Molecular characterization of Cryptosporidium spp. from patients with diarrhoea in Lusaka, Zambia

Cryptosporidium is a major etiological agent of diarrhoeal diseases among children and immune-compromised individuals in sub-Saharan African countries. We conducted a study to determine the prevalence and genetic characteristics of Cryptosporidium spp. in stool samples from patients with diarrhoea who presented at the University Teaching Hospital in Lusaka, Zambia. Cryptosporidium species and subtypes from 71 microscopically confirmed cryptosporidiosis stool samples collected between 2017 and 2019 were determined by polymerase chain reaction followed by partial sequencing of the small subunit rRNA and 60-kDa glycoprotein (gp60) gene. Additionally, data for the period between 2014 and 2019 were reviewed and analysed for cryptosporidiosis seasonal and age distribution. Cryptosporidium was more prevalent in the rainy season. The highest number of cases was reported among the 1–4 year age group. By sequence analysis of the 71 positive isolates, Cryptosporidium hominis (n = 42; 59.2%), C. parvum (n = 27; 38%), C. felis (n = 1; 1.4%), and C. meleagridis (n = 1; 1.4%) were identified. Four C. hominis subtype families (Ia, Ib, Id, and Ie) and three C. parvum subtype families (IIc, IIe, and IIs) were identified. The most frequent subtypes were IeA11G3T3 (n = 20; 28.2%), IIcA5G3 (n = 12; 16.9%), IIeA12G1 (n = 11; 15.5%) and IaA30R3 (n = 10; 14.1%). The observed species/subtypes of C. hominis and C. parvum indicated that the infection was mainly transmitted through the anthroponotic route. The identification of C. felis and C. meleagridis suggests that an atypical zoonotic transmission cycle also exists.


Introduction
Cryptosporidium is one of the most prevalent protozoan parasites causing diarrhoea in humans, but it has been largely neglected [29]. It is a major cause of diarrhoeal diseases in children in developing countries and is associated with occasional outbreaks among adults in developed countries [6,24]. Cryptosporidium is also an opportunistic parasite, which has been associated with diarrhoea in immune-compromised individuals and is a major cause of morbidity and mortality among such populations [26,28,30]. In immune-competent individuals, the infection is either asymptomatic or may cause selflimiting diarrhoea that normally resolves within a week or two [26,28]. However, in immune-compromised individuals or children, diarrhoea is usually severe and life-threatening, lasting for more than two weeks if not effectively managed [28]. Cryptosporidiosis is usually associated with poor hygiene and is transmitted through contaminated food or water via the fecal-oral route [30].
More than 30 species of Cryptosporidium have been identified worldwide, with C. hominis and C. parvum accounting for most of the infections in humans [36]. Cryptosporidium hominis primarily infects humans, whereas C. parvum has both zoonotic and anthroponotic genotypes [30,36]. These species can be further classified into subtype families and subtypes based on the 60 kDa glycoprotein (gp60) gene sequence. Some subtype families such as C. parvum IIc and IIe are predominantly found in humans, especially in lower-income countries, and are considered to be anthroponotic [17,37]. Other less common Cryptosporidium species that are primarily animal infective pathogens, but that can also occasionally infect humans include C. meleagridis, C. felis, C. canis, C. ubiquitum, C. cuniculus, C. viatorum, and C. muris [9].
While Cryptosporidium spp. have been documented from both asymptomatic and symptomatic people in Zambia [27,32,33], data on subtype characterisation of the species are limited. In this study, we aimed to document Cryptosporidium species and subtypes circulating in patients with diarrhoea at the University Teaching Hospital in Lusaka, Zambia. Additionally, the prevalence and seasonal distribution of Cryptosporidium were determined.

Ethics statement
This study was approved by the University of Zambia Biomedical Research Ethics Committee (IRB00001131). Further clearance to conduct this study and publish the findings was given by the National Health Research Authority.

Study population and Cryptosporidium sample collection
This study was conducted at the University Teaching Hospital (UTH), a tertiary-level government hospital located in Lusaka, Zambia. Data for 19,033 patients who had submitted stool samples for routine parasitological investigations over 5 years (January 2014-May 2019) were reviewed and analysed. The cryptosporidiosis case prevalence, age, and seasonal distribution were determined. Fisher's exact test was used to determine differences in the prevalence of cryptosporidiosis by age-group and sex. A p-value below 0.05 was considered statistically significant. Monthly precipitation data at Lusaka airport during the study period were obtained from SASSCAL WeatherNet (www.sasscalweathernet.org). All 19,033 samples were routinely examined for Cryptosporidium oocysts by microscopic examination of modified Ziehl-Neelsen-stained stool smears [13]. Between 2017 and 2019, 71 microscopically confirmed Cryptosporidium spp.-positive faecal samples were stored at À20°C before the molecular characterisation of species and subtypes. Detection of other parasites was done using the formol-ether concentration method. Briefly, about 1 g of stool was mixed with 10% formalin and was sieved through gauze into a centrifuge tube to which 3 mL of ether were added. After vigorous mixing, it was centrifuged at 3000 rpm, as previously described [1], and the deposit was microscopically examined for the presence of intestinal parasites.

