Diagnostic Microbiology and Infectious Disease 79 (2014) 149–154
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Evaluation of a multiplex PCR assay for simultaneous detection of bacterial and viral enteropathogens in stool samples of paediatric patients☆ Manuela Onori a,⁎, Luana Coltella a, Livia Mancinelli a, Marta Argentieri b, Donato Menichella c, Alberto Villani d, Annalisa Grandin d, Diletta Valentini d, Massimiliano Raponi c, Cristina Russo a a
Department of Laboratory Medicine, Virology Unit, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy Department of Laboratory Medicine, Bacteriology Unit, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy c Medical Direction, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy d Paediatric and Infectious Disease Unit, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy b
a r t i c l e
i n f o
Article history: Received 9 October 2013 Received in revised form 4 February 2014 Accepted 5 February 2014 Available online 22 February 2014 Keywords: Multiplex PCR Enteric pathogens Paediatric patients
a b s t r a c t We evaluated a multiplex PCR assay, the Seeplex Diarrhoea ACE detection, that simultaneously detects 15 enteric pathogens, including Salmonella spp., Shigella spp., Vibrio spp., toxin B producer Clostridium difficile, Campylobacter spp., Clostridium perfringens, Yersinia enterocolitica, Aeromonas spp., Escherichia coli O157:H7, verocytotoxin-producing Escherichia coli, adenovirus, Group A rotavirus, norovirus GI and GII, and astrovirus. We compared this assay with clinical methods routinely used in our laboratory, for detecting enteropathogens in stool samples collected from 245 paediatric patients with suspected infectious gastroenteritis. We recovered 61 bacterial pathogens and 121 enteric viruses with our laboratory assays, while we detected 78 bacteria and 167 viruses with the molecular assay. We calculated specificity and sensitivity for both methods after analysis of discordant results and demonstrated greater sensitivity for multiplex PCR than for our routine methods, with the exception of Salmonella spp. and toxigenic C. difficile detection. The multiplex PCR assay proved to be a reliable tool to directly detect the most common enteropathogens in stool samples but with some limitations. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Acute gastroenteritis is one of the most common causes of death amongst infants and children in developing countries (Thapar and Sanderson, 2004). In Europe, most cases are mild to moderately severe, and fatal outcomes are rare. About 40% of acute diarrhoeal episodes in the first 5 years of life are caused by rotaviruses, while 30% are caused by other viruses, primarily noroviruses and adenoviruses (Van Damme et al., 2007). A bacterial pathogen can be identified in stool in about 20% of children with gastroenteric infections, while parasites are detected in less than 5% of cases (Koletzko and Osterrieder, 2009). Currently, a national surveillance system that is able to determine the incidence of different etiologic agent of gastroenteritis is not in place in Italy. However, the surveillance of Acute Gastroenteritis is included in the official surveillance program of infectious disease (Italian National Surveillance System of Infectious Diseases) and in the laboratory-based surveillance network for enteric pathogens (as Enter-net Italia and RotaNet-Italia). Rotavirus represents the principal etiologic agent in ☆ Transparency declaration: All authors report that they have no potential conflicts of interest to declare. ⁎ Corresponding author. Tel.: +39-06-68592206; fax: +39-06-68592218. E-mail address: [email protected] (M. Onori). http://dx.doi.org/10.1016/j.diagmicrobio.2014.02.004 0732-8893/© 2014 Elsevier Inc. All rights reserved.
