Compliance with ethical standards
The study was approved by the University Hospital of the West Indies/University of the West Indies/Faculty of Medical Sciences (UHWI/UWI/FMS) Ethics committee (Ethics no. ECP 266 13/14). Human samples were assigned a unique study to ensure anonymity and were tested in an ISO 15189:2012 accredited laboratory which adhered to the established standards of the Ministry of Health, Jamaica. As such, all methods were carried out in accordance with FMS Ethics committee guidelines and regulations. Informed consent was obtained from all patients and pet owners for sample collection. No pertinent identifying information was revealed.
Sample collection and parasitological investigation
Seventy-seven (77) human fecal samples were collected from symptomatic patients presenting to the GI clinic at the (UHWI) in Kingston, Jamaica. Patients’ ages raged between 18-60 years old. All patients presented with diarrhea and abdominal pain as major symptoms.
One hundred and twenty-eight samples from asymptomatic and symptomatic dogs were collected from 20 veterinary practices across Jamaica. These were transported to the Department of Microbiology, UHWI for parasitological investigation. If analyses were delayed, samples were stored at 4ºC until investigation was performed. Formalin-ether concentration technique (FECT) was conducted on all human samples and the zinc sulfate (ZnSO4) centrifugation technique was used for the detection of hookworm eggs in dogs 27. Parasite stages were observed using microscopy following iodine staining. Fresh samples were sub-sampled (at least 1000 ml) and stored in 85% Alcohol for DNA extraction and PCR amplification.
Genomic DNA extraction and PCR
Genomic DNA was extracted using the QIAamp Fast DNA Stool Kit® (Qiagen, Hilden, Germany). Prior to DNA extraction, fresh samples were stored in 85% alcohol at -20 ºC until DNA extraction was performed. For DNA extraction, 200 ml of stool were added to 1000 ml stool lysis buffer in a PowerBead® Tube and heated for 10 min at 95ºC, then vortexed for 3 minutes. This was followed by homogenization using a mechanical shaker at 5000 rpm for 20s to disrupt Ancylostoma eggs for complete cell lysis to release DNA. The remaining protocol specified by the Stool Kit® was followed and the extracted Ancylostoma-specific DNA was stored at -20ºC until PCR was performed.
A fragment of the first and second ITS regions were amplified using the forward RTHW1F (5-GAT GAG CAT TGC WTG AAT GCC G-3) and reverse RTHW1R (5-GCA AGT RCC GTT CGA CAA ACA G-3) primers as described previously 28. These primers, designed by Traub et al. (2008), amplify a 380 bp and 480 bp fragment, respectively of the ITS1, 5.8S and ITS2 regions of Necator americanus and Ancylostoma spp. Amplification conditions were set for an initial denaturation at 95 ºC for 15 min, followed by 35 cycles of 95 ºC for 1 min, 54 ºC for 2 min, and 72 ºC for 3 min, and final extension at 72 ºC for 7 min. The PCR products were analyzed using 2% agarose gel electrophoresis stained with gel red and visualized using a UV transilluminator box.
DNA sequencing
All amplicons were purified using the PCR and Gel Band purification kit (Illustra GFX, GE Healthcare, Austria) and stored at -20ºC until DNA sequencing was performed. Sequencing of the PCR products was performed using the BigDye Terminator dye (V3.1, Applied Biosystems, Austria) strictly following the manufacturer’s instructions. Sequences were obtained from both strands and analyzed on an ABI automated sequencer (Applied Biosystems, Austria). Consensus sequences were created using GeneDoc® (version 2.7.000) and compared to reference sequences from GenBank® by multiple alignment using CLUSTAL W®.