Advertisement
Research Article

Distribution and Clinical Manifestations of Cryptosporidium Species and Subtypes in HIV/AIDS Patients in Ethiopia

  • Haileeyesus Adamu,

    Affiliations: Division of Foodborne, Waterborne and Environmental Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America, Addis Ababa University, Addis Ababa, Ethiopia

    X
  • Beyene Petros,

    Affiliation: Addis Ababa University, Addis Ababa, Ethiopia

    X
  • Guoqing Zhang,

    Affiliation: Division of Global HIV/AIDS, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America

    X
  • Hailu Kassa,

    Affiliation: Department of Public and Allied Health, Bowling Green State University (BGSU), Bowling Green, Ohio, United States of America

    X
  • Said Amer,

    Affiliations: Division of Foodborne, Waterborne and Environmental Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America, Department of Zoology, Faculty of Science, Kafr El sheikh University, Kafe El Sheikh, Egypt

    X
  • Jianbin Ye,

    Affiliations: Division of Foodborne, Waterborne and Environmental Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America, State Key Laboratory of Bioreactor Engineering, School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai, People's Republic of China

    X
  • Yaoyu Feng mail,

    yyfeng@ecust.edu.cn (YF); lxiao@cdc.gov (LX)

    Affiliation: State Key Laboratory of Bioreactor Engineering, School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai, People's Republic of China

    X
  • Lihua Xiao mail

    yyfeng@ecust.edu.cn (YF); lxiao@cdc.gov (LX)

    Affiliation: Division of Foodborne, Waterborne and Environmental Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America

    X
  • Published: April 17, 2014
  • DOI: 10.1371/journal.pntd.0002831

Abstract

Background

Cryptosporidiosis is an important cause for chronic diarrhea and death in HIV/AIDS patients. Among common Cryptosporidium species in humans, C. parvum is responsible for most zoonotic infections in industrialized nations. Nevertheless, the clinical significance of C. parvum and role of zoonotic transmission in cryptosporidiosis epidemiology in developing countries remain unclear.

Methodology/Principal Findings

In this cross-sectional study, 520 HIV/AIDS patients were examined for Cryptosporidium presence in stool samples using genotyping and subtyping techniques. Altogether, 140 (26.9%) patients were positive for Cryptosporidium spp. by PCR-RFLP analysis of the small subunit rRNA gene, belonging to C. parvum (92 patients), C. hominis (25 patients), C. viatorum (10 patients), C. felis (5 patients), C. meleagridis (3 patients), C. canis (2 patients), C. xiaoi (2 patients), and mixture of C. parvum and C. hominis (1 patient). Sequence analyses of the 60 kDa glycoprotein gene revealed a high genetic diversity within the 82 C. parvum and 19 C. hominis specimens subtyped, including C. parvum zoonotic subtype families IIa (71) and IId (5) and anthroponotic subtype families IIc (2), IIb (1), IIe (1) and If-like (2), and C. hominis subtype families Id (13), Ie (5), and Ib (1). Overall, Cryptosporidium infection was associated with the occurrence of diarrhea and vomiting. Diarrhea was attributable mostly to C. parvum subtype family IIa and C. hominis, whereas vomiting was largely attributable to C. hominis and rare Cryptosporidium species. Calf contact was identified as a significant risk factor for infection with Cryptosporidium spp., especially C. parvum subtype family IIa.

Conclusions/Significance

Results of the study indicate that C. parvum is a major cause of cryptosporidiosis in HIV-positive patients and zoonotic transmission is important in cryptosporidiosis epidemiology in Ethiopia. In addition, they confirm that different Cryptosporidium species and subtypes are linked to different clinical manifestations.

Author Summary

The disease burden of Cryptosporidium parvum and role of zoonotic transmission in cryptosporidiosis epidemiology are poorly understood in developing countries. In this study, we examined the distribution and clinical manifestations of Cryptosporidium species and subtypes in HIV/AIDS patients in Addis Ababa, Ethiopia. Using molecular diagnostic tools, we detected Cryptosporidium infection in 26.9% of 520 HIV/AIDS patients studied. We have shown a very high diversity of Cryptosporidium species and subtypes in these patients, but unlike in other developing countries, C. parvum is overwhelmingly the dominant species in the study community, responsible for ~65% Cryptosporidium infections. The common occurrence of C. parvum zoonotic subtype family IIa, combined with calf contact as a significant risk factor, suggest that zoonotic transmission is important in cryptosporidiosis epidemiology in HIV/AIDS patients in Ethiopia. We have also shown that different Cryptosporidium species and subtypes are linked to different clinical manifestations. Improved hygiene and avoidance of calf contact should be advocated to reduce cryptosporidiosis transmission in HIV/AIDS patients in the study setting.

