An Evaluation of Molecular Diagnostic Tools for the Detection and Differentiation of Human-Pathogenic Cryptosporidium spp

The performance of 10 commonly used genotyping tools in the detection and differentiation of 7 human-pathogenic Cryptosporidium spp. (C. hominis, C. parvum, C. meleagridis, C. felis, C. canis, C. muris and Cryptosporidium pig genotype I) was evaluated. All 3 SU rRNA genebased tools could amplify the DNA of 7 Cryptosporidium spp. efficiently. However, the tools based on the antigens TRAP-C1, TRAP-C2 and COWP genes, the housekeeping genes HSP70 and DHFR, or a genomic sequence, failed to detect the DNA of C. felis, C. canis, Cryptosporidium pig genotype I, and C. muris. With the exception of 1 tool based on the TRAP-C2 gene, the PCR-RFLP or the PCR sequencing tools evaluated in this study could differentiate C. hominis, C. parvum and C. meleagridis from each other, and 2 SSU rRNA gene-based tools could differentiate all 7 Cryptosporidium spp. Thus, a thorough understanding of the strength and weakness of each technique is needed when using molecular diagnostic tool in epidemiological investigations of human cryptosporidiosis. Numerous molecular techniques have been developed for the differentiation of Cryptosporidium genotypes or species, mostly based on PCR or DNA sequencing of genes coding for the oocyst wall protein (COWP) [6,8], a 70 kDa heat shock protein (HSP70) [4], dihydrofolate reductase (DHFR) [3], thrombospondin-related adhesive proteins (TRAP) [1,9], small-subunit rRNA [10,11,13], and a genomic sequence [5]. Most of these tools are designed to detect and differentiate C. hominis and C. parvum in clinical specimens. Because other Cryptosporidium species (C. meleagridis, C. felis, C. canis, C. muris, and Cryptosporidium pig genotype I) can also infect humans [2,7,12,13], molecular techniques used in the analysis of clinical samples should be able to detect and differentiate all humanpathogenic Cryptosporidium spp. Currently, it is not clear whether most of the Cryptosporidium genotyping techniques have the ability to detect and differentiate human-pathogenic Cryptosporidium spp. beyond C. parvum and C. hominis. In this study, we compared and evaluated 10 commonly used molecular diagnostic tools for the detection and differentiation of 7 human-pathogenic Cryptosporidium species. MATERIALS AND METHODS Cryptosporidium species used in this study. All Cryptosporidium DNA used in this study were extracted using QIAamp DNA Stool mini kit (Qiagen, Valencia, Calif.), and have been identified to species by the SSU rRNA gene-based PCR-RFLP tool of Xiao et al. [13]. Two samples each of 7 Cryptosporidium spp. (C. hominis, C. parvum, Cryptosporidium pig genotype I, C. canis, C. meleagridis, C. felis and C. muris) were used in the initial evaluation, and another 20 samples each of C. canis and C. felis and 8 samples of C. muris were used in the secondary evaluation of potential species differentiation tools. With the exception of C. muris samples, all these samples were from HIV patients in Peru. Three isolates of C. muris samples were from humans, and 7 isolates were from mice, a bactrian camel, a mountain goat, and a Dolichotis patagonum. PCR. All PCR mixtures consisted of 1 ll DNA (;50 ng), 100 nM (primary PCR) or 200 nM (secondary PCR) primers, 0.2 mM deoxynucleoside triphosphate (Perkin-Elmer, Foster City, Calif.), 3 mM MgCI2, 400 ng/ll of non-acetylated bovine serum albumin (Sigma, St. Louis, M.O.), 1 3 PCR buffer (Applied Biosystems, Branchburg, NJ), and 2.5 U Taq DNA polymerase (Promega, Madison, Wis.); total volume was 100 ll. A negative control consisting of the PCR mixture without the DNA template was included in each experiment. DNA amplification was carried out in a Perkin-Elmer 9700 GeneAmp themocycler using the cycling condition described in original publications [1,3,4,5,6,8,9,10,11,13]. When poor results were obtained with some primers, PCR was repeated without non-acetylated bovine serum albumin or using original PCR conditions. PCR products were visualized by 1.5% agarose gel electrophoresis. Restriction enzyme digestion. Restriction fragment length polymorphism (RFLP) analysis for each tool was carried out using enzymes described in the original publications [1,3,4,5,6,8,9,10,11,13] (Table 1). Briefly, 5 ll PCR product was digested with Rsa I, Hae III, BstE II,Mwo I,Mlu I, Bpm I, Alu I, Taq I, Ssp I (all from New England BioLabs, Beverly, Mass.), BpuA 1 (Roche Applied Science, Indianapolis, IN), Dra II (Roche Applied Science), or Vsp I (Promega, Madison, Wis.) under conditions recommended by the manufacturers. The digested products were fractioned by agarose gel electrophoresis and visualized by ethidium bromide staining. DNA sequencing and data analysis. PCR products of the SSU rRNA gene-based tool by Ward et al. [11] in the initial evaluation and all positive PCR products from the antigens (TRAP-C2 and COWP) and housekeeping (DHFR) gene-based tools were sequenced on an ABI 3100 autosequencer (Perkin-Elmer, Foster City, Calif.) in both directions. Distinct sequences generated in this study were deposited in the GenBank database under accession numbers AY391720, AY391721, AY391722, AY391723, AY391724, AY391725, AY391726, AY391727, and AY391728. RESULTS AND DISCUSSION Detection and differentiation of Cryptosporidium spp. by SSU rRNA gene-based tools. Three SSU rRNA gene-based PCR-RFLP or PCR sequencing tools [10,11,13] were evaluated. All 3 tools effectively amplified the DNA of all 7 species in the initial evaluation (Fig. 1, Table 2). When more samples of C. felis, C. canis and C. muris were used in the secondary evaluation, all of them were amplified by the Xiao tool [13], and 18 of 20 C. felis, all 20 C. canis, and 7 of 8 C. muris samples were amplified by the Sturbaum tool [10], whereas all 20 C. felis, 19 of 20 C. canis and 7 of 8 C. muris were amplified by the Ward tool [11] (Table 2). RFLP analyses with the 2 restriction enzymes, Vsp I and Dra II, are used for species differentiation in the Sturbaum tool [10]. The combination of RFLP analysis with these two enzymes could only differentiate C. hominis and C. muris from the others. Other species evaluated shared 1 of the 2 other banding patterns (Fig. 2). The tool by Xiao et al. [13] uses a combination of RFLP analysis with Ssp I and Vsp I to differentiate species (Fig. 3). All 7 Cryptosporidium spp. could be differentiated from each other by banding patterns using these 2 enzymes (Fig. 3). No restriction enzyme analysis is included in the Ward tool [11]. DNA sequencing of secondary PCR products resulted in unique sequences for all species examined, even though it was difficult to sequence C. felis samples due to Corresponding author: L. Xiao. Telephone: 770-488-4840; Fax: 770-4884454; Email: lxiao@cdc.gov 542 J. EUKARYOT. MICROBIOL. , 2003