hospitalized children affected by acute diarrhea with an age b5 years (Palumbo et al., 2010). Norovirus is the major responsible virus of outbreak (Colomba et al., 2006; Di Bartolo et al., 2011), and with regard to sporadic enteritis, the few studies performed have reported prevalence rates from 2.1% to 18.6% (Medici et al., 2006). For bacteria, Salmonella is the most frequently isolated bacteria in enteric infections, while verotoxin-producing Escherichia coli (VTEC) is only sporadically reported (Dionisi et al., 2011). The annual rate of acute gastrointestinal illness observed in Italy falls within the range of incidence reported in other industrialized countries (Scavia et al., 2012); however, estimates of the incidence of enteric infections normally suffer from large underreporting (Rimoldi et al., 2011). The clinical presentation of patients with acute gastroenteritis symptoms is generally not indicative of a specific pathogen, and laboratory diagnosis is often difficult due to the long list of potential enteropathogens. Moreover, conventional diagnostic procedures for bacteria detection require time, expert microbiologists, and vital bacteria because based on culture procedures, the immunochromatographic detection of enteric viral antigens has limited sensitivity. Molecular techniques, such as PCR, are widely used for diagnosing infectious disease. However, singleplex PCR is unsuitable for this type of investigation, due to the wide variety of pathogens that can
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potentially be associated with diarrhoea. Multiplex PCR systems, which allow the simultaneous amplification of several targets, are promising diagnostic tools for detection of enteropathogens (Platts-Mills et al., 2012). Several papers describe molecular approaches for the simultaneous detection of multiple gastrointestinal bacteria (Cunningham et al., 2010; de Boer et al., 2010; Liu et al., 2012) and/or viruses (Khamrin et al., 2011; Liu et al., 2011; Wolffs et al., 2011). In this study, we compared the performance of a multiplex PCR assay (Seeplex® Diarrhea ACE Detection; Seegene, Seoul, Korea), which simultaneously detects 10 bacterial species and 5 viruses, with our routine clinical methods for investigating the aetiology of gastrointestinal infections in children with diarrhoea. 2. Materials and methods 2.1. Specimens A total of 245 liquid stools samples were collected from 245 paediatric patients, aged between 1 month and 16 years, admitted for presumptive infectious diarrhoea to the Paediatric and Infectious Disease Unit of Bambino Gesù Children's Hospital in Rome, Italy, between April 2010 and August 2011. Stool samples were immediately processed for routine detection of bacteria and viruses and for multiplex PCR. None of the children enrolled in this study were vaccinated for rotavirus. Informed consent was obtained from patients enrolled in the study. This study was conducted with the approval of the Ethics Committee of Bambino Gesù Children's Hospital. 2.2. Routine assays for detection of bacteria Stool samples were spread on selective media shown in Table 1. Species identification was carried out using the systems listed in Table 1. Evaluation of Clostridium difficile toxin B was performed by real-time PCR (GeneXpert C. difficile; Cepheid, Sunnyvale, CA, USA), employing a laboratory-validated procedure: a 10-μL loop of isolated colonies grown on selective media was suspended directly in 2 mL of sample reagent. 2.3. Routine assays for detection of viruses Immunochromatographic assays were used for direct detection of viral antigens in faecal samples. Antigens of group A rotavirus, adenovirus serotypes 40 and 41, and norovirus genotypes I and II were detected, respectively, using Rota-Strip C1001 (Coris BioConcept, Gembloux, Belgium), Adeno-Strip C1002 (Coris BioConcept) and RIDA®QUICK Norovirus (R-Biopharm AG, Darmstadt, Germany). In our routine testing, no diagnostic tool was available for recovery of astrovirus.