Introduction

Cryptosporidium is an important protozoan parasite affecting HIV/AIDS patients, causing diarrhea, wasting syndrome, and reduced life quality [1]. Since specific therapy or vaccine for the control of this parasite is not yet available, preventing infections depends on avoiding exposure to the parasite and maintaining immune competence. In industrialized nations, access to highly active antiretroviral therapy (HAART) has significantly reduced the morbidity and mortality by cryptosporidiosis [2][4]. Nonetheless, cryptosporidiosis is still a major threat to AIDS patients who do not have access to HAART, especially in developing countries [5][8]. In industrialized nations, transmission of cryptosporidiosis via contaminated drinking and recreational water and contact with infected farm animals remains a major public health problem in both HIV-positive and immunocompetent persons [9][11].

The use of molecular epidemiologic tools has provided new insights into the diversity of Cryptosporidium species infecting humans and animals [12]. So far, 26 Cryptosporidium species have been described [13][17]. Most human cases are caused by C. hominis and C. parvum [12]. The latter also infects some other mammals, notably calves and lambs, and is responsible for most zoonotic infections in humans. Several other Cryptosporidium species are seen in humans at lower frequency, including C. meleagridis, C. felis, C. canis, C. ubiquitum, and C. cuniculus [12]. More recently, a new species, C. viatorum, has been described in 10 travelers returning to Great Britain from the Indian subcontinent [17]. This species appears to be a human-specific pathogen and has since been found in 2 Swedish travelers to Africa and Latin America [18]. In children and HIV-positive persons, differences in clinical manifestations have been observed among different Cryptosporidium species, especially between C. hominis and C. parvum, with the former more virulent than the latter [19][21]. In addition, infections with C. parvum were associated with chronic diarrhea and vomiting in HIV-positive persons more frequently than was C. hominis [21].

Sequence characterization of the 60-kDa glycoprotein (gp60) gene has been commonly used in subtyping C. hominis and C. parvum [12]. Differences have been observed in host specificity among C. parvum subtype families and in virulence among C. hominis subtype families. Thus, C. parvum subtype family IIa is commonly found in calves, IId is mostly found in lambs and goat kids, whereas IIc is mostly found in humans. Within C. hominis, studies in Peru have shown that subtype family Ib was more virulent than other subtype families in children, whereas subtype family Id was more virulent than subtype families Ia and Ie in HIV-positive patients [20], [21].

Cryptosporidiosis is endemic in Ethiopia; occurrence rates of 7.6% to 43.6% were reported in HIV/AIDS patients [22][26]. Some potential risk factors for cryptosporidiosis occurrence included contamination of drinking water, contact with calves, living in overcrowded households with many family members, and poor personal hygiene. Thus far, only one study has genetically characterized Cryptosporidium spp. from Ethiopia. In the study, 39 of the 41 specimens genotyped had C. parvum, one had C. hominis, and one had both species. All 12 C. parvum specimens subtyped by sequence analysis of the gp60 gene belonged to the subtype family IIa [22].

In the present study, we examined the occurrence of Cryptosporidium infection in HIV/AIDS patients in Ethiopia and characterized Cryptosporidium spp. at the species, subtype family, and subtype levels. We also examined the association between clinical manifestations and infections with specific Cryptosporidium species and subtype families. Data generated from the study have clearly shown a dominance of C. parvum in the study population, importance of zoonotic transmission in the epidemiology of cryptosporidiosis in Ethiopia, and differences in clinical manifestations among Cryptosporidium species and subtypes.

Materials and Methods

Ethical statement

The research protocol was approved by the Ethical Clearance Committee of the Addis Ababa University. All study participants had given written informed consent before enrollment into the study. When the study participant was a child, written consent was obtained from his or her parent or guardian. Researchers at the Centers for Disease Control and Prevention (CDC) had no contact with patients and no access to personal identifiers. Laboratory work on the study specimens was covered under CDC IRB protocol No. 990115: “Use of residual human specimens for the determination of frequency of genotypes or sub-types of pathogenic parasites”.

