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Molecular Screening for Gastrointestinal Pathogens among Food Workers and Housemaids in Qatar

Haitham Ghunaim, Ayeda Ahmed, Raja’a Dalloul , and Marwan Abu-Madi Biomedical Science program, Department of Health Sciences, Qatar University. Molecular Screening for Gastrointestinal Pathogens among Food Workers and Housemaids in Qatar. 10 -1. 10 -2. 10 -3. 10 -4. 10 -5. 10 -6.

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Molecular Screening for Gastrointestinal Pathogens among Food Workers and Housemaids in Qatar

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  1. Haitham Ghunaim, Ayeda Ahmed, Raja’a Dalloul, and Marwan Abu-Madi Biomedical Science program, Department of Health Sciences, Qatar University Molecular Screening for Gastrointestinal Pathogens among Food Workers and Housemaids in Qatar 10-1 10-2 10-3 10-4 10-5 10-6 Abstract Materials and Methods Worldwide adoption of RT-PCR machines have lowered the cost of conducting such assays to where it became the standard diagnostic tool in detection of enteric pathogens. Herein, we developed a molecular method to screen the stool specimens from food workers and housemaids in Qatar for the presence of several common enteric bacteria and parasites using RT-PCR. A total of 200 samples were collected from apparently healthy subjects during a routine health check-up. The stools were screened for the presence of Salmonella spp., Shigella spp., Campylobacter jejuni, Entamoeba hystolytica, Cryptosporidium parvum, and Giardialamblia using multiplex RT-PCR. For each of Salmonella and Campylobacter only one sample was found to be positive (0.5% prevalence), while two samples were positive for Shigella,Cryptosporidium, and Entamoeba (1% prevalence), and 14 for Giardia (7% prevalence).These initial results confirm our previous finding that food workers in Qatar are shedding several important enteric pathogens and their role in spreading the infection requires further investigation. A total of 200 stool samples were collected from food workers and housemaids arriving in Qatar at the medical commission and kept frozen. DNA was extracted from the samples using QIagenQIampstool kit (Qiagen, Germany) following manufacturer’s instructions with minor modifications. DNA quantity and quality were determined using spectrophotometry and agarose gel electropheresis. Samples were examined for the presence of parasites and bacteria by real-time multiplex PCR using Applied Biosystems Cycler 7500 (Fig 1). The various primer/probe sets for detecting bacterial pathogens were designed based on sequence data available through National Center for Biotechnology Information (NCBI) databases. To determine the limit of detection, specificity, and sensitivity of the assays a set of positive controls of pure bacterial cultures (ATCC) and light microscopy-confirmed parasitic samples were used (Fig 2). Results The first step was to optimize reaction conditions. Primers/probe sets were confirmed to detect the targeted pathogen in a singleplex and multiplex reactions (Fig 1). Determining the LOD for each primer/probe set by serial dilution. DNA extracted from bacterial culture with known CFU counts was serially diluted. The LOD for Campylobacter, Salmonella, and Shigellawas 3.5×105, 4.5×105, and 3×105CFU/ml respectively. A total of 21 samples tested positive for intestinal pathogens, of which 8.5% were infected with intestinal parasites and 2% with intestinal bacteria (Table1). The percentage of intestinal pathogens distributed in the table shown. The samples were determined to be positive based on the signal intensity (Fig 1), in which the samples were tested for the presence of Cryptosporidium (C), Giardia (G), and E. histolytica (E) by RT-PCR. Table1: Prevalence of targeted pathogens in stool samples collected from food workers Background Infectious gastroenteritis remains a major public health concern worldwide as it affects millions of people across the five cotenants (1). Food workers are one of the main sources through which gastrointestinal pathogens spread. The presence of the carrier state in these workers has been demonstrated to be directly related to several outbreaks (2). The Center for Disease Control estimates that out of the 76 million foodborne cases that occur every year in the US, up to 20% are directly linked to food workers.Our previous research using microscopy showed that food workers in Qatar are commonly infected with gastrointestinal parasitessuch as protozoa and helminthes (3). Co-infection was common with some worker harboring up to 7 different parasites simultaneously (3). Detection of pathogens using molecular screening approach (MSA) has significantly better sensitivity and specificity compared to traditional diagnostic methods (4). Herein, we developed a molecular method to screen the stool specimens from food workers and housemaids in Qatar for the presence of several common enteric bacteria and parasites using RT-PCR. The targeted pathogens are commonly associated with foodborne illness. Fig.1. Detection of G. lambliausing RT-PCR 3 4 5 6 2 1 Dilution Over-night growth Tube 1-6 Tube 1-6 Tube 1-6 Inoculation DNA Extraction Conclusion Grow over-night RT-PCR The use of RT-PCR as a standard molecular screening techniques for the detection of gastrointestinal pathogens especially in food workers arriving in Qatar instead of traditional methods has proven successful. The introduction of this technique might play a significant role in limiting the transmission of gastrointestinal infections. Colony count Fig.2. Determination of the limit of detection of bacterial pathogens. A total of 200 stools samples were collected from food workers and housemaids from the Medical Commission in Qatar. The majority of the positive samples were females with 58% prevalence, while only 42% of the samples testing positive in this study came from male workers (Fig.3a). Out of 15 different nationality, intestinal pathogens were positive among 7 different nationals (Fig.3b). Objectives Acknowledgement • Design and validate RT-PCR primer/probe set suitable for multiplex reaction to detect three different bacteria or parasite simultaneously. • Determine the limit of detection of the primers/probe set for each pathogen. • Determine the specificity of each primer/probe set. • Screen archived stool samples for the presence of target pathogens. This work was funded by Qatar National Research Fund Undergraduate Research Experience program (UREP14-025-3-10). References a b (1) Fletcher, S.M., et al., Enteric protozoa in the developed world: a public health perspective. Clinical microbiology reviews, 2012. 25(3): p. 420-49. (2) Porter, C.K., et al., The Incidence and gastrointestinal infectious risk of functional gastrointestinal disorders in a healthy US adult population. Am J Gastroenterol, 2011. 106(1): p. 130-8. (3) Abu-Madi, M.A., J.M. Behnke, and A. Ismail, Patterns of infection with intestinal parasites in Qatar among food handlers and housemaids from different geographical regions of origin.Actatropica, 2008. 106(3): p. 213-20. (4) Verweij, J.J., et al., Simultaneous detection of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum in fecal samples by using multiplex real-time PCR. J ClinMicrobiol, 2004. 42(3): p. 1220-3. Fig.3. Analysis of the gender and the nationalities of the subjects. (a) Male: female ratio. (b) nationalities of the infected workers.

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