2.4. Multiplex PCR assay Stool samples were pre-treated by dissolving 1 mL of faeces in 5 mL of phosphate-buffered saline, followed by centrifugation at 4000 × g for 30 min. The supernatant (500 μL) was used for extraction of DNA/RNA using the NucliSENS® easyMAG® System (bioMérieux, Marcy l'Etoile, France). Extracted nucleic acids were eluted in a final volume of 100 μL. Reverse transcription was performed using the GeneAmp® RNA PCR kit (Applied Biosystems, Carlsbad, CA, USA), according to the manufacturer's instructions. Thermal cycling parameters were: 25 °C for 10 min, 42 °C for 60 min, and 95 °C for 5 min. The Seeplex® Diarrhea ACE Detection assay is composed of 3 separate primer mixtures (B1, B2, and V) in 3 reaction tubes and is able to identify Salmonella spp. (Salmonella bongori and Salmonella enterica), Shigella spp. (Shigella flexneri, Shigella boydii, Shigella sonnei, and Shigella dysenteriae), Vibrio spp. (Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus), C. difficile toxin B, Campylobacter spp. (Campylobacter jejuni and Campylobacter coli), Clostridium perfringens toxin, Yersinia enterocolitica, Aeromonas spp. (Aeromonas salmonicida, Aeromonas sobria, Aeromonas bivalvum, and Aeromonas hydrophila), E. coli O157:H7, VTEC, group A rotavirus, adenovirus serotypes 40 and 41, astrovirus, and norovirus GI and GII. PCR inhibitory effects were assessed by co-amplification of an internal control included in the primer mixtures. Multiplex PCR was performed according to the manufacturer's instructions. The detection limit declared by the manufacturer is 100 copies/reaction. PCR products were detected by the ScreenTape system (Lab901, Loanhead, UK), according to the manufacturer's instructions. The numerical value ascribed by the ScreenTape system to a positive signal in the detection step represents the intensity value (i.v.) relative to the marker. Manufacturer's instructions do not indicate a range or a breakpoint value to assign a positive result.
2.5. Confirmatory tests All extracts for which discordant results were obtained by multiplex PCR and our routine methods were retested using a home-brew singleplex PCR (Table 2). The primers employed for the home-brew PCR were selected according to the literature, as the multiplex assay does not specify the target region for each pathogen. Primers and probes were synthesized by Sigma Aldrich (Saint Louis, MO, USA) and Applied Biosystems, respectively. Reaction mixtures for Salmonella spp., Shigella spp., Yersinia enterocolitica, Aeromonas spp., VTEC, C. difficile, and C. perfringens (final volume 25 μL) contained: 10.75 μL of sterile water, 2.5 μL of 10× buffer solution, 1.5 μL MgCl2, 1.25 μL of DMSO, 2 μL of dNTP mix (10 mmol/L), 2 μL each of forward and reverse primers (10 μmol/L), 0.5 μL of Taq DNA polymerase, and 2.5 μL of template DNA. Reaction
Table 1 Media, growth conditions, and identification systems used for the detection of bacterial pathogens from stool samples by routine methods. Pathogen
Media
Growth conditions
Identification systems
Time of detection
Salmonella spp.
Hektoen enteric agar (bioMérieux) CHROMagar Salmonella (BD, Franklin Lakes, NJ, USA) after 24 h of enrichment in selenite broth (Copan Italia, Brescia, Italy) Hektoen enteric agar (bioMérieux) Campylosel agar (bioMérieux) Thiosulfate Citrate Bile Salts Sucrose agar (Biolife, Milan, Italy) MacConkey agar (bioMérieux) Cefsulodina-irgasan-novobiocina agar (bioMérieux) Aeromonas Yersinia agar (BD) Sulphite polimixin sulphadiazine agar (Liofilchem, TE, Italy) CLO agar (bioMérieux) CHROMagar O157 (BD) Sorbitol MacConkey (Biolife)
35–37 °C aerobic
VITEK® 2 system (bioMérieux)
24–48 h
35–37 °C aerobic 42 °C microaerophilic 35–37 °C aerobic
VITEK® 2 system API-Campy system (bioMérieux) VITEK® 2 system
24 h 48–72 h 24–48 h
30 °C aerobic 30 °C aerobic 35–37 °C anaerobic 35–37 °C anaerobic 35–37 °C aerobic
VITEK® 2 system VITEK® 2 system RapID™ System (Remel, Lenexa, KS, USA) RapID™ System ImmunoCard STAT!® EHEC (Meridian Bioscience Inc.)
24 h 24 h 7d 7d 24 h
Shigella spp. Campylobacter spp. Vibrio spp. Y. enterocolitica Aeromonas spp. C. perfringens C. difficile E. coli O157:H7
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Table 2 Primers and probes used for analysis of discordant results. Target
PCR method
Primers sequence
Target gene
Amplicon size
Reference
Salmonella spp.