Study population and enrollment

This study was cross-sectional in nature. It was conducted between September 2009 and December 2011 in Addis Ababa, Ethiopia. A total of 520 HIV/AIDS patients were recruited from in-patients (hospitalized) and outpatients attending the Tikur Anbessa Hospital, Addis Ababa University, and patients referred to the study by attending physicians. The criteria for inclusion in the study were documented HIV infection, the ability to provide informed consent by the patient or the guardian, and willingness to provide one stool specimen. A structured questionnaire was used to collect CD4+ cell counts, demographic data, clinical symptoms, HAART history, antibiotics usage, and animal exposure history. Each participant was asked to provide a single fresh stool specimen.

DNA extraction

Stool specimens were stored in 2.5% potassium dichromate at 4°C and shipped to the CDC laboratory in Atlanta for screening and molecular characterization of Cryptosporidium spp. by PCR. After washing the stool specimens twice with distilled water, genomic DNA was extracted from 0.5 ml of fecal materials using a FastDNA SPIN Kit for Soil (MP Biomedicals, Solon. OH) and eluted in 100 µl of reagent-grade water following the manufacturer-recommended procedures. DNA was stored at −80°C until analyzed by PCR.

Cryptosporidium species detection, genotyping and subtyping

Cryptosporidium oocysts present in the specimens were detected by nested PCR amplification of an approximate 830 bp fragment of the small subunit (SSU) rRNA gene as described previously [27]. Cryptosporidium species were determined by restriction fragment length polymorphism (RFLP) analysis of the secondary PCR products using endonucleases SspI and VspI [27]. PCR products and restriction fragments were subjected to electrophoresis in 1.5% and 2% agarose gels, respectively, and visualized after staining with GelRed (Biotium Inc., Hayward, CA). All secondary PCR products from species other than C. parvum were sequenced to confirm the identification. Specimens that contained C. parvum or C. hominis were further subtyped by DNA sequencing of the nested PCR product of the gp60 gene [28]. Each specimen was analyzed at least twice by PCR at each locus using 2 µl of the DNA extraction per PCR. As a positive control, C. baileyi DNA was used in SSU rRNA PCR and C. hominis DNA was used in gp60 PCR. A negative control using DNase-free water was also included in each PCR run.

Sequence analysis

PCR products were sequenced using the forward and reverse primers of the secondary PCR. An intermediary sequencing primer gp60-R3 [5′-GAG ATA TAT CTT GTT GCG-3′] was also used in the sequencing of gp60 PCR products. DNA sequencing was done using the ABI BigDye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and an ABI3130 Genetic Analyzer (Applied Biosystems). Sequence accuracy was confirmed by sequencing of two PCR products from each positive specimen. Nucleotide sequences obtained were aligned with reference sequences using the ClustalX 1.81 package (http://www.clustal.org/) to identify Cryptosporidium species and C. parvum subtypes. Subtypes of C. parvum and C. hominis were named based on the established nomenclature system [29]. Unique sequences generated in this study were deposited in GenBank under accession numbers AB830575 to AB830590.

Statistical analysis

Data from the study were analyzed using the SPSS 20.0 for Windows software (IBM Corp, Armonk, NY) at three levels of parasite categorization: presence of Cryptosporidium, species of Cryptosporidium, and subtype families of C. parvum and C. hominis. Data from persons infected with low-frequency species were pooled based on their genetic similarities. Univariate and multivariate logistic regression modeling were used to analyze the association between Cryptosporidium infection and clinical symptoms or risk-factors while adjusting for potential confounders when the sample size was sufficient. For comparisons at the species or subtype family level, separate models and different subsets of the main dataset were run to examine the effect of each species or subtype family, with Cryptosporidium-negative as the referent. The Hosmer-Lemeshow test was used to assess the goodness-of-fit of each multivariate logistic regression model. The strength of the associations was estimated by odds ratios (OR) and 95% confidence intervals (CI). The association was considered statistically significant when the P value obtained was smaller than 0.05.

