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CC BY-NC-ND 4.0 · J Lab Physicians 2019; 11(03): 275-280
DOI: 10.4103/JLP.JLP_66_18
Original Article

Multiplex PCR assay for simultaneous detection and differentiation of Entamoeba histolytica, Giardia lamblia, and Salmonella spp. in the municipality-supplied drinking water

Prem Shankar
Department of Laboratory Medicine, Division of Clinical Microbiology and Molecular Medicine, All India Institute of Medical Sciences, New Delhi, India
,
Jyotsna Mishra
Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA
,
Vijaya Bharti
Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, USA
,
Deepak Parashar
Department of Obstetrics and Gynecology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA
,
Sarman Singh
Department of Laboratory Medicine, Division of Clinical Microbiology and Molecular Medicine, All India Institute of Medical Sciences, New Delhi, India
› Author Affiliations Financial support and sponsorship Nil
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Abstract

BACKGROUND: The contamination with Entamoeba histolytica, Giardia lamblia, and Salmonella spp. in drinking water is the most prevalent in Indian subcontinent, but often difficult to detect all these pathogens from the drinking water.

MATERIALS AND METHODS: A multiplex polymerase chain reaction (mPCR) method was developed to detect contamination of municipality-supplied drinking water with E. histolytica, G. lamblia, and Salmonella spp. The primers were designed to target small subunit of 16S rRNA type gene of E. histolytica and G. lamblia, and invasive A gene of Salmonella typhimurium. The optimized mPCR assay was applied on 158 municipality-supplied drinking water samples collected from Delhi.

RESULTS: Out of total 158 water samples, 89 (56.32%) were found positive for the targeted pathogens by mPCR while conventional methods could be detected only in 11 (6.96%) samples. The mPCR assay showed 100% sensitivity and specificity for these pathogens in comparison with culture and microscopic detection. Of the 89 mPCR-positive samples, G. lamblia, E. histolytica, and Salmonella spp. were present in 35 (22.15%), 26 (16.45%), and 28 (17.72%), respectively. Nine (5.69%) samples were positive for both E. histolytica and G. lamblia, 10 (6.32%) were positive for G. lamblia and Salmonella spp., and 8 (5.06%) had Salmonella spp. and E. histolytica. Nonetheless, 3 (1.89%) samples were positive for all three pathogens.

CONCLUSIONS: The present assay is an alternative to conventional methods to serve as highly sensitive, specific, and economical means for water quality surveillance to detect the outbreak caused by E. histolytica, G. lamblia, and Salmonella spp. pathogens.

Key words

Drinking water - Entamoeba histolytica - Giardia lamblia - multiplex polymerase chain reaction - Salmonella species


Publication History

Received: 03 May 2018

Accepted: 24 August 2019

Article published online:
07 April 2020

© 2019.

Thieme Medical and Scientific Publishers Private Ltd.
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  • References

