TABLE 2

Examples of multiplex pathogen detection approaches including parasitic targets m

Assay formatCombined assay targetsSample selection criterionNo. of samples testedCommercial availabilityDescriptionReference(s)
Multiplex real–time PCR a E. histolytica, C. parvum sensu lato, l G. lamblia, and PhHVMicroscopy–positive stool samples112In–houseFirst multiplex real–time PCR described for detection of diarrhea–causing protozoa; has been adapted in many diagnostic laboratories since then 79
Multiplex PCR array E. histolytica, E. dispar, Cryptosporidium, and G. lamblia Control DNA only In-houseSimultaneous detection, species differentiation, and genotyping after hybridization of a multiplex PCR in a microarray format 447
Multiplex real-time PCR b G. lamblia, Cryptosporidium, and E. histolytica Antigen- and PCR-positive stool samples129In-housePrimers designed from the COWP gene should be able to detect C. parvum, C. hominis, and C. meleagridis but not other species of Cryptosporidium 448
Multiplex real-time PCR c E. histolytica, C. parvum sensu lato, G. lamblia, and PhHVGastrointestinal complaints956In-houseHigher detection rates found for Giardia and Cryptosporidium with PCR, and no additional parasites found with microscopy in a general practitioner patient population 104
Multiplex real-time PCR N. americanus, A. duodenale, O. bifurcum, and PhHVNo selection, population based339In-houseGood correlation found between DNA load and egg/larval counts 328
Multiplex real-time PCR d E. histolytica, C. parvum sensu lato, G. lamblia, and PhHV; S. stercoralis and PhHVStool samples from travelers2,591In-housePCR for the targets used outperformed expert microscopy; strikingly, even in travelers, not many additional parasites were found by microscopy 107
Multiplex real-time PCR d , e E. histolytica, C. parvum sensu lato, G. lamblia, and PhHV; D. fragilis and PhHVStool samples from patients with gastrointestinal complaints397In-houseIncreased detection rate of D. fragilis with PCR (31%) compared to that with microscopy (17%) 27
Multiplex real-time PCR b G. lamblia, Salmonella enterica, Campylobacter jejuni, and PhHVStool samples from patients with gastrointestinal complaints13,974In-houseImplementation of Giardia PCR in a routine diagnostic laboratory 106
Multiplex real-time PCR f , g A. duodenale, N. americanus, A. lumbricoides, S. stercoralis, and PhHVNo selection, population based1,312In-houseLarge albendazole placebo-controlled trial on the effect of STH infections on allergy, atherosclerosis, and malaria; effect of treatment was monitored by quantitative real-time PCR 319, 321, 333, 449
Multiplex nested PCR-RFLPMicrosporidia, Cyclospora, and Cryptosporidium Controls In-houseConventional and nested PCR are not very practical in a routine setting 450
Multiplex tandem real-time PCR E. histolytica, Cryptosporidium, G. lamblia, and D. fragilis Stool samples from patients with gastrointestinal complaints472AusDiagnostics, Beaconsfield, AustraliaAutomated system of multiplex preamplification followed by target-specific real-time PCR 451
Multiplex PCR using Luminex beads b , f , g , h , i E. histolytica, Cryptosporidium, G. lamblia, A. duodenale, N. americanus, A. lumbricoides, and S. stercoralis Microscopy- and/or PCR-positive stool samples319In-houseLuminex detection of multiplex PCR products using primers and probes that were described previously as real-time TaqMan-based assays 322
Multiplex PCR using Luminex beads C. cayetanensis, C. belli, E. bieneusi, and E. intestinalis Microscopy- and/or PCR-positive stool samples234In-houseLuminex detection of multiplex PCR products using primers and probes that were described previously as real-time TaqMan-based assays 193
Multiplex real-time PCR f , g , h A. duodenale, N. americanus, A. lumbricoides, S. stercoralis, and PhHVStool samples from patients with gastrointestinal complaints78In-houseApplication of multiplex real-time PCR in Malaysia for STH infection, finding higher detection rates with PCR, especially for A. lumbricoides and S. stercoralis 320
Multiplex real-time PCR c , e , j E. histolytica, E. dispar, C. parvum sensu lato, and G. lamblia; E. bieneusi and Encephalitozoon spp.; D. fragilis; Blastocystis HIV positive96In-houseAlthough a high prevalence of intestinal parasites was detected, there was no association between any of the parasites and the presence of diarrhea 160
Multiplex tandem real-time PCR Campylobacter, Clostridium difficile, Salmonella, Cryptosporidium, G. lamblia, Shigella, adenovirus 40/41, and norovirus Cryptosporidium-positive samples and negative-control samples267AusDiagnosticsExtension of the no. of targets n 452
Multiplex real-time PCR c , f , g , h E. histolytica, Cryptosporidium, and G. lamblia; A. duodenale, N. americanus, A. lumbricoides, and S. stercoralis Stool samples from patients with gastrointestinal complaints229In-houseApplication of multiplex real-time PCR in Malaysia for STHs and diarrhea-causing protozoa 161
