TABLE 2.

Examples of multiplex pathogen detection approaches including parasitic targets^m

Assay format	Combined assay targets	Sample selection criterion	No. of samples tested	Commercial availability	Description	Reference(s)
Multiplex real–time PCRa	E. histolytica, C. parvum sensu lato,l G. lamblia, and PhHV	Microscopy–positive stool samples	112	In–house	First 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-house	Simultaneous detection, species differentiation, and genotyping after hybridization of a multiplex PCR in a microarray format	447
Multiplex real-time PCRb	G. lamblia, Cryptosporidium, and E. histolytica	Antigen- and PCR-positive stool samples	129	In-house	Primers 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 PCRc	E. histolytica, C. parvum sensu lato, G. lamblia, and PhHV	Gastrointestinal complaints	956	In-house	Higher 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 PhHV	No selection, population based	339	In-house	Good correlation found between DNA load and egg/larval counts	328
Multiplex real-time PCRd	E. histolytica, C. parvum sensu lato, G. lamblia, and PhHV; S. stercoralis and PhHV	Stool samples from travelers	2,591	In-house	PCR for the targets used outperformed expert microscopy; strikingly, even in travelers, not many additional parasites were found by microscopy	107
Multiplex real-time PCRd,e	E. histolytica, C. parvum sensu lato, G. lamblia, and PhHV; D. fragilis and PhHV	Stool samples from patients with gastrointestinal complaints	397	In-house	Increased detection rate of D. fragilis with PCR (31%) compared to that with microscopy (17%)	27
Multiplex real-time PCRb	G. lamblia, Salmonella enterica, Campylobacter jejuni, and PhHV	Stool samples from patients with gastrointestinal complaints	13,974	In-house	Implementation of Giardia PCR in a routine diagnostic laboratory	106
Multiplex real-time PCRf,g	A. duodenale, N. americanus, A. lumbricoides, S. stercoralis, and PhHV	No selection, population based	1,312	In-house	Large 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-RFLP	Microsporidia, Cyclospora, and Cryptosporidium	Controls		In-house	Conventional 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 complaints	472	AusDiagnostics, Beaconsfield, Australia	Automated system of multiplex preamplification followed by target-specific real-time PCR	451
Multiplex PCR using Luminex beadsb,f,g,h,i	E. histolytica, Cryptosporidium, G. lamblia, A. duodenale, N. americanus, A. lumbricoides, and S. stercoralis	Microscopy- and/or PCR-positive stool samples	319	In-house	Luminex 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 samples	234	In-house	Luminex detection of multiplex PCR products using primers and probes that were described previously as real-time TaqMan-based assays	193
Multiplex real-time PCRf,g,h	A. duodenale, N. americanus, A. lumbricoides, S. stercoralis, and PhHV	Stool samples from patients with gastrointestinal complaints	78	In-house	Application 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 PCRc,e,j	E. histolytica, E. dispar, C. parvum sensu lato, and G. lamblia; E. bieneusi and Encephalitozoon spp.; D. fragilis; Blastocystis	HIV positive	96	In-house	Although 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 samples	267	AusDiagnostics	Extension of the no. of targetsn	452
Multiplex real-time PCRc,f,g,h	E. histolytica, Cryptosporidium, and G. lamblia; A. duodenale, N. americanus, A. lumbricoides, and S. stercoralis	Stool samples from patients with gastrointestinal complaints	229	In-house	Application of multiplex real-time PCR in Malaysia for STHs and diarrhea-causing protozoa	161
Multiplex PCR	E. histolytica, astrovirus, calicivirus, and EIEC	Stool samples from patients with gastrointestinal complaints	103	In-house	Conventional PCR is not very practical in a routine setting with large numbers of samples	453
Multiplex real-time PCRc,e	E. histolytica, C. parvum sensu lato, and G. lamblia; E. dispar; D. fragilis	Stool samples from patients with gastrointestinal complaints	889	In-house	PCR 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 sapovirus	Stool samples from patients with gastrointestinal complaints	86	In-house	A 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 PCRd	E. histolytica, C. parvum sensu lato, G. lamblia, and PhHV	Stool samples from patients with gastrointestinal complaints	396	In-house	Microscopy 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 PhHV	Microscopy-, culture-, and/or immunoassay-positive stool samples	109	In-house	A 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 panelf,i	E. histolytica, Cryptosporidium, G. lamblia, A. duodenale, N. americanus, A. lumbricoides, S. stercoralis, and T. trichiura	No selection, population based	525	In-house	Parallel 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 rotavirus	Stool samples from patients with gastrointestinal complaints	1,758	CE 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 A	Stool samples from patients with gastrointestinal complaints	901	FDA 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 A	Stool samples from diarrheic patients	440	FDA 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 A	Stool samples from patients with gastrointestinal complaints	393	FDA 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 PCRf,g,j,k	A. duodenale, N. americanus, A. lumbricoides, S. stercoralis, and PhHV; Schistosoma and PhHV	Stool samples from HIV-positive patients	153	In-house	Improved 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

^aPCR primers and probe for Cryptosporidium are described in reference 158.

^bPCR primers and probe for Giardia are described in reference 79.

^cPCR primers and probes for E. histolytica, Cryptosporidium, and Giardia are described in reference 79.

^dPCR primers and probes for E. histolytica and Giardia are described in reference 79.

^ePCR primers and probe for D. fragilis are described in reference 128.

^fPCR primers and probes for N. americanus and A. duodenale are described in reference 328.

^gPCR primers and probe for S. stercoralis are described in reference 354.

^hPCR primers and probe for A. lumbricoides are described in reference 319.

ⁱPCR primers and probe for E. histolytica are described in reference 448.

^jPCR primers and probes for E. bieneusi and Encephalitozoon spp. are described in reference 457.

^kPCR primers and probe for Schistosoma are described in reference 458.

^lNomenclature 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.

^mAbbreviations: 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.

ⁿAs described in reference 451.