End-point PCR
ITS
312 bp
(Park et al., 2006)
Shigella spp. (chromosome and plasmid)
End-point PCR
5′-TATAGCCCCATCGTGTAGTCAGAAC-3′ F 5′-TGCGGCTGGATCACCTCCTT-3′ R 5′-AGCTCAGGCAATGAAACTTTGAC-3′ F 5′-TGGGCTTGATATTCCGATAAGTC-3′ R 5′-CTCGGCACGTTTTAATAGTCTGG-3′ F 5′-GTGGAGAGCTGAAGTTTCTCTGC-3′ R 5′-CACGTGTCACAATGGCATAT-3′ F 5′-GGCTTCATGCTCTCGAGTT-3′ R 5′-VIC-AGACGCAATACCGC-TAMRA-3′ Probe 5′-GTTAATGCTGTCTTCATTTGGAGC-3′ F 5′-GACATCCCAATCACTACTGACTTC-3′ R 5′-CTACTTTTGCCGGCGAGCGG-3′ F 5′-TGATTCCCGAAGGCACTCCC-3′ R 5′-ACAGGTACCTTTAGCCAATC-3′ F 5′-AATCTTTCTGTAGCAGCAGC-3′ R 5′-GGATTTGGAATAACTATAGG-3′ F 5′-CTGCAGATGTTTTACTAAGC-3′ R 5′-GGAAAAGAGAATGGTTTTATTAA-3′ F 5′-ATCTTTAGTTATAACTTTGACATCTTT-3′ R 5′-ACCCTGTAACGAAGTTTGAC-3′ F 5′-ATCTCATGCGACTCTTGAC-3′ R 5′-TTAACCACACCCACGGCAGT-3′ F 5′-GCTCTGGATGCATCTCTGGT-3′ R 5′-ATACCACTATGATGCAGATTA-3′ F 5′-TCATCATCACCATAGAAAGAG-3′ R 5′-GACGGVGCRACTACATGGT-3′ F 5′-GTCCAATTCATNCCTGGTGG-3′ R 5′-AACTGTGCCTTGGAATCATC-3′ F 5′-TAAGGTTAAAGCCCCGTTT-3′ R
virF
628 bp
(Vidal et al., 2005)
ipaH
933 bp
16S rRNA
~ 115 bp
(Leblanc-Maridor et al., 2011)
YST
145 bp
(Gómez-Duarte et al., 2009)
16S rRNA
~935 bp
(Lee et al., 2002)
cpe
199 bp
(Miwa et al., 1998)
tcdB
160 bp
(Lemee et al., 2004)
VT1
135 bp
(El Sayed and El-Adrosy, 2007)
VT2
346 bp
RNA pol
310 bp
(Vinjé and Koopmans, 1996)
VP6
382 bp
(Iturriza Gómara et al., 2002)
Fiber protein
499 bp
(La Rosa et al., 2011)
Campylobacter spp.
Real-time PCR
Y. enterocolitica
End-point PCR
Aeromonas spp.
End-point PCR
C. perfringens
Nested-PCR
C. difficile toxin B
End-point PCR
E. coli Shiga toxin 1-2
End-point PCR
Norovirus
End-point PCR
Rotavirus
End-point PCR
Adenovirus
End-point PCR
ITS = Salmonella internal transcribed spacer region of 16S-23S rRNA; virF = Shigella virulence regolatory gene; ipaH = Shigella invasion plasmid antigen gene; 16S rRNA = Campylobacter conserved regions of 16S rDNA gene sequences; YST = Yersinia heat-stable enterotoxin gene; 16S rRNA = Aeromonas conserved regions of 16S rDNA gene sequences; cpe = C. perfringens enterotoxin gene; tcdB = internal fragment of the C. difficile toxin B gene; VT1/VT2 = E. coli verotoxin gene; RNA pol = norovirus RNA polymerase gene; VP6 = rotavirus middle-layer protein; fiber protein of adenovirus.