Results

Study patients and Cryptosporidium occurrence

Among the 520 HIV/AIDS patients who participated in this study, 276 (53.1%) were males and 244 (46.9%) were females. The median age of the study participants was 41 years (range: 7 months to 86 years), and the mean CD4+ cell count was 278 cells/µL. Almost one-third (32.9%) of the study patients were bedridden and hospitalized. Of the 520 stool specimens examined in this study, 140 (26.9%) were positive for Cryptosporidium by the SSU rRNA-based PCR technique (Table 1). There were no significant differences in prevalence of Cryptosporidium infection among age groups and between the male and female gender (Table 1). No significant association between hospitalization and Cryptosporidium infection was found; percentages of Cryptosporidium infection in hospitalized and non-hospitalized patients were 35.7% and 37.4% (P = 0.83). In addition, no significant difference was observed in the occurrence of vomiting, diarrhea, age, HAART, antibiotic use, animal contact and CD4 between inpatients and outpatients. There was also no association between infections with Cryptosporidium spp. or any specific species and hospitalization rates (data not shown).

thumbnail

Table 1. Occurrence of Cryptosporidium spp. (n = 140 cases) in 520 HIV/AIDS patients in Ethiopia by age and gender*.

doi:10.1371/journal.pntd.0002831.t001

Cryptosporidium species and C. hominis and C. parvum subtypes

Species determination by RFLP was successful for 128/140 Cryptosporidium-positive specimens (Table 2). Cryptosporidium parvum (n = 92) and C. hominis (n = 25) were the species most frequently detected, followed by C. felis (n = 5), C. meleagridis (n = 3), C. canis (n = 2), and C. parvum and C. hominis co-infection (n = 1). All SSU rRNA PCR products from non-C. parvum specimens were sequenced, confirming the RFLP results. In addition, 12 specimens demonstrated a RFLP pattern that was similar to C. parvum, but with a slightly smaller upper SspI band. DNA sequences from two of the specimens were identical to a reference sequence of C. xiaoi (GenBank accession no. JQ413443) and those from 10 specimens were identical to a reference sequence (GenBank accession no. HM485434) of C. viatorum, a recently described species in humans [17].

thumbnail

Table 2. Distribution of Cryptosporidium species and C. parvum and C. hominis subtypes in HIV/AIDS patients in Ethiopia (n = 140).

doi:10.1371/journal.pntd.0002831.t002

Subtype family data were obtained from 82 (89.1%) of the 92 participants with C. parvum and showed the presence of subtype families IIa, IIb, IIc, IId, IIe, and a new subtype family genetically related to If in 71, 1, 2, 5, 1, and 2 persons, respectively. In contrast, 19 (76%) of the 25 participants with C. hominis were successfully subtyped and showed the presence of subtype families Ib, Id, and Ie in 1, 13, and 5 persons, respectively. Within C. parvum, 8 subtypes were from the subtype family IIa. This was followed by subtype families IId with 4, If-like with 2 subtypes, and IIb, IIc, and IIe with 1 subtype each. Thus, participants in the study were infected with 14 subtypes of C. parvum. The most frequently detected subtype was IIaA15G2R1, which was seen in 60 patients. Five subtypes were identified in C. hominis, including IdA20, IdA26, IdA24, IeA11G3T3, and IbA10G2 in 10, 2, 1, 5, and 1 patient, respectively (Table 2).

Cryptosporidium species/subtypes and clinical manifestations

Cryptosporidium infection was significantly associated with diarrhea in univariate analysis (P<0.001), especially in patients with C. parvum and C. hominis (P<0.001 and P = 0.012 respectively). Vomiting was also more often seen in Cryptosporidium-positive than Cryptosporidium-negative patients (63.6% versus 51.8%; P = 0.018). Patients with C. hominis or rare species (C. meleagridis/C. felis/C. canis and C. xiaoi) were more likely to have vomiting compared with Cryptosporidium-negative patients (P = 0.057 and P = 0.027, respectively; Table 3).

thumbnail

Table 3. Association with diarrhea and vomiting by Cryptosporidium species and of C. parvum and C. hominis subtype in HIV/AIDS patients in Ethiopia.

doi:10.1371/journal.pntd.0002831.t003

Diarrhea was significantly associated with infections with C. parvum subtype family IIa compared with Cryptosporidium-negative patients (78.9%; P = 0.003). However, there were no significant differences in diarrhea occurrence between patients infected with C. hominis subtype families and Cryptosporidium-negative patients. Patients infected with different C. parvum and C. hominis subtype families also did not show significant differences in the occurrence of vomiting compared to Cryptosporidium-negative patients (P>0.05; Table 3).

Multivariable modeling was attempted for diarrhea and vomiting occurrence in order to adjust for age, gender, type of patients and clinical parameters (HAART and CD4+); however, it did not reveal major differences compared with the crude OR (Table 3).

