Cryptosporidium species and subtypes in diarrheal children and HIV-infected persons in Ebonyi and Nsukka , Nigeria

Introduction: Cryptosporidiosis is a common disease of children and immune-compromised persons. This study evaluated the diversity and distribution of Cryptosporidium species in diarrheal children and HIV-infected persons on highly active antiretroviral therapy (HAART) and those not on HAART. Methodology: A total of 394 fecal specimens were collected from patients attending clinics in Nsukka and Ebonyi, Nigeria. Detection and identification of Cryptosporidium species were conducted by PCR-RFLP of the small subunit (SSU) rRNA gene, whereas subtyping was done by sequence analysis of the 60 kDa glycoprotein (gp60) gene. Results: Twenty-five (6.3%) specimens yielded four Cryptosporidium species, including C. hominis, C. parvum, C. felis, and C. viatorum. C. hominis was the most dominant species with 48.0% occurrence and three identified subtype families: Ia (six specimens), Ib (three specimens), Ie (two specimens), and one un-subtyped species. C. parvum had 44.0% occurrence and two subtype families: IIc (eight specimens) and IIe (three specimens), while C. felis and C. viatorum each had 4.0% occurrence. There were significant differences in Cryptosporidium species distribution between age groups in children and HIV-infected persons, between suburban and urban areas, and between low and high CD4 cell counts in HIV-infected patients. There were no significant differences in infection rate and species distribution between HIV-infected patients on HAART and those not on HAART. Conclusions: The results from this study show that there is a high diversity of Cryptosporidium spp. in humans in Ebonyi and Nsukka, Nigeria, and that all the C. parvum subtypes identified are most likely anthroponotic in origin.


Introduction
Cryptosporidium is a protozoan parasite that has been implicated in diarrheal illness in children and immune-compromised individuals such as HIVinfected persons [1,2]. The parasite can be zoonotic or anthroponotic in origin and has been implicated in diarrhea outbreaks in different parts of the world [3][4][5]. Infections in HIV + patients have been associated with low CD4 + cell counts (< 200 cells/mm 3 ), lack of access to highly active antiretroviral therapy (HAART), and poor hygiene, while infections in children are associated mostly with young age and poor hygiene [4,6,7].
The use of molecular diagnostic tools has greatly enhanced the identification of infection sources and transmission routes of Cryptosporidium [3].
Genotyping and subtyping of Cryptosporidium species are done mostly by polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) analysis of the small subunit (SSU) rRNA gene and the sequencing of the 60 kDa glycoprotein gene (gp60). This has led to identification of about 20 Cryptosporidium species and genotypes in humans, including C. parvum, C. hominis, C. meleagridis, C. canis, C. felis, and the recently identified C. viatorum. Among them, C. parvum and C. hominis are most common. These Cryptosporidium species have been found in both HIV + patients and children.
Molecular characterizations of Cryptosporidium spp. in a small number of HIV-infected persons and children in southwestern and mid-western states have provided some information on Cryptosporidium species and subtypes in Nigeria [4,6,[8][9][10][11]. However, no information is available on the species and subtypes circulating in humans in south-eastern Nigeria. In this study, a molecular epidemiological study of Cryptosporidium spp. in HIV-infected patients and diarrheal children in Ebonyi and Nsukka, south-eastern Nigeria, was conducted.

Specimens
Fecal specimens were collected from 394 patients including diarrheal children and HIV-positive individuals attending clinics at 7 healthcare facilities: were diarrheal children under 6 years of age from urban and suburban areas of Ebonyi state, while 251 were HIV-infected persons with diarrhea or related illness from Ebonyi (n = 115) and Nsukka (n = 136). Data on CD4 + cell counts, history of HAART in HIV + persons were obtained from each patient. Ethical permission and informed consent were obtained in each hospital/clinic where specimens were collected. The study protocols were approved by the hospital's research ethics committees in line with the Nigerian Ministry of Health guidelines. The fecal specimens were preserved in 2.5% potassium dichromate at 4°C and shipped to the Centers for Disease Control and Prevention (CDC), Atlanta, USA for molecular analysis. Information on patients' personal details was kept strictly confidential.

DNA extraction
The fecal specimens were washed twice with distilled water by centrifugation. DNA was extracted from 0.2 mL of the concentrates using the FastDNA SPIN kit for soil (MP Biologicals, Irvine, USA). The extracted DNA was stored at -20°C prior to polymerase chain reaction (PCR) analysis.

PCR detection of Cryptosporidium species and RFLP analysis
The extracted DNA was analyzed for Cryptosporidium spp. by nested PCR analysis of the SSU rRNA gene according to Xiao et al. [12]. RFLP analysis of the secondary PCR products using restriction enzymes SspI (New England Biolab, Ipswich, USA) and VspI (Promega, Madison, USA) was used to identify Cryptosporidium species. DNA sequencing was used to confirm the diagnosis of C. felis and C. viatorum. Subtyping was done by DNA sequence analysis of the gp60 gene as described by Alves et al. [13].