mixtures for rotavirus, adenovirus, and norovirus (final volume 25 μL) contained: 12.7 μL of sterile water, 2.5 μL of 10× buffer solution, 2 μL MgCl2, 0.5 μL of dNTP mix (10 mmol/L), 2 μL each of forward and reverse primers (10 μmol/L), 0.3 μL of Taq DNA polymerase, and 3 μL of template DNA. The PCR products were analysed by electrophoresis on 2.2% agarose gel (Flash gel; Lonza, Basel Switzerland). The real-time PCR for Campylobacter spp. was performed in an ABI PRISM® 7300 Sequence Detection System (Applied Biosystems). Reaction mixtures (final volume 50 μL) contained: 25 μL of 2× Taqman Universal PCR Mastermix (Applied Biosystems), containing AmpliTaq Gold™ DNA polymerase, dNTPs, Passive reference (ROX), and optimized buffer components including 5 mmol/L MgCl2, 17.55 μL of sterile water, 0.5 μL of 20 μmol/L Camp Probe, 0.45 μL of 100 μmol/L Camp Forward primer, 1.5 μL of 10 μmol/L Camp Reverse primer, and 5 μL of template DNA. In each reaction was included a concordant positive and a concordant negative sample to confirm home-brew reliability. The PCR methods were performed according to published protocols (El Sayed and El-Adrosy, 2007; Gómez-Duarte et al., 2009; Iturriza Gómara et al., 2002; La Rosa et al., 2011; Leblanc-Maridor et al., 2011; Lee et al., 2002; Lemee et al., 2004; Miwa et al., 1998; Park et al., 2006; Vidal et al., 2005; Vinjé and Koopmans, 1996). No limit of detection was calculated for each singleplex PCR assay. 2.6. Sensitivity and specificity After the analysis of discordant results, the sensitivity and specificity of both the Seeplex® Diarrhea ACE Detection assay and of our routine methods were determined. The sensitivity was calculated as the number of positive results obtained by either multiplex PCR or routine methods, divided by the number of total positive results (concordant positive results plus positive results by
confirmatory testing). The specificity was calculated as the number of negative results obtained by either multiplex PCR or by routine methods divided by the number of total negative results (concordant negative results plus negative results by confirmatory test). 3. Results Out of 245 stool samples processed, our routine methods detected 61 bacteria (16 Salmonella spp., 5 Campylobacter spp., 13 C. perfringens, 25 toxigenic C. difficile, and 2 VTEC, 1 of which was E. coli O157:H7) and 121 viruses (7 norovirus, 98 rotavirus, and 16 adenovirus). These methods allowed for the diagnosis of bacterial diarrhoea in 43/245 (17.6%) cases, viral diarrhoea in 106/245 (43.2%) cases, and bacterial-viral co-infections in 14/245 (5.7%) cases. Therefore, at least 1 pathogen responsible for infectious gastroenteritis was detected in 163/245 patients (66.5%). No aetiological agent was detected in 82/245 (33.5%) cases. Multiplex PCR detected 78 bacteria (9 Salmonella spp., 3 Shigella spp., 19 Campylobacter spp., 3 Y. enterocolitica, 5 Aeromonas spp., 25 C. perfringens, 11 toxigenic C. difficile, and 3 VTEC, 1 of which was E. coli O157:H7) and 167 viruses (11 astrovirus, 4 norovirus GI, 18 norovirus GII, 110 rotavirus, and 24 adenovirus). The molecular method allowed for the diagnosis of bacterial diarrhoea in 36/245 (14.7%) cases, viral diarrhoea in 121/245 (49.4%) cases, and bacterial-viral co-infections in 30/245 (12.2%) cases. Therefore, at least 1 pathogen responsible for infectious gastroenteritis was detected in 187/245 patients (76.3%). No aetiological agent was detected in 58/245 (23.7%) cases. Forty-four stool samples resulted concordant negative, positive results were obtained for each pathogen by both methods, and concordant and discordant results are shown in Table 3. All discordant samples were investigated further by confirmatory tests (Table 4) and the following results were confirmed positive: 6/7 Salmonella spp., 1/3 Shigella spp., 16/16 Campylobacter spp., 3/3 Y.