Multiplex PCR E. histolytica, astrovirus, calicivirus, and EIECStool samples from patients with gastrointestinal complaints103In-houseConventional PCR is not very practical in a routine setting with large numbers of samples 453
Multiplex real-time PCR c , e E. histolytica, C. parvum sensu lato, and G. lamblia; E. dispar; D. fragilis Stool samples from patients with gastrointestinal complaints889In-housePCR showed higher detection rates of the parasites targeted; no additional pathogens were found by FECT-microscopy 162
TaqMan open array Cryptosporidium, G. lamblia, Campylobacter spp., Clostridium difficile, Salmonella, Vibrio parahaemolyticus, diarrheagenic Escherichia coli (EHEC), Shigella, Yersinia enterocolitica, Listeria monocytogenes, adenovirus, astrovirus, norovirus GI, norovirus GII, rotavirus, and sapovirusStool samples from patients with gastrointestinal complaints86In-houseA DNA sample with PCR mix is distributed by hydrophilic forces into 64 through-holes, in which primers and a probe are spotted by the manufacturer; in this way, up to 64 PCRs per sample can be performed for 48 samples simultaneously 87, 454
Multiplex real-time PCR d E. histolytica, C. parvum sensu lato, G. lamblia, and PhHVStool samples from patients with gastrointestinal complaints396In-houseMicroscopy showed low sensitivity and specificity compared to real-time PCR, especially for detection of E. histolytica 87
TaqMan array card Cryptosporidium, G. lamblia, Ascaris lumbricoides, Trichuris trichiura Campylobacter jejuni-C. coli, Clostridium difficile, Salmonella, Vibrio cholerae, diarrheagenic Escherichia coli (EAEC, ETEC, EPEC, and STEC), Shigella/EIEC, adenovirus, astrovirus, norovirus GII, rotavirus, sapovirus, and PhHVMicroscopy-, culture-, and/or immunoassay-positive stool samples109In-houseA DNA sample with PCR mix is distributed by centrifugation into 42 wells of a microfluidic card in which primers and probe are spotted by the manufacturer 309
TaqMan panel f , i E. histolytica, Cryptosporidium, G. lamblia, A. duodenale, N. americanus, A. lumbricoides, S. stercoralis, and T. trichiura No selection, population based525In-houseParallel testing of 8 singleplex PCR mixtures that were prealiquoted and stored frozen until use; good correlation between egg counts and DNA load; all Ascaris PCR-positive cases tested negative 3 wk after treatment 18
Multiplex real-time PCR E. histolytica, Cryptosporidium, and G. lamblia; Campylobacter coli-C. jejuni, E. coli (VTEC), Salmonella spp., Shigella spp., E. coli (EIEC), Y. enterocolitica, and C. difficile; norovirus GI, norovirus GII, and IC; adenovirus, astrovirus, and rotavirusStool samples from patients with gastrointestinal complaints1,758CE labeled (Fast-Track Diagnostics, Luxembourg)Enhanced rate of detection of diarrhea-causing pathogens using a broad approach due to more sensitive detection of pathogens and detection of pathogens that were not requested 413
Multiplex PCR using Luminex beads E. histolytica, Cryptosporidium, G. lamblia, Salmonella spp., Shigella spp., Campylobacter spp., E. coli O157, ETEC, STEC, C. difficile, Y. enterocolitica, V. cholerae, norovirus GI, norovirus GII, adenovirus 40/41, and rotavirus AStool samples from patients with gastrointestinal complaints901FDA cleared, CE labeled (Luminex)Multicenter study comparing standard routine procedures including culture, antigen tests, and real-time PCR with Luminex detection of multiplex PCR products of 15 viral, bacterial, and parasitic pathogens (Luminex xTAG GPP) 423
Multiplex PCR using Luminex beads E. histolytica, Cryptosporidium, G. lamblia, Salmonella spp., Shigella spp., Campylobacter spp., E. coli O157, ETEC, STEC, C. difficile, Y. enterocolitica, V. cholerae, norovirus GI, norovirus GII, adenovirus 40/41, and rotavirus AStool samples from diarrheic patients440FDA cleared, CE labeled (Luminex)Luminex xTAG GPP was compared with standard routine procedures which did not include real-time PCR; no further analysis of discrepant results 422
Multiplex PCR using Luminex beads E. histolytica, Cryptosporidium, G. lamblia, Salmonella spp., Shigella spp., Campylobacter spp., E. coli O157, ETEC, STEC, C. difficile, Y. enterocolitica, V. cholerae, norovirus GI, norovirus GII, adenovirus 40/41, rotavirus AStool samples from patients with gastrointestinal complaints393FDA cleared, CE labeled (Luminex)Luminex xTAG GPP was compared to standard routine procedures including culture, antigen tests, and real-time PCR; 5 of 6 E. histolytica-positive results by xTAG GPP could not be confirmed 455
Multiplex real-time PCR f , g , j , k A. duodenale, N. americanus, A. lumbricoides, S. stercoralis, and PhHV; Schistosoma and PhHVStool samples from HIV-positive patients153In-houseImproved detection of helminth infections using real-time PCR; discrepancies in risk factor analysis between qualitative data from PCR and microscopy; this was resolved by using quantitative data from PCR 456
  • a PCR primers and probe for Cryptosporidium are described in reference 158.