Cryptosporidium species/subtypes and risk factors

After controlling for other potential risk factors, history of any contact with animals was associated with overall Cryptosporidium infections (OR = 1.6; P = 0.04), and with C. parvum (OR = 2.5; P = 0.002) and its subtype family IIa (OR = 2.1; P = 0.02) in particular. This association was mostly due to contact with calves (OR = 1.6 and P = 0.02 for overall Cryptosporidium infection; OR = 1.8 and P = 0.01 for C. parvum; and OR = 2.0 and P = 0.01 for C. parvum subtype family IIa; Table 4). However, there was no significant association between Cryptosporidium infection and age, gender, HAART history, CD4+ cell counts, antibiotics use, or type of patients (P = 0.56, 0.13, 0.59, 0.90, 0.84, and 0.83, respectively). No significant association was found between Cryptosporidium infection and CD4+ cell counts or HAART history at the species and subtype levels (Tables S1 and S2).

thumbnail

Table 4. Association between animal contact and infection with Cryptosporidium species or C. parvum and C. hominis subtypes in HIV/AIDS patients in Ethiopia*.

doi:10.1371/journal.pntd.0002831.t004

Discussion

The present findings showed that (1) Ethiopian HIV/AIDS patients were infected with a diverse population of Cryptosporidium species, including the unusual species C. viatorum and C. xiaoi; (2) C. parvum was the most frequently detected species; and (3) Cryptosporidium species or subtype families were associated with different clinical manifestations. The dominance of C. parvum in this study is in agreement with the previous observation in a small study in Ethiopia [22]. It is, however, in sharp contrast with studies of human cryptosporidiosis in other developing countries where C. hominis dominates [12], [30], [31]. In Europe, the two species are almost evenly distributed, with C. parvum being more prevalent in some reports [32] and C. hominis in others [33]. Data from this study also indicate that the recently established species C. viatorum is more widely distributed than believed, and humans can be infected occasionally with the sheep and goat parasite C. xiaoi. As expected, the prevalence of cryptosporidiosis in HIV+ patients (26.9%) in this study is substantially higher than in the largely healthy persons (8.7%) in a previous study conducted in the same area [22].

Differences in geographical distribution of C. parvum and C. hominis are generally considered a reflection of differences in infection sources and transmission routes [12]. C. hominis is transmitted almost exclusively among humans, whereas C. parvum, especially its IIa subtype family, is more likely transmitted zoonotically. In industrialized nations, C. parvum infections have often been linked to contact with farm animals, and C. hominis infections to contact with children with diarrhea [34]. Both species have been associated with drinking-water outbreaks [33]. The predominance of C. hominis in most developing countries suggests that anthroponotic transmission is more important than zoonotic transmission in cryptosporidiosis epidemiology in developing countries in general [12]. In contrast, the dominance of C. parvum IIa subtype family in Ethiopia in HIV/AIDS patients suggests that unlike in other developing countries, Cryptosporidium infection in Ethiopia is mostly transmitted zoonotically. Previously, it was shown that that small numbers of C. parvum infections seen in humans in developing countries were mostly caused by the anthroponotic subtype family IIc [12].

Results of the risk factor analysis support the role of zoonotic transmission in cryptosporidiosis epidemiology in HIV/AIDS patients in Ethiopia. Even though the present study was performed in an urban area, more than 50% of patients had contact with animals, as households in the study area usually have farm animals living inside the residence. Thus, in this study, animal contact, especially with calves, was a significant risk factor for Cryptosporidium, especially C. parvum and its IIa subtype family. The distribution of C. parvum subtypes in this study reinforces the likely occurrence of zoonotic transmission, as the majority of C. parvum infections were caused by IIa subtypes (71/82 specimens subtyped), especially its IIaA15G2R1 subtype (60/82 specimens subtyped), which is a dominant C. parvum subtype in calves around the world [12]. In contrast, the anthroponotic C. parvum subtype family IIc was seen in only 2/82 of C. parvum infections.

In addition to IIa, C. parvum subtype family IId was also identified in five Ethiopian HIV/AIDS patients. Subtype family IId of C. parvum is generally considered a sheep and goat parasite [35], but has been found at high frequency in calves in China, Egypt, and Sweden [36][38]. In this study, only one patient infected with IId had contact with sheep in this study. Genotyping and subtyping studies of domestic animals from the study area and additional case-control-studies are needed to support the conclusion on the importance of zoonotic transmission in cryptosporidiosis epidemiology in Ethiopia.

