Abstract:
This study evaluated the epidemiology of cryptosporidiosis in Ebonyi and Enugu states. A total of 955 human faecal samples were collected and analysed for Cryptosporidium infection. Of the 955 samples, 561 were analysed by direct wet mount and modified Ziehl Neelsen staining techniques, while 394 samples were analysed by molecular methods. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were used to identify Cryptosporidium species, while sequencing of gp60 PCR products was used to identify subtypes. Real-time PCR analysis of 18S rRNA gene was used to determine intensity of infection. The results showed that routine wet mount alone could not identify Cryptosporidium as the technique could not differentiate the parasite from yeasts and 56 samples out of the 561 (10%) were scored positive for yeast-like organisms. However, the application of modified Ziehl Neelsen staining detected Cryptosporidium in 25 out of the 56 specimen with yeast-like organisms, giving an overall prevalence of 4.5% for Cryptosporidium and prevalence values of 2% and 7.3%, in samples from Enugu and Abakaliki, respectively. Application of PCR gave a prevalence value of 6.3% and four Cryptosporidium spp. (C. hominis, C. parvum, C. felis and C. viatorum) were identified by RFLP. C. hominis had the highest occurrence, accounting for 48% of the species identified, followed by C. parvum, with 44%, whereas C. felis and C. viatorum each had 4% occurrence. Molecular subtyping identified seven C. hominis subtypes in three subtype families (Ia, Ib and Ie) and three C. parvum subtypes in two subtype families (IIc and IIe). One novel C. hominis strain, which did not match any of the known subtypes, was also identified. Children had a significantly (p < 0.05) higher C. hominis infection rate (62.5%) compared to C. parvum (37.5%). Infection rates of HIV-infected patients on HAART (7.1%) and those not on HAART (6.5%) did not differ significantly (p > 0.05). Real-Time PCR revealed lower infectivity of C. parvum subtype family IIc and amino acid analysis of the gp60 gene revealed lack of tryptophan in the IIc subtype family. This study shows that molecular techniques are more sensitive for detection of Cryptosporidium species in clinical specimens and buttresses the need for the accurate diagnosis of every suspected yeast-like infection as may be detected in wet examination of faecal specimens. The study further showed a high diversity of Cryptosporidium spp. in humans in Ebonyi and Enugu, Nigeria and that most of the C. parvum subtypes were anthroponotic in origin.