Sequence analysis
DNA sequencing of the secondary PCR products of SSU rRNA and gp60 genes was carried out using the BigDye Terminator Sequencing Kit (Applied Biosystems, Foster City, USA) and an ABI Prism 3130 Genetic Analyzer (Applied Biosystems, Foster City, USA). The nucleotide sequences were assembled and edited using ChromasPro version 1.7.5 software (Technelysium, South Brisbane, Australia). To confirm the species identification and subtypes, the consensus sequences were aligned with each other and reference sequences of each gene downloaded from GenBank using ClustalX (http://www.clustal.org/). Cryptosporidium subtypes were named based on the established naming terminology [3].

Statistical analysis
The association of Cryptosporidium infections with CD4 + cell counts and HAART history in HIV-infected persons was determined by χ 2 analysis. The difference in infection rates between urban and sub-urban areas or between HIV-infected patients in Ebonyi, Nsukka and diarrheal children were evaluated using one-way ANOVA. All statistical analyses were performed using SPSS version 16 at the 95% confidence level.

Occurrence of Cryptosporidium spp. in HIV + persons and diarrheal children in Ebonyi and Nsukka
A total of 25 of the 394 specimens screened in this study were positive for Cryptosporidium spp., giving a general prevalence of 6.3%. The 25 positive samples comprised 20 of 258 samples examined in Ebonyi and 5 of 136 samples from Nsukka, thus giving prevalence values of 7.8% and 3.7% for Ebonyi and Nsukka, respectively. Within Ebonyi, there was a significantly (p < 0.05) higher prevalence of Cryptosporidium in the suburban area (13.6%) than in the urban area (4.7%).
Of the 394 samples, 251 were from HIV + persons, while 143 were from children. Infection rates in the HIV + persons and children were 6.8% and 5.6%, respectively. All the infected children were under 2 years of age, and no infection was observed in older children ( Figure 1). Among the HIV + population, infection rates were higher in Ebonyi (4.7%) than in Nsukka (3.7%).

Occurrence of Cryptosporidium infection in HIV + patients receiving HAART and those not receiving HAART
Review of patients' clinical histories showed that 47 of 115 HIV + patients in Ebonyi were on HAART, while 68 were not. Of the 47 patients on HAART, 5 (10.6%) were infected with Cryptosporidium as opposed to 7 of 68 (10.3%) patients who were not on HAART. Similarly, in Nsukka, 3 of 67 (4.5%) HIV + patients on HAART were infected, while 2 of 69 (2.9%) of patients not on HAART were infected ( Figure 4). Therefore, a total of 8/113 or 7.1% of HIV-infected persons on HAART were co-infected with Cryptosporidium, while 9/138 or 6.5% of non-HAART patients were co-infected. These differences were not statistically significant (p > 0.05). Of the 8 patients who were on HAART and infected with Cryptosporidium, 7 were residents of suburban areas, while 1 patient was a resident of an urban area.
A total of 10 Cryptosporidium subtypes were identified, including 7 C. hominis subtypes (IaA18R3, IaA25R3, IaA27R4, IaA29R3, IbA10G2, IbA13G3, and IeA11G3T3) and 3 C. parvum subtypes (IIeA10G1, IIcA5G3b and IIcA5G3k). One C. hominis, which could not be matched with any known subtype, was also identified from Ebonyi (Table 1). Among the C. hominis subtypes, IaA18R3 had the highest occurrence, followed by IbA13G3, with 3 and 2 positive samples in HIV + patients placed on HAART and those not placed HAART therapy at different study locations (Nsukka and Ebonyi respectively). The two bars on the left represent those placed on HAART therapy, whereas the two right bars represent those who were not placed on HAART in the study locations.  respectively, while the other subtypes occurred in only 1 sample each. Among the C. parvum subtypes, on the other hand, IIeA10G1 and IIcA5G3b were identified in 3 specimens each, while IIcA5G3k was identified in 2 specimens (Table 1).