In agreement with previous observations elsewhere [19][21], data from the study suggest that different Cryptosporidium species and subtypes are linked to different manifestations of cryptosporidiosis. As expected, cryptosporidiosis in our study was significantly associated with the occurrence of diarrhea. This association, however, was largely attributable to C. parvum and C. hominis; other species, including the newly described C. viatorum, were less pathogenic than these two species. Likewise, the significant association between Cryptosporidium infection and the occurrence of vomiting was also largely attributable to C. hominis and some less frequent species (C. meleagridis, C. canis, C. felis and C. xiaoi); C. parvum and C. viatorum were largely not associated with vomiting. Within C. parvum, the IIa subtype family also appeared to be more associated with the occurrence of diarrhea than other C. parvum subtype families. We did not observe any effect of CD4+ cell counts and HAART on the occurrence of cryptosporidiosis in HIV/AIDS patients in this study. This was probably largely the result of severe overall immunodeficiency in the study population, as reflected by the low mean CD4+ cell counts (278 cells/µL) and very high hospitalization rate (32.9%) and occurrence of cryptosporidiosis (26.9%).

In conclusion, Ethiopian HIV/AIDS patients with low CD4+ cell counts had an extremely high occurrence of Cryptosporidium infection, even when they were on HAART. Although the majority of cryptosporidiosis cases were caused by C. parvum, there was a high diversity of Cryptosporidium species, with a significant number of cases caused by the newly recognized C. viatorum. These Cryptosporidium spp. and C. parvum subtypes were linked to different clinical manifestations. Therefore, improved hygiene and avoidance of calf contact among this population should be advocated to reduce the occurrence of Cryptosporidium infections, especially those caused by C. parvum IIa subtypes of calves.

Supporting Information

Checklist S1.

STROBE checklist.

doi:10.1371/journal.pntd.0002831.s001

(PDF)

Table S1.

Association between CD4+ cell count and infection with Cryptosporidium species or C. parvum and C. hominis subtypes in HIV/AIDS patients in Ethiopia*.

doi:10.1371/journal.pntd.0002831.s002

(DOCX)

Table S2.

Association between HAART history and infection with Cryptosporidium species or C. parvum and C. hominis subtypes in HIV/AIDS patients in Ethiopia*.

doi:10.1371/journal.pntd.0002831.s003

(DOCX)

Acknowledgments

We thank the Tikur Anbessa Hospital for supporting the sample collection and CD4 cell count, and Na Li, Hongwei Zhang, Theresa Dearen, and Dawn Roellig for technical assistance. We also thank the anonymous reviewer for suggestions on statistical analyses of the data.

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Author Contributions

Conceived and designed the experiments: HA LX BP HK. Performed the experiments: HA SA JY. Analyzed the data: HA GZ LX. Contributed reagents/materials/analysis tools: LX YF JY. Wrote the paper: HA LX YF.

