabhu T, Chandran A, Shivananda PG (1999)

Weber R, Ledergerber B, Zbinden R, Altwegg M, Pfyffer GE, et al. (1999)

Navin TR, Weber R, Vugia DJ, Rimland D, Roberts JM, et al. (1999)

Manabe YC, Clark DP, Moore RD, Lumadue JA, Dahlman HR, et al. (1998)

Hung CC, Tsaihong JC, Lee YT, Deng HY, Hsiao WH, et al. (2007)

Pozio E, Rezza G, Boschini A, Pezzotti P, Tamburrini A, et al. (1997)

Abubakar I, Aliyu SH, Arumugam C, Usman NK, Hunter PR (2007)

Rossignol JF, Ayoub A, Ayers MS (2001)

Rossignol JF (2006)

Savioli L, Smith H, Thompson A (2006)

Hommer V, Eichholz J, Petry F (2003)

Rossignol JF (2010)

Assefa S, Erko B, Medhin G, Assefa Z, Shimelis T (2009)

Chintu C, Luo C, Baboo S, Khumalo-Ngwenya B, Mathewson J, et al. (1995)

Mwachari C, Batchelor BI, Paul J, Waiyaki PG, Gilks CF (1998)

Gumbo T, Sarbah S, Gangaidzo IT, Ortega Y, Sterling CR, et al. (1999)

Lebbad M, Norrgren H, NauclÃ©r A, Dias F, Andersson S, et al. (2001)

Leav BA, Mackay MR, Anyanwu A, O' Connor RM, Cevallos AM, et al. (2002)

Houpt ER, Bushen OY, Sam NE, Kohli A, Asgharpour A, et al. (2005)

Tumwine JK, Kekitiinwa A, Bakeera-Kitaka S, Ndeezi G, Downing R, et al. (2005)

Sarfati C, Bourgeois A, Menotti J, Liegeois F, Moyou-Somo R, et al. (2006)

Awole M, Gebre-Selassie S, Kassa T, Kibru G (2003)

Samie A, Bessong PO, Obi CL, Sevilleja JE, Stroup S, et al. (2006)

Ibrahim A, Ikeh E, Malu A, Okeke E, Damen J (2007) Intestinal parasitosis in Human Immunodeficiency Virus (HIV) infected adults with chronic diarrhoea at Jos University Teaching Hospital, Nigeria. Internet J Para Dis 2: 1.

Adesiji YO, Lawal RO, Taiwo SS, Fayemiwo SF, Adeyeba OA (2007)

Essid R, Mousli M, Aoun K, Abdelmalek R, Mellouli F, et al. (2008)

Blanco MA, Iborra A, Vargas A, Nsie E, MbÃ¡ L, et al. (2009)

Getaneh A, Medhin G, Shimelis T (2010)

Morgan U, Weber R, Xiao L, Sulaiman I, Thompson RC, et al. (2000)

Gatei W, Greensill J, Ashford RW, Cuevas LE, Parry CM, et al. (2003)

Ryan UM, Samarasinghe B, Read C, Buddle JR, Robertson ID, et al. (2003)

(2009)

Chhin S, Harwell JI, Bell JD, Rozycki G, Ellman T, et al. (2006)

Kurniawan A, Karyadi T, Dwintasari SW, Sari IP, Yunihastuti E, et al. (2009)

Mukhopadhya A, Ramakrishna BS, Kang G, Pulimood AB, Mathan MM, et al. (1999)

Mohandas, Sehgal R, Sud A, Malla N (2002)

Kumar SS, Ananthan S, Lakshmi P (2002)

Kumar SS, Ananthan S, Saravanan P (2002)

Muthusamy D, Rao SS, Ramani S, Monica B, Banerjee I, et al. (2006)

Dwivedi KK, Prasad G, Saini S, Mahajan S, Lal S, et al. (2007)

Rao Ajjampur SS, Asirvatham JR, Muthusamy D, Gladstone BP, Abraham OC, et al. (2007)

Ramakrishnan K, Shenbagarathai R, Uma A, Kavitha K, Rajendran R, et al. (2007)