  • 1 Clasen T, Roberts I, Rabie T, Schmidt W, Cairncross S. Interventions to improve water quality for preventing diarrhoea. Cochrane Database Syst Rev 2006;(3):CD004794.
  • 2 Guerrant RL, Hughes JM, Lima NL, Crane J. Diarrhea in developed and developing countries: Magnitude, special settings, and etiologies. Rev Infect Dis 1990;12 Suppl 1:S41-50.
  • 3 Thompson RC. Giardiasis as a re-emerging infectious disease and its zoonotic potential. Int J Parasitol 2000;30:1259-67.
  • 4 Amin OM. Seasonal prevalence of intestinal parasites in the United States during 2000. Am J Trop Med Hyg 2002;66:799-803.
  • 5 Singh S. Biological contamination of drinking water in New Delhi. Isolation of free living amoebas as a highly sensitive index of water contamination. Water Int2000;25:403-9.
  • 6 Rose JB, Slifko TR. Giardia, Cryptosporidium, and Cyclospora and their impact on foods: A review. J Food Prot 1999;62:1059-70.
  • 7 Gouws PA, Visser M, Brözel VS. A polymerase chain reaction procedure for the detection of Salmonella spp. Within 24 hours. J Food Prot 1998;61:1039-42.
  • 8 Keister DB. Axenic culture of Giardia lamblia in TYI-S-33 medium supplemented with bile. Trans R Soc Trop Med Hyg 1983;77:487-8.
  • 9 Roach PD, Olson ME, Whitley G, Wallis PM. water-borne Giardia cysts and Cryptosporidium oocysts in the Yukon, Canada. Appl Environ Microbiol 1993;59:67-73.
  • 10 Mahbubani MH, Schaefer FW 3rd, Jones DD, Bej AK. Detection of Giardia in environmental waters by immuno-PCR amplification methods. Curr Microbiol 1998;36:107-13.
  • 11 Ljungström I, Castor B. Immune response to Giardia lamblia in a water-borne outbreak of Giardiasis in Sweden. J Med Microbiol 1992;36:347-52.
  • 12 Thompson RC, Hopkins RM, Homan WL. Nomenclature and genetic groupings of Giardia infecting mammals. Parasitol Today 2000;16:210-3.
  • 13 Wallis PM, Matson D, Jones M, Jamieson J. Application of monitoring data for Giardia and Cryptosporidium to boil water advisories. Risk Anal 2001;21:1077-85.
  • 14 Petri WA Jr., Singh U. Diagnosis and management of amebiasis. Clin Infect Dis 1999;29:1117-25.
  • 15 Lee CY, Panicker G, Bej AK. Detection of pathogenic bacteria in shellfish using multiplex PCR followed by CovaLink NH microwell plate sandwich hybridization. J Microbiol Methods 2003;53:199-209.
  • 16 Everest P, Wain J, Roberts M, Rook G, Dougan G. The molecular mechanisms of severe typhoid fever. Trends Microbiol 2001;9:316-20.
  • 17 Bhunia AK. Food-borne Microbial Pathogens: Mechanisms and Pathogenesis. New York: Springer: Science; 2008.
  • 18 Mason WJ, Blevins JS, Beenken K, Wibowo N, Ojha N, Smeltzer MS. Multiplex PCR protocol for the diagnosis of staphylococcal infection. J Clin Microbiol 2001;39:3332-8.
  • 19 Wang G, Clark CG, Rodgers FG. Detection in Escherichia coli of the genes encoding the major virulence factors, the genes defining the O157:H7 serotype, and components of the type 2 Shiga toxin family by multiplex PCR. J Clin Microbiol 2002;40:3613-9.
  • 20 Fritsch EF, Maniatis T, Sambrook J. Molecular Cloning: A Laboratory Manual. New York: CSHL Press; 1989.
  • 21 Dusserre E, Ginevra C, Hallier-Soulier S, Vandenesch F, Festoc G, Etienne J, et al. A PCR-based method for monitoring Legionella pneumophila in water samples detects viable but noncultivable legionellae that can recover their cultivability. Appl Environ Microbiol 2008;74:4817-24.
  • 22 Morio F, Corvec S, Caroff N, Le Gallou F, Drugeon H, Reynaud A. Real-time PCR assay for the detection and quantification of Legionella pneumophila in environmental water samples: Utility for daily practice. Int J Hyg Environ Health 2008;211:403-11.
  • 23 Verheyen J, Timmen-Wego M, Laudien R, Boussaad I, Sen S, Koc A, et al. Detection of adenoviruses and rotaviruses in drinking water sources used in rural areas of Benin, West Africa. Appl Environ Microbiol 2009;75:2798-801.
  • 24 Abd E-Haleem D, Kheiralla ZH, Zaki S, Rushdy AA, Abd-El-Rahiem W. Multiplex-PCR and PCR-RFLP assays to monitor water quality against pathogenic bacteria. J Environ Monit 2003; 5:865-70.
  • 25 Rai AK, Chakravorty R, Paul J. Detection of Giardia entamoeba and Cryptosporidium in unprocessed food items from Northern India. World J Microbiol Biotechnol 2008;24:2879-87.
  • 26 Webster KA, Smith HV, Giles M, Dawson L, Robertson LJ. Detection of Cryptosporidium parvum oocysts in faeces: Comparison of conventional coproscopical methods and the polymerase chain reaction. Vet Parasitol 1996;61:5-13.
  • 27 Verweij JJ, Schinkel J, Laeijendecker D, van Rooyen MA, van Lieshout L, Polderman AM. Real-time PCR for the detection of Giardia lamblia. Mol Cell Probes 2003;17:223-5.