Detection of Giardia spp. with formalin/ether concentration in Brassica oleracea (cabbage) and Lactuca sativa (lettuce)

Abstract

Methods to detect protozoa are needed for food safety monitoring. We evaluated protocols to recover Giardia spp. cysts in Brassica oleracea (cabbage) and Lactuca sativa (lettuce) and then detection was performed by concentrating with formalin/ether solutions and microscopy or immunofluorescence or DNA amplification via PCR. To evaluate this methodology, G. duodenalis cysts were inoculated in triplicate (10 cysts) in 35-g samples of lettuce and cabbage. The method obtaining the highest percentage of recovery in cabbage was sulfamic acid solution plus stirring with stomacher (47.7% ± 7.5). For lettuce, the best method was glycine solution plus stirring with stomacher (46.6% ± 5.3). Inter-observer agreement was of 0.99. Giardia was detected by amplifying specific sequences for the DNA coding SSU rRNA. In 27 lettuce samples and 27 cabbage samples, obtained from supermarkets and street vendors, two lettuce samples (7.4%) and one cabbage sample (3.7%) were positive for Giardia via PCR assay and were sequenced, determining that they were two of assemblage B and one of lettuce to assemblage E. This method is proposed to detect Giardia in vegetables by PCR detection, enabling public health authorities to identify genotypes circulating in food, which will help to establish measures that reduce outbreaks of parasitic diseases associated with contaminated food.

Frecuencia de parasitosis intestinal en escuelas primarias en Veracruz, México

“…Hay estudios que han identificado ensambles patógenos de Giardia spp. en verduras, como el repollo y la lechuga; 28 pero es posible que la falta de tipificación de este parásito sea porque no se utilizó un método específico para su identificación.…”

Food protozoa safety assessment and risk in school restaurants in Armenia, Colombia

“…For food, live, and surface samples, the DNeasy Blood & Tissue Extraction Kit (Qiagen, Germany) with mechanical lysis and zirconium beads were applied five times for DNA extraction after agitation in a stomacher 400‐W BagMixer (Interscience, France) at 260 rpm for 30 s with glycine 1 M (Biorad, Estados Unidos) and pH of 5.5 and a wash solution (PBS 1X, 100 μl of Tween 80, Merck, Germany, and 10 g of sulfamic acid—Merck, Germany) at pH of 7.5, respectively (Cook et al, ), and a formalin–ether concentration method for water eluates was applied (also for inert and live surface samples), as described (Hernández‐Arango, Pinto, Muñoz‐Sanchez, Lora‐Suarez, & Gómez‐Marín, ; Lora‐Suarez, Rivera, Triviño‐Valencia, & Gomez‐Marin, ; Triviño‐Valencia, Lora, Zuluaga, & Gomez‐Marin, ). For stools, 1 g of sample was diluted in 5 ml of sterile saline solution at 0.9% and filtered through gauze.…”