References

  1. 1. Agholi M, Hatam GR, Motazedian MH (2013) HIV/AIDS-associated opportunistic protozoal diarrhea. AIDS Res Hum Retroviruses 29: 35–41. doi: 10.1089/aid.2012.0119
  2. 2. Ives NJ, Gazzard BG, Easterbrook PJ (2001) The changing pattern of AIDS-defining illnesses with the introduction of highly active antiretroviral therapy (HAART) in a London clinic. J Infect 42: 134–139. doi: 10.1053/jinf.2001.0810
  3. 3. Miao YM, Awad-El-Kariem FM, Franzen C, Ellis DS, Muller A, et al. (2000) Eradication of cryptosporidia and microsporidia following successful antiretroviral therapy. J Acquir Immune Defic Syndr 25: 124–129. doi: 10.1097/00126334-200010010-00006
  4. 4. Zardi EM, Picardi A, Afeltra A (2005) Treatment of cryptosporidiosis in immunocompromised hosts - Still an open question. Chemotherapy 51: 193–196. doi: 10.1159/000086920
  5. 5. Da Silva CV, Ferreira MS, Borges AS, Costa-Cruz JM (2005) Intestinal parasitic infections in HIV/AIDS patients: Experience at a teaching hospital in central Brazil. Scand J Infect Dis 37: 211–215.
  6. 6. Leitch GJ, He Q (2012) Cryptosporidiosis-an overview. J Biomed Res 25: 1–16. doi: 10.1016/s1674-8301(11)60001-8
  7. 7. Certad G, Arenas-Pinto A, Pocaterra L, Ferrara G, Castro J, et al. (2005) Cryptosporidiosis in HIV-infected Venezuelan adults is strongly associated with acute or chronic diarrhea. Am J Trop Med Hyg 73: 54–57.
  8. 8. Werneck-Silva AL, Prado IB (2009) Gastroduodenal opportunistic infections and dyspepsia in HIV-infected patients in the era of Highly Active Antiretroviral Therapy. J Gastroenterol Hepatol 24: 135–139. doi: 10.1111/j.1440-1746.2008.05700.x
  9. 9. Baldursson S, Karanis P (2011) Waterborne transmission of protozoan parasites: review of worldwide outbreaks - an update 2004–2010. Water Res 45: 6603–6614. doi: 10.1016/j.watres.2011.10.013
  10. 10. Yoder JS, Beach MJ (2010) Cryptosporidium surveillance and risk factors in the United States. Exp Parasitol 124: 31–39. doi: 10.1016/j.exppara.2009.09.020
  11. 11. Chalmers RM (2012) Waterborne outbreaks of cryptosporidiosis. Ann Ist Super Sanita 48: 429–446. doi: 10.4415/ann_12_04_10
  12. 12. Xiao L (2010) Molecular epidemiology of cryptosporidiosis: an update. Exp Parasitol 124: 80–89. doi: 10.1016/j.exppara.2009.03.018
  13. 13. Fayer R, Santín M (2009) Cryptosporidium xiaoi n. sp. (Apicomplexa: Cryptosporidiidae) in sheep (Ovis aries). Vet Parasitol 164: 192–200. doi: 10.1016/j.vetpar.2009.05.011
  14. 14. Fayer R, Santín M, Macarisin D (2010) Cryptosporidium ubiquitum n. sp. in animals and humans. Vet Parasitol 172: 23–32. doi: 10.1016/j.vetpar.2010.04.028
  15. 15. Plutzer J, Karanis P (2009) Genetic polymorphism in Cryptosporidium species: An update. Vet Parasitol 165: 187–199. doi: 10.1016/j.vetpar.2009.07.003
  16. 16. Ren X, Zhao J, Zhang L, Ning C, Jian F, et al. (2012) Cryptosporidium tyzzeri n. sp. (Apicomplexa: Cryptosporidiidae) in domestic mice (Mus musculus). Exp Parasitol 130: 274–281. doi: 10.1016/j.exppara.2011.07.012
  17. 17. Elwin K, Hadfield SJ, Robinson G, Crouch ND, Chalmers RM (2012) Cryptosporidium viatorum n. sp. (Apicomplexa: Cryptosporidiidae) among travellers returning to Great Britain from the Indian subcontinent, 2007–2011. Int J Parasitol 42: 675–682. doi: 10.1016/j.ijpara.2012.04.016
  18. 18. Insulander M, Silverlås C, Lebbad M, Karlsson L, Mattsson JG, et al. (2013) Molecular epidemiology and clinical manifestations of human cryptosporidiosis in Sweden. Epidemiol Infect 141-: 1009–1020. doi: 10.1017/s0950268812001665
  19. 19. Hunter PR, Hughes S, Woodhouse S, Raj N, Syed Q, et al. (2004) Health sequelae of human cryptosporidiosis in immunocompetent patients. Clin Infect Dis 39: 504–510. doi: 10.1086/422649
  20. 20. Cama VA, Bern C, Roberts J, Cabrera L, Sterling CR, et al. (2008) Cryptosporidium species and subtypes and clinical manifestations in children, Peru. Emerg Infect Dis 14: 1567–1574. doi: 10.3201/eid1410.071273