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Table 3 Summary of results obtained with routine methods and multiplex PCR assay. Pathogen
Routine methods
Multiplex PCR
Concordant results
Discordant results
Salmonella spp. Shigella spp. Campylobacter spp. Vibrio spp. Y. enterocolitica Aeromonas spp. C. perfringens C. difficile toxin B VTEC Astrovirus Norovirus Rotavirus Adenovirus
16 0 5 0 0 0 13 25 2 7 98 16
9 3 19 0 3 5 25 11 3 11 22 (4 GI; 18 GII) 110 24
9 4 7 7 2 6 91 12
7 3 16 3 5 24 22 1 17 26 16
enterocolitica, 1/5 Aeromonas spp., 16/24 C. perfringens enterotoxin (CPE) producers, 18/22 toxigenic C. difficile, 14/17 norovirus, 18/26 rotavirus, and 9/16 adenovirus. Among the not confirmed results, those resulted positive only by multiplex PCR, with the exception of VTEC and rotavirus, showed an i.v. b20, specifically: 2/3 Shigella spp. (i.v. 18 and 6), 4/5 Aeromonas spp. (i.v. 19, 18, 15, and 17), 1/4 C. difficile (i.v. 12), 2/16 norovirus (i.v. 9 and 13), and 5/12 adenovirus (i.v. 5, 5, 11, 9, and 17). After analysis of discordant results, multiplex PCR was found to have a higher level of sensitivity than our routine detection methods for common enteric pathogens, with the exception of Salmonella spp. and toxigenic C. difficile (Table 5). Evaluation of sensitivity is unreliable for Shigella spp., Y. enterocolitica, and Aeromonas spp., due to the lack of positive results by culture methods and the few or no positive results by multiplex PCR. The use of multiplex PCR did not always result in a significant improvement in specificity.
4. Discussion We evaluated the Seeplex® Diarrhea ACE Detection assay for the detection of enteropathogens in stool samples from paediatric patients with diarrhoea, comparing results obtained versus our routine methods. Analyzing our data by patients, between the traditional and the molecular method, we obtained an overall concordance for 133 samples (89 positive and 44 negative), resulting in a 54.3% of patients that would have received the same diagnosis. For 45/245 patients (18.4%), the result was partially concordant and for 67/245 (27.3%), the result was completely discordant.
Table 4 Summary of analysis of discordant results. Pathogen (n)
Salmonella spp. (7) Shigella spp. (3) Campylobacter spp. (16) Y. enterocolitica (3) Aeromonas spp. (5) C. perfringens (24) C. difficile toxin B (22) VTEC (1) Norovirus (17) Rotavirus (26) Adenovirus (16)
Positive results Routine methods
Multiplex PCR
7 0 1 0 0 6 18 0 1 7 4
0 3 15 3 5 18 4 1 16 19 12
Confirmed positive results (home-made PCR) 6 1 16 (1/1 + 15/15)a 3 1 10 (1/6 + 9/18)a 18 (15/18 + 3/4)a 0 14 (0/1 + 14/16)a 18 (4/7 + 14/19)a 9 (2/4 + 7/12)a
a Numbers in parentheses are related to specimens confirmed positive by homebrew PCR, referred to routine methods and multiplex PCR, respectively.