Kaushik K, Khurana S, Wanchu A, Malla N (2008)

Tuli L, Gulati AK, Sundar S, Mohapatra TM (2008)

Kulkarni SV, Kairon R, Sane SS, Padmawar PS, Kale VA, et al. (2009)

Zali MR, Mehr AJ, Rezaian M, Meamar AR, Vaziri S, et al. (2004)

Meamar AR, Rezaian M, Mohraz M, Zahabiun F, Hadighi R, et al. (2007)

Taherkhani H, Fallah M, Jadidian K, Vaziri S (2007)

Daryani A, Sharif M, Meigouni M, Mahmoudi FB, Rafiei A, et al. (2009)

Guk SM, Seo M, Park YK, Oh MD, Choe KW, et al. (2005)

Kamel AG, Maning N, Arulmainathan S, Murad S, Nasuruddin A, et al. (1994)

Lim YA, Rohela M, Sim BL, Jamaiah I, Nurbayah M (2005)

Lim YA, Iqbal A, Surin J, Sim BL, Jex AR, et al. (2011)

Zaidah AR, Chan YY, Asma HS, Abdullah S, Nurhaslindawati AR, et al. (2008)

Asma I, Johari S, Sim BL, Lim YA (2011)

Ghimire P, Sapkota D, Manandhar SP (2004) Cryptosporidiosis: opportunistic infection in HIV/AIDS patients in Nepal. J Trop Med Parasitol 27: 7-10.

Moolasart P, Eampokalap B, Ratanasrithong M, Kanthasing P, Tansupaswaskul S, et al. (1995)

Manatsathit S, Tansupasawasdikul S, Wanachiwanawin D, Setawarin S, Suwanagool P, et al. (1996)

Punpoowong B, Viriyavejakul P, Riganti M, Pongponaratn E, Chaisri U, et al. (1998)

Uga S, Kunaruk N, Rai SK, Watanabe M (1998)

Wanke CA, Cohan D, Thummakul T, Jongwuitiwes S, Grayson ML, et al. (1999)

Waywa D, Kongkriengdaj S, Chaidatch S, Tiengrim S, Kowadisaiburana B, et al. (2001)

Saksirisampant W, Eampokalap B, Rattanasrithong M, Likanonsakul S, Wiwanitkit V, et al. (2002)

Pinlaor S, Mootsikapun P, Pinlaor P, Pipitgool V, Tuangnadee R (2005)

Srisuphanunt M, Suvedyathavorn V, Suputtamongkol Y, Arnantapunpong S, Wiwanitkit V, et al. (2008)

Nuchjangreed C, Boonrod K, Ongerth J, Karanis P (2008)

Che Ghani M, Abdullah MM, Hashim MB (1984) A case of cryptosporidiosis in a young man presenting with bloody diarrhea. J Malay Soc Hlth 4: 80-81.

Yu JR, Seo M (2004)

Tiangtip R, Jongwutiwes S (2002)

Meamar AR, Rezaian M, Mohraz M, Zahabiun F, Hadighi R, et al. (2006)

Meamar AR, Guyot K, Certad G, Dei-Cas E, Mohraz M, et al. (2007)

Iqbal A, Lim YA, Surin J, Sim BL (2012)

Asma I, Lim YAL, Johari S, Sim BLH (2012) Molecular characterization of Cryptosporidium in immunocompromized patients in Malaysia. Unpublished Data.

Connolly GM, Dryden MS, Shanson DC, Gazzard BG (1988)

Connolly GM, Ellis DS, Williams JE, Tovey G, Gazzard BG (1991)

Ravn P, Lundgren JD, Kjaeldgaard P, Holten-Anderson W, HÃ¸jlyng N, et al. (1991)

Cotte L, Rabodonirina M, Piens MA, Perreard M, Mojon M, et al. (1993)

McGowan I, Hawkins AS, Weller IV (1993)

De Jose MI, Garcia MJ, Ramos JT, Martin P, Hernandez T, et al. (1996)

Matos O, TomÃ¡s A, Aguiar P, Casemore D, Antunes F (1998)