  21. 21. Cama VA, Ross JM, Crawford S, Kawai V, Chavez-Valdez R, et al. (2007) Differences in clinical manifestations among Cryptosporidium species and subtypes in HIV-infected persons. J Infect Dis 196: 684–691. doi: 10.1086/519842
  22. 22. Adamu H, Petros B, Hailu A, Petry F (2010) Molecular characterization of Cryptosporidium isolates from humans in Ethiopia. Acta Trop 115: 77–83. doi: 10.1016/j.actatropica.2010.02.003
  23. 23. Fisseha B, Petros B, WoldeMichael T (1998) Cryptosporidium and other parasites in Ethiopian AIDS patients with chronic diarrhoea. East Afr Med J 75: 100–101.
  24. 24. Tadesse A, Kassu A (2005) Intestinal parasite isolates in AIDS patients with chronic diarrhea in Gondar Teaching Hospital, North west Ethiopia. Ethiop Med J 43: 93–96.
  25. 25. Mengesha B (1994) Cryptosporidiosis among medical patients with the acquired immunodeficiency syndrome in Tikur Anbessa Teaching Hospital, Ethiopia. East Afr Med J 71: 376–378.
  26. 26. Alemu A, Shiferaw Y, Getnet G, Yalew A, Addis Z (2011) Opportunistic and other intestinal parasites among HIV/AIDS patients attending Gambi Higher Clinic in Bahir Dar City, North West Ethiopia. Asian Pac J Trop Med 4: 661–665. doi: 10.1016/s1995-7645(11)60168-5
  27. 27. Jiang J, Alderisio KA, Xiao L (2005) Distribution of Cryptosporidium genotypes in storm event water samples from three watersheds in New York. Appl Environ Microbiol 71: 4446–4454. doi: 10.1128/aem.71.8.4446-4454.2005
  28. 28. Feng Y, Li N, Duan L, Xiao L (2009) Cryptosporidium genotype and subtype distribution in raw wastewater in Shanghai, China: Evidence for possible unique Cryptosporidium hominis transmission. J Clin Microbiol 47: 153–157. doi: 10.1128/jcm.01777-08
  29. 29. Sulaiman IM, Hira PR, Zhou L, Al-Ali FM, Al-Shelahi FA, et al. (2005) Unique endemicity of cryptosporidiosis in children in Kuwait. J Clin Microbiol 43: 2805–2809. doi: 10.1128/jcm.43.6.2805-2809.2005
  30. 30. Molloy SF, Smith HV, Kirwan P, Nichols RAB, Asaolu SO, et al. (2010) Identification of a high diversity of Cryptosporidium species, genotypes and subtypes in a pediatric population in Nigeria. Am J Trop Med Hyg 82: 608–613. doi: 10.4269/ajtmh.2010.09-0624
  31. 31. Gatei W, Wamae CN, Mbae C, Waruru A, Mulinge E, et al. (2006) Cryptosporidiosis: prevalence, genotype analysis, and symptoms associated with infections in children in Kenya. Am J Trop Med Hyg 75: 78–82.
  32. 32. Zintl A, Proctor AF, Read C, Dewaal T, Shanaghy N, et al. (2009) The prevalence of Cryptosporidium species and subtypes in human faecal samples in Ireland. Epidemiol Infect 137: 270–277. doi: 10.1017/s0950268808000769
  33. 33. Chalmers RM, Smith R, Elwin K, Clifton-Hadley FA, Giles M (2011) Epidemiology of anthroponotic and zoonotic human cryptosporidiosis in England and Wales, 2004–2006. Epidemiol Infect 139: 700–712. doi: 10.1017/s0950268810001688
  34. 34. Chalmers RM, Katzer F (2013) Looking for Cryptosporidium: the application of advances in detection and diagnosis. Trends Parasitol 29: 237–251. doi: 10.1016/j.pt.2013.03.001
  35. 35. Quilez J, Vergara-Castiblanco C, Monteagudo L, Del Cacho E, Sanchez-Acedo C (2013) Host-association of C. parvum populations infecting domestic ruminants in Spain. Appl Environ Microbiol 79: 5363–5371. doi: 10.1128/aem.01168-13
  36. 36. Amer S, Zidan S, Feng Y, Adamu H, Li N, et al. (2013) Identity and public health potential of Cryptosporidium spp. in water buffalo calves in Egypt. Vet Parasitol 191: 123–127. doi: 10.1016/j.vetpar.2012.08.015
  37. 37. Silverlas C, Bosaeus-Reineck H, Naslund K, Bjorkman C (2012) Is there a need for improved Cryptosporidium diagnostics in Swedish calves? Int J Parasitol 43: 155–161. doi: 10.1016/j.ijpara.2012.10.009
  38. 38. Wang R, Wang H, Sun Y, Zhang L, Jian F, et al. (2011) Characteristics of Cryptosporidium transmission in preweaned dairy cattle in Henan, China. J Clin Microbiol 49: 1077–1082. doi: 10.1128/jcm.02194-10