Discussion
Cryptosporidium infections in children and immune-compromised individuals such as HIVinfected persons have been reported from many parts of the world, including southwestern and midwestern Nigeria [4][5][6]8,10,[14][15][16]. This study was conducted in southeastern Nigeria. The results showed relatively high rates of Cryptosporidium infection in diarrheal children and HIV-infected persons in Ebonyi and Nsukka, southeastern Nigeria, with a significantly (p < 0.05) higher infection rate in Ebonyi (7.8%) than in Nsukka (3.7%). The difference in Cryptosporidium infection rate between Ebonyi and Nsukka is probably due to the fact that residents of Ebonyi have more frequent contact with bodies of water, such as streams and rivers, which are commonly contaminated by sewage and domestic wastewater, as transmission of Cryptosporidium spp. is often associated with human and animal fecal contamination of water sources [3,[17][18][19][20].
Cryptosporidium infections have been associated with low immunity, especially in HIV-infected persons [8,14]. In this study, HIV-infected persons with CD4 + cell counts < 200 cells/mm 3 had a significantly (p < 0.05) higher infection rate (9.7%) than those with CD4 + cells > 200 cells/mm 3 (5.2%). This finding is consistent with the reports of Ayinmode et al. and Adamu et al. [5,6] from similar studies conducted in western Nigeria and Ethiopia, respectively. In this study, however, one of the HIV-infected persons, with CD4 + cell count of 542 cells/mm 3 , was infected with Cryptosporidium. CD4 + cell counts of 500 cells/mm 3 are considered as the baseline for immunological competence in humans [21]. The finding in this study, therefore, of Cryptosporidium infection in a patient who could be regarded as immune competent, supports previous suggestions by some investigators that Cryptosporidium can infect immune-competent individuals [22].
HAART is used to manage HIV infection by reducing viral load, improving immunological status, and reducing opportunistic infections associated with HIV infection [21,23]. In this study, no significant (p > 0.05) difference in infection rate was found between HIV-infected persons on HAART and those not on HAART. This finding is consistent with the reports of Akinbo et al. and Ayinmode et al. [6,11] that HIVinfected patients on HAART can have Cryptosporidium infection.
Review of the clinical records of the HIV-infected persons on HAART co-infected with Cryptosporidium in this study revealed that they were placed on a threedrug regimen comprising nucleoside/nucleotide reverse transcriptase inhibitors (NRTI) and non-nucleoside reverse transcriptase inhibitors (NNRTI). None of the patients had HIV-protease inhibitors in their treatment regimen. Of the 8 patients on HAART and co-infected with Cryptosporidium, 5 had been on the drugs for over 2 years, 2 for about 1 year and the remaining 1 patient for 5 weeks prior to sampling. The investigation also revealed that the hospitals from which the specimens were collected in this study used HIV-protease inhibitors as the second line of drugs for management of HIV + patients, only in the event of treatment failure. Protease inhibitors have been reported to reduce the occurrence of opportunistic infections in HIV-infected persons [21,22]. It is therefore possible that Cryptosporidium infection of HIV-infected persons on HAART seen in this study could be attributable to the non-inclusion of HIV-protease inhibitors in their treatment regimen. The occurrence of infection in these patients could also be attributable to non-adherence to treatment as is quite common in Nigeria.
A high diversity of Cryptosporidium species/subtypes was observed in this study. Although C. hominis and C. parvum were the dominant Cryptosporidium species, one case each of C. viatorum and C. felis was found. The latter two had been previously seen in HIV-infected patients in Ibadan and Benin City, Nigeria [6,8,11]. Within the two dominant species, 6 C. hominis subtypes (IaA27R4, IaA18R3, IaA25R3, IbA10G2, IbA13G3, and IeA11G3T3) in 3 subtype families (Ia, Ib and Ie) and 3 C. parvum subtypes (IIcA5G3b, IIcA5G3k and IIeA10G1) in 2 subtype families (IIc & IIe) were seen diarrheal children and HIV-infected persons in Ebonyi. Similarly, 2 C. parvum subtypes (IIcA5G3b and IIcA5G3k) and 1 C. hominis subtype (IaA29R3) were among the small number of Cryptosporidium-positive specimens from HIV-infected persons in Nsukka. The high diversity of Cryptosporidium subtypes observed in this study is consistent with previous studies in western Nigeria and other African countries [5,6,8,10,11]. Some of the subtypes seen in this study, such as C. hominis subtypes IeA11G3T3, IbA13G3, IbA10G2, and IaA25R3 and C. parvum subtypes IIcA5G3b and IIcA5G3k, have been reported in studies conducted in Benin city, Edo, midwestern Nigeria and Oyo state, southwestern Nigeria [6,8,10]. Others such as C. hominis subtypes IaA18R3, IaA27R4, and IaA29R3 and C. parvum subtype IIeA10G1 represent unique subtypes seen in this study.
In this study, a higher occurrence of C. parvum, C. felis, and C. viatorum was seen in suburban areas, whereas C. hominis occurred more frequently in the urban area. The differences in Cryptosporidium infection rates, species, and subtype distribution between geographic locations or between urban and suburban areas are probably indications of differences in transmission rates in different settings within Nigeria. Nevertheless, anthroponotic transmission appears to be important in cryptosporidiosis epidemiology, as most Cryptosporidium infections were caused by the largely human-specific C. hominis and the anthroponotic C. parvum subtype families IIc and IIe [3]. This is consistent with reports of earlier epidemiological studies in Nigeria and other developing countries. More epidemiological studies are needed to identify reasons for the differences in the transmission of Cryptosporidium species and subtypes seen in this study among patient populations and geographic areas.

Conclusions
The high diversity of Cryptosporidium species and subtypes observed in this study (including unique Cryptosporidium hominis possibly lacking the gp60 gene [unsubtyped], C. hominis subtypes IaA18R3, IaA27R4, and IaA29R3, and C. parvum subtype IIeA10G1) is indicative of the need for consistent surveillance for zoonotic parasite in the region. The study further revealed that Cryptosporidium transmission in study area is probably anthroponotic in nature. Hence, further studies are needed to identify the exact route(s) of Cryptosporidium transmission in the studied areas, which will aid in controlling of the spread of the infectious agent.