Extensive and complex sequence diversity in mitochondrial cytochrome c oxidase subunit 1 within Necator americanus from Colombia revealed by SSCP-coupled sequencing.

In spite of the human health importance of hookworms, there has been only a small number of studies of genetic variability within Necator americanus, and none in South America. In the present study, we investigated sequence variability in a 395-bp region of the mitochondrial cytochrome c oxidase subunit 1 gene among N. americanus individuals (n=100) from humans of six villages in Colombia, employing a mutation scanning-coupled sequencing approach. Haplotypic diversity within N. americanus varied from 0.95 to 1.0 (0.9743+/-0.0068) and nucleotide diversity from 0.025 to 0.045 (0.0257+/-0.013). Nucleotide variation was reflected in nine amino acid alterations over 132 positions. The network of cox1 haplotypes (n=59) displayed a complex relationship with no apparent association between haplotype and geographical origin in Colombia. The extensive haplotypic variability detected suggests different epidemiological or disease characteristics for distinct population variants of N. americanus.

Capillary electrophoretic analysis of fragment length polymorphism in ribosomal markers of Cryptosporidium from humans.

Cryptosporidium oocyst DNA samples (n=80) from humans with cryptosporidiosis in Australia and the UK were characterized genetically and categorized by capillary electrophoretic (CE) analysis of part of the small subunit gene (pSSU; approximately 300bp) and second internal transcribed spacer (pITS-2; approximately 230bp) of nuclear ribosomal DNA. The amplicons were heat denatured and subjected to capillary electrophoresis in LPA matrix (Amersham) in a MegaBACEtrade mark 1000 system (Amersham). The chromatograms captured were stored electronically and then analysed using MegaBACEtrade mark Fragment Profiler software. Using reference DNA control samples representing Cryptosporidium hominis and Cryptosporidium parvum, particular peaks in the profiles were defined for their specific identification and differentiation. The two species could be readily differentiated based on their profile in the pSSU, the peak differences being associated with a nucleotide difference of <1.7%. While no variation was detectable in the pSSU profiles within each species, significant intraspecific variability in the positions of peaks in the pITS-2 chromatograms was displayed. For the 80 samples subjected to CE analysis of the pITS-2, four different genetic variants (genotypes) were detected within C. hominis and seven within C. parvum. Based on CE analysis of either pSSU and pITS-2 amplicons, it was readily possible to detect both species in 'mixed samples'. This CE method is time- and cost-effective, and may find applicability as a tool for the high throughput analysis of oocyst DNA samples for epidemiological surveys and for the monitoring of cryptosporidiosis outbreaks.