Brandonisio O, Maggi P, Panaro MA, Lisi S, Andriola A, et al. (1999)

BÃ¼yÃ¼kbaba Boral O, Uysal H, Alan S, Nazlican O (2004)

Llorente MT, Clavel A, GoÃ±i MP, Varea M, Seral C, et al. (2007)

Pedersen C, Danner S, Lazzarin A, Glauser MP, Weber R, et al. (1996)

Karanis P, Kourenti C, Smith H (2007)

CacciÃ² S, Pinter E, Fantini R, Mezzaroma I, Pozio E (2002)

Del Chierico F, Onori M, Di Bella S, Bordi E, Petrosillo N, et al. (2011)

Bonnin A, Fourmaux MN, Dubremetz JF, Nelson RG, Gobet P, et al. (1996)

CacciÃ² S, Homan W, Camilli R, Traldi G, Kortbeek T, et al. (2000)

Guyot K, Follet-Dumoulin A, LeliÃ¨vre E, Sarfati C, Rabodonirina M, et al. (2001)

Alves M, Matos O, Antunes F (2003)

Alves M, Xiao L, Antunes F, Matos O (2006)

Matos O, Alves M, Xiao L, Cama V, Antunes F (2004)

O'Brien E, McInnes L, Ryan U (2008)

Pedraza-DÃaz S, Amar C, Iversen AM, Stanley PJ, McLauchlin J (2001)

Leoni F, Amar C, Nichols G, Pedraza-DÃaz S, McLauchlin J (2006)

Inungu JN, Morse AA, Gordon C (2000)

Spencer KL, Soave R, Acosta A, Gellin B, Prince A, et al. (1997)

Colford JM Jr, Tager IB, Hirozawa AM, Lemp GF, Aragon T, et al. (1996)

Sorvillo FJ, Lieb LE, Kerndt PR, Ash LR (1994)

Escobedo AA, NÃºÃ±ez FA (1999)

Widmer G, Tzipori S, Fichtenbaum CJ, Griffiths JK (1998)

Pieniazek NJ, Bornay-Llinares FJ, Slemenda SB, da Silva AJ, Moura IN, et al. (1999)

Sulaiman IM, Xiao L, Yang C, Escalante L, Moore A, et al. (1998)

Gatei W, Barrett D, Lindo JF, Eldemire-Shearer D, Cama V, et al. (2008)

Sauda FC, Zamarioli LA, Ebner Filho W, Mello Lde B (1993)

Chacin-Bonilla L, Guanipa N, Cano G, Raleigh X, Quijada L (1992)

Cimerman S, Cimerman B, Lewi DS (1999)

Caraballo A, Orozco A, MuÃ±oz L (2001) Intestinal parasitic infections in human immunodeficiency virus (HIV) positive individuals in Southeastern Venezuela. Bol Chil Parasitol 56: 3-4.

Botero JH, CastaÃ±o A, Montoya MN, Ocampo NE, Hurtado MI, et al. (2003)

Ribeiro PC, Pile E, Queiroz MM, Norberg AN, TenÃ³rio JR (2004)

Silva CV, Ferreira MS, Borges AS, Costa-Cruz JM (2005)

Certad G, Arenas-Pinto A, Pocaterra L, Ferrara G, Castro J, et al. (2005)

Neira-Otero P, MuÃ±oz-SaldÃas N, Sanchez-Moreno M, Rosales-Lombardo MJ (2005)

JÃºnior LG, Souza LR (2007)

Bachur TP, Vale JM, CoÃªlho IC, Queiroz TR, Chaves Cde S (2008)

Brantley KR, Williams RK, Silva TMJ, Sistrom M, Thielman NM, et al. (2003)

Cama V, Gilman RH, Vivar A, Ticona E, Ortega Y, et al. (2006)

AraÃºjo AJ, Kanamura HY, Almeida ME, Gomes AH, Pinto TH, et al. (2008)

Lucca P, De Gaspari EN, Bozzoli LM, Funada MR, Silva SO, et al. (2009)