  • b PCR primers and probe for Giardia are described in reference 79.

  • c PCR primers and probes for E. histolytica, Cryptosporidium, and Giardia are described in reference 79.

  • d PCR primers and probes for E. histolytica and Giardia are described in reference 79.

  • e PCR primers and probe for D. fragilis are described in reference 128.

  • f PCR primers and probes for N. americanus and A. duodenale are described in reference 328.

  • g PCR primers and probe for S. stercoralis are described in reference 354.

  • h PCR primers and probe for A. lumbricoides are described in reference 319.

  • i PCR primers and probe for E. histolytica are described in reference 448.

  • j PCR primers and probes for E. bieneusi and Encephalitozoon spp. are described in reference 457.

  • k PCR primers and probe for Schistosoma are described in reference 458.

  • l Nomenclature of the sequence from GenBank used for the design of primers and probes was for C. parvum, which was later separated into C. hominis and C. parvum.

  • m Abbreviations: RFLP, restriction fragment length polymorphism; SSU, small subunit; ITS, internal transcribed spacer; STH, soil-transmitted helminth; GPP, gastrointestinal pathogen panel; COWP, Cryptosporidium oocyst wall protein; FECT, formol ethyl-acetate concentration technique; EIEC, enteroinvasive Escherichia coli; EHEC, enterohemorrhagic E. coli; EAEC, enteroaggregative E. coli; ETEC, enterotoxigenic E. coli; EPEC enteropathogenic E. coli; STEC, Shiga-toxigenic E. coli; VTEC, verotoxin-producing Escherichia coli; IC, internal control; CE, European Community; PhHV, phocine herpesvirus.

  • n As described in reference 451.