DNA sequencing and species/subtypes identification
All PCR-positive samples were treated with ExoSAP-IT (Thermo Fisher Scientific) before sequencing to remove excess primers and dNTPs, according to the manufacturer's instructions. PCR products were then analysed on a SeqStudio genetic analyzer using an ABI BigDye3.1 Terminator Cycle Sequencing Kit (Applied Biosystems, Carlsbad, CA, USA). The nucleotide sequences obtained for the SSU rRNA genes were then used to search the GenBank nucleotide database for any similarities using Basic Local Alignment Search Tool (BLASTn) software (National Center for Biotechnology Information, NCBI) for species identification. Top hit sequences with high similarity (>98%) were considered to be the assigned species. The gp60 sequences were used for the assignment of the subtypes by counting the repeat number by ApE editor (by Davis M. Wayne; https://jorgensen.biology.utah.edu/wayned/ ape/). The gp60 sequences were aligned with the representative sequences of Cryptosporidium subtypes by using ClustalW, and a neighbour-joining tree with 1000 bootstrap replications was created using Molecular Evolutionary Genetics Analysis (MEGA) software version 7 [19]. The obtained sequences were deposited in GenBank (SSU rRNA of C. parvum; MH816914, MH816918; SSU rRNA of C. hominis; MH816925, MH816915; SSU rRNA of C. felis; MH816917; SSU rRNA of C. meleagridis; MT549157; gp60 of C. parvum; MN904699-MN904725; gp60 of C. hominis; MN904655-MN904698).
The confirmed cryptosporidiosis cases were also analysed by month in relation to the average monthly rainfall (mm) in Lusaka (Fig. 1). The number of cryptosporidiosis cases varied across time, with some months recording fewer than 10 cases. The highest prevalence was observed toward the end of the rainy seasons of 2015 and 2019. A notable increase in the number of patients with cryptosporidiosis was observed between January 2019 and April 2019. The number of patients with cryptosporidiosis was 5.6 times higher than what was recorded in the same period between 2015 and 2018 (78 cases vs. 14 average cases, respectively).
Information on age, HIV infection status, and the year of cryptosporidiosis detection were obtained, where available. Among the 51 cases with demographic data, the median age was 4 years (range: 4 months to 61 years). Thirty samples (58.8%) were from male patients and 15 samples (29.4%) were from HIV-positive patients. No significant associations between Cryptosporidium subtypes and patient age group, sex, year of cryptosporidiosis detection, or HIV infection status were observed.
Sequencing results and phylogenetic tree analysis based on SSU rRNA and gp60 Seventy-one microscopically Cryptosporidium-positive samples stored from 2017 to 2019 were analysed for species and subtype identification using the SSU rRNA and gp60 PCR, respectively. Out of 71 samples, 60 samples were successfully sequenced for SSU rRNA genes. Based on the SSU rRNA sequence analysis, one C. felis (GenBank: MH816917) and one C. meleagridis (MT549157) were obtained, and these two samples were not amplified by our gp60 targeting primer. In all, 58 samples were amplified for both SSU rRNA and gp60, and species identified in SSU rRNA agreed with the gp60 genotype families (genotype family I for C. hominis and genotype family II for C. parvum). Eleven samples were not amplified for the SSU rRNA gene, but the species could be identified based on the gp60 sequence ( Table 2).