If our laboratory were to use only the Seeplex Diarrhea ACE Detection, we would obtain a 10.6% (26/245) of positive patients more than if only routine methods were used, with a total of 76.3% versus 66.5% of patients with at least a positivity for a gastrointestinal pathogen. A causative agent was not found in 44/245 (18%) of the presumed infectious gastroenteritis cases; however, we did not evaluate parasitic enteric pathogens (Cryptosporidium spp., Giardia lamblia, Entamoeba histolytica, etc.) or emerging viruses related to gastroenteritis, such as Aichi virus, parechovirus, enterovirus, and human bocavirus (Pham et al., 2010; Van Damme et al., 2007). The analysis of discordant results revealed that Salmonella spp. was detected more frequently by routine culture methods, most likely due to the use of selective enrichment broth, which increases bacterial load and recovery. The unique unconfirmed sample may be the result of a bacterial load that was so low that it could not be detected even by singleplex PCR. Using routine culture methods, there were no positive results for Shigella spp., Y. enterocolitica, and Aeromonas spp. Singleplex PCR confirmed positive only those samples that were assigned a high intensity value by the detection system. Our results confirmed the limitations of detection by culture methods for Campylobacter spp. (Bessède et al., 2011), as 15/15 stool samples that were shown to be positive only by multiplex PCR were confirmed by real-time PCR. Since CPE-producing C. perfringens type A is 1 of the most common food poisoning agents and is implicated in 5–15% of cases of antimicrobial drug-associated diarrhoea and sporadic diarrhoea (Heikinheimo et al., 2006; Morris and Fernández-Miyakawa, 2009), the 24 discordant stool samples were tested by singleplex PCR targeting cpe enterotoxin. Culture methods detected 6/24 C. perfringens, with no information about toxin production and only 1 was revealed to be a CPE producer by singleplex PCR. On the other hand, toxin-producing C. perfringens were detected by multiplex PCR in 18/ 24 discordant sample, and of these, 9 were confirmed to be CPE producers. The target toxin is not specified in the multiplex PCR assay, but according to our results, we assume that this toxin is not necessarily CPE. For toxigenic C. difficile, as well as for Salmonella spp., the routine method proved to be the most sensitive. This is likely because the assay for the detection of toxin B was performed on isolated colonies grown on selective agar, resulting in a higher DNA load with no interference by the DNA from other pathogens or cells. In 3/18 stool samples that were positive only by routine methods, home-brew PCR did not confirm C. difficile toxin B, likely due to a low load of toxin B DNA in these samples. Similarly, 1/4 stool samples that were positive only by multiplex PCR were not confirmed by singleplex PCR, likely a result of a nonspecific amplification (i.v. 12) in the multiplex PCR. Among the 3/4 samples positive by multiplex PCR and confirmed by home-brew singleplex PCR, 2 were positive by culture for nontoxigenic C. difficile. Therefore, it is possible that toxigenic and nontoxigenic populations coexisted in the same culture and that the assay to detect toxin was performed on an isolated non-toxigenic strain. The third sample could have given a negative result by culture method because of low bacterial viability. The only 1 sample discordant for VTEC was confirmed to be negative by home-brew PCR. This result is likely due to crossreactivity in the multiplex PCR. We obtained 11/245 stool samples positive for astrovirus by multiplex PCR, but no concordance evaluation was possible. Norovirus was confirmed by home-brew PCR in 14/16 stool samples that were positive only by multiplex PCR, supporting claims of low sensitivity for the immunoassay currently in use (86%); the 2/16 stool samples, which resulted negative, were the only samples with a very low intensity value. The unique stool sample that was positive only by immunochromatographic assay and not confirmed was likely due to a
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Table 5 Sensitivity and specificity of Seeplex® Diarrhea Ace Detection multiplex PCR results for each target, after analysis of discordant results. Pathogens
Salmonella spp. Shigella spp. Campylobacter spp. Vibrio spp. Y. enterocolitica Aeromonas spp. C. perfringens C. difficile toxin B VTEC Astrovirus Norovirus Rotavirus Adenovirus
Routine methods
Multiplex PCR
Sensitivity %
Specificity %
Sensitivity %
Specificity %
100.00 (15/15) 0 (0/1) 25.00 (5/20) nv 0 (0/3) 0 (0/1) 47.06 (8/17) 88.00 (22/25) 100.00 (2/2) nv 30.00 (6/20) 87.16 (95/109) 58.33 (14/24)
99.57 (1/230) 100.00 (0/244) 100.00 (0/225) nv 100.00 (0/242) 100.00 (0/244) 97.81 (5/228) 98.64 (3/220) 100.00 (0/243) nv 99.56 (1/225) 97.79 (3/136) 99.10 (2/221)
60.00 (9/15) 100.00 (1/1) 95.00 (19/20) nv 100.00 (3/3) 100.00 (1/1) 94.12 (16/17) