Identification of Cryptosporidium Species Infecting Humans in Tunisia

Samples.

From 2006 through 2007, 708 stools samples were collected from 633 apparently immunocompetent children less than five years of age (477 living in farming areas in northeastern Tunisia and 156 originally from Tunis City) and from 75 patients hospitalized for immunodeficiency (39 HIV-infected patients and 36 children with primary immunodeficiency). Informed consent was obtained from parents and adult patients before stool collection.

Fresh stool specimens (549 formed and 159 diarrheal) were examined for Cryptosporidium spp. oocysts (Table 1). Microscopic examination was carried out on smears of fecal concentrates (formalinethyl acetate concentration) after staining with the modified Ziehl-Neelsen technique.⁸ When Cryptosporidium oocysts were identified microscopically, an aliquot of the stool specimen was diluted [v/v] in distilled water, placed into tubes, and stored frozen for molecular analysis.

Extraction of DNA.

DNA was extracted from samples using the QIAamp DNA Stool MiniKit (Qiagen Inc., Hilden, Germany) according to the manufacturer’s recommendations using approximately 200 μL of diluted stools for the first buffer step. After quantification, extracted DNA was frozen at −20°C until analyzed.

Analysis by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP).

A Cryptosporidium genus-specific nested PCR was used to amplify a 214-basepair fragment of the Cryptosporidium 18S ribosomal RNA gene encompassing the polymorphic region between nucleotides 179 and 271 as described.⁹ The first PCR amplification was performed with forward primer SCL1 (5′-CTGGTTGATC-CTGCCAGTAG-3′) and reverse primer CBP-DIAGR (5′-TAAGGTGCTGAAGGAGTAAGG-3′) complementary to nucleotides 4–23 and 1016–1036, respectively (GenBank accession no. AF093489). The second-round PCR was carried out using forward primer SCL2 (5′-CAGTTATAGTT-TACTTGATAATC-3′) and reverse primer SCR2 (5′-CAATACCCTACCGTCTAAAG-3′) complementary to nucleotides 106–128 and 299–318 respectively. The PCR amplification was performed in a 25-μL volume containing 5 μL of DNA template, 10× PCR buffer, 2.5 mM MgCl₂, 0.2 mM of each dNTP, 0.2 μM of each primer, and 1.25 units of Ampli-Taq Gold Polymerase (Roche, Indianapolis, IN). Cycling conditions were an initial denaturation at 94°C for 5 minutes, followed by 30 cycles of a three-step program (94°C for 30 seconds, 60°C for 34 seconds, and 72°C for 90 seconds). The reactions were then held at 72°C for 10 minutes (PXE 0.2 Thermal cycler; Thermo Fisher Scientific Inc., Waltham, MA).

The second-round PCR conditions were as described above using slight modifications. Briefly, 5 μL of the initial PCR product was used as DNA template and was amplified with 0.4 μM of each primer. Amplification included 30 cycles at 94°C for 30 seconds, 52°C for 45 seconds, and 72°C for 60 seconds. Amplified products were separated by electrophoresis on a 2% agarose gel and visualized after staining with ethidium bromide.

A DNA sample testing positive (kindly provided by Dr. Francis Derouin, Laboratoire de Parasitologie-Mycologie, Université Paris VII, Paris, France) was used as positive control. Oocyst-positive samples from which no specific DNA was amplified were retested at a 1:10 dilution and checked for inhibition by spiking these samples with 1 μL of the positive control.

Restriction assays.

Amplified product was initially digested with the restriction endonuclease Taq I as reported.⁹ The reaction contained 5 μL PCR product, 3 μL of the recommended 10× buffer, 2 units of enzyme, and distilled water to give a volume of 30 μL. Reactions were incubated for 2 hours at 65°C. Fragments were separated by electrophoresis on a 3% agarose gel in 1× Tris-borate-EDTA buffer, and the fragments were visualized after staining with ethidium bromide. The products that were digested by Taq I were digested with Ase I to differentiate C. hominis from C. parvum. In cases of negative results, the restriction enzymes Mse I, Bst UI, and Ssp I were used to identify other species.⁹ All restriction assays were conducted according to the manufacturer’s recommendations (New England Biolabs, Hitchin, United Kingdom).

Sequencing.

Three second-round PCR products identified as C. hominis, C. parvum, and C. meleagridis by the restriction profile were purified using the Wizad Purification Kit (Promega, Charbonnières, France). The PCR products were then sequenced using an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA). Sequences were compared with those available in the GenBank database with the BLASTN program (http://www.ncbi.nlm.nih.gov/BLAST).

Statistical analysis.

Chi-square and Fisher exact tests were used to compare percentages (Epi-Info Software, version 6.04; Centers for Disease Control and Prevention, Atlanta, GA).

Detection of Cryptosporidium spp.

Among 708 samples examined by microscopy, 19 (2.7%) were positive for Cryptosporidium spp. oocysts. Oocysts were found in 10 (6.3%) diarrheal stools and 9 (1.6%) formed stools (P < 0.01) (Table 1). Positive samples were collected from 11 immunocompetent children (7 living in rural communities and 4 in urban communities) and from 8 immunocompromised patients (6 HIV-infected patients and 2 children with cellular immunodeficiency caused by defective expression of major histocompatibility complex class II molecules). Prevalence was significantly higher in immunocompromised patients than in immunocompetent children (10.7% versus 1.7%; P < 0.01) (Table 1). In this latter pediatric population, there was no significant difference between urban and rural prevalence rates (2.5% versus 1.4%) (Table 2).

Species identification.

Nested PCR analysis of microscopy-positive samples confirmed the presence of Cryptosporidium species. The RFLP analysis yielded typical restriction patterns for C. parvum in 8 cases, C. hominis in 7 cases, and C. meleagridis in 4 cases (Figure 1). Sequences of 3 samples identified as C. parvum, C. hominis, and C. meleagridis have been stored in GenBank under the accession nos. EU675852, EU675853, and EU675854, respectively. These samples showed 100% similarity with C. parvum sequence (GenBank accession no. AF308600) and 99% similarity with C. hominis sequence (GenBank accession no. DQ388386) and C. meleagridis sequence (GenBank accession no. AF112574), respectively. Mutations were not detected around restriction enzyme sites (Figure 2).

Cryptosporidium parvum and C. hominis were responsible for 15 cases of Cryptosporidium spp. infections (78.9%). Their distribution was not different on the basis of stool consistency (five cases with C. parvum and four cases with C. hominis in formed stools versus three cases from each species in diarrheic specimens). Their distribution was not different by immune status (five and three cases of C. parvum and C. hominis, respectively, in immunocompetent children versus three and four cases, respectively, in immunocompromised persons). Cryptosporidium hominis was more prevalent in children from urban areas than in children from rural areas (1.9% versus 0%; P < 0.05), and C. parvum was found with similar prevalence rates in the two populations (0.6% versus 0.8%) (Table 2).

Cryptosporidium meleagridis was identified only in children from rural areas; three immunocompetent non-diarrheic children and a four-year-old diarrheic immunocompromised child. In rural areas, no significant difference between the prevalence rate of C. meleagridis and that of C. parvum was found (0.6% versus 0.8%) (Table 2).