Discussion
Cryptosporidium was identified as the most common parasite among patients with diarrhoea diagnosed at the parasitology laboratory of the UTH, a tertiary-level hospital in Lusaka, Zambia during the studied period. The sequence analysis revealed that C. hominis and C. parvum were both common in the patients. Several studies have reported Cryptosporidium as a major cause of moderate-severe diarrhoea among young children in developing countries [18,34]. In the current study,   high Cryptosporidium prevalence was observed in the 1-4 year age group, emphasising the importance of Cryptosporidium as an aetiological agent of diarrhoea in this age group. When analysed by gender, the prevalence of cryptosporidiosis was significantly higher in males than in females in the 1-4 year age group. Similar findings were also documented in other countries including Nigeria, Guinea-Bissau, and Ghana [3,7,22], although the cause of this gender bias is unknown. In contrast, females in the 20-29 year age group were more likely to have cryptosporidiosis than males in our study. One possible explanation for the observed trend is the high risk of exposure to Cryptosporidium among women when caring for their infected children. According to Kimani et al., gender is one of the determinants of Cryptosporidium infection as women are more likely to become infected than men [16]. Although data on HIV positivity and immune status among our analysed population were not available, the higher prevalence of HIV in the adult female population in Zambia [5] may also explain the higher prevalence of Cryptosporidium in the female 20-29 year age group in the current study. Analysis of Cryptosporidium cases by month revealed that the infection was more common during the rainy season. This observation corroborates the results of several studies in African countries that have shown similar seasonality [14,33,35]. Previous studies have reported an association between rainfall and diarrhoeal disease outbreaks [21,31]. Similarly, we observed a higher number of cryptosporidiosis cases towards the end of the rainy season, which peaked in March for the years 2015, 2018, and 2019. This is possibly due to the contamination of water sources with human waste during flash flooding. However, despite having less precipitation in the year 2018/2019, more cases were recorded compared to those in the previous years. The high number of cases could have been attributed to water shortages causing people to consume contaminated water from unclean sources.
Although previous studies had reported high cryptosporidiosis prevalence in Zambia [15,25], molecular studies on Cryptosporidium species or subtypes associated with the disease were not reported. Microscopic identification of Cryptosporidium at the species level is impossible as the species are morphologically similar, and hence can only be differentiated with molecular tools. The current study is the first to our knowledge to characterise Cryptosporidium species and subtypes among patients with diarrhoea in Zambia. We utilised SSU rRNA gene sequencing for species identification and gp60 sequencing for subtyping of C. parvum and C. hominis. Although C. parvum and C. hominis are classified in distinct species for their biological and sequence differences [23], it is well known that some gp60 alleles in these species are phylogenetically closely related [39]. Incongruence of the gp60 loci topology was also observed in our analysed phylogenetic tree. A possible cause of the discordance may be recombination events between species and subtypes at the gp60 locus [12,20,24]. The gp60 subtypes are known to be associated with host tropism [24], virulence [4], and geographical distribution [11,24], and thus provide useful information to understand the molecular epidemiology of cryptosporidiosis. In this study, of the four species detected, C. hominis was the most dominant species (59.2%). Subtype IeA11G3T3 was the most prevalent subtype (28.2%), with minimal sequence diversity. Three other subtypes of C. hominis, IaA30R3, IbA9G3, and IdA21, also showed low sequence variation within the analysed samples, suggesting that limited lineages of C. hominis were circulating in Lusaka. Subtype IIcA5G3 was the most prevalent (16.9%) in C. parvum and sequences were further classified into IIcA5G3a and IIcA5G3b. The IIcA5G3a subtype has been almost exclusively reported in humans [24]. A recent whole-genome analysis revealed that subtype IIc-a was clustered differently from zoonotic subtypes, and thus was referred to as C. p. anthroponosum compared to zoonotic subtypes C. p. parvum. It is speculated that C. p. anthroponosum resulted from recent genomic introgression events and is considered to be human-adapted C. parvum [24]. The documentation of the IIc-a subtype family in our study confirms earlier reports that it is mostly found in low-income countries and other African countries [10,17,35]. The IIc-b and IIe subtypes detected in the current study were also considered to be anthroponotic [36]. The major zoonotic subtype families IIa and IId have cosmopolitan distributions, with more cases being reported in industrialised countries [9,11]. However, none of these zoonotic subtypes were detected in our study. In a neighbouring country, Malawi, four C. hominis subtypes (Ia, Ib, Id, and Ie), and two C. parvum subtypes (IIc and IIe) have been reported, and were all consistent with the subtype family identified in our study. Subtype IIs found in one sample in our study is a rare subtype that had been reported only from a human faecal sample in Sweden (GenBank KU852720). The host tropism, transmission cycle, and distribution of this subtype are largely unknown. Except for IIs, all subtypes of C. parvum and C. hominis detected in our study had also been reported in other African countries such as Ghana, Kenya, South Africa, or Nigeria [35]. Thus, like the cases in most of the other African countries, the human-to-human anthroponotic transmission route seems to be the major route of Cryptosporidium spp. infection in the urban setting of Zambia. We analysed samples from patients in urban areas with limited or no animal contact; hence, further studies in peri-urban and rural populations of Zambia where human-animal contact is high should be conducted to analyse the nation-wide epidemiological features of this disease. The detection of the potentially zoonotic species, C. felis and C. meleagridis, is a noteworthy finding that warrants further investigations, particularly as their transmission dynamics remain unknown [8,9].
In our population, no associations between any particular subtype and the patient's age, sex, year of cryptosporidiosis detection, or HIV status were observed. The high number of cryptosporidiosis cases observed in the 2018/2019 rainy season seems to be the result of infections originating from different sources. Since our sequence analysis targeted only patients with diarrhoea, further studies will be needed to assess the genetic heterogeneity of the Cryptosporidium parasite in non-diarrhoeal cases and animals in Zambia.

Conclusions
Our study shows that C. hominis and C. parvum are the most common aetiological agents of cryptosporidiosis among diarrhoeal cases presenting at the UTH. The high prevalence in children (1-4 years) calls for more targeted interventions to control cryptosporidiosis in this age group. Cryptosporidium hominis subtypes Ia, Ib, Id, Ie, and C. parvum subtypes IIc, IIe and IIs were identified by sequencing for the first time in patients with diarrhoea in Zambia. Identification of the rare zoonotic species C. felis and C. meleagridis suggests that both anthroponotic and zoonotic transmission routes were responsible for cryptosporidiosis transmission in the studied population.

Conflicts of interest
The authors declare that they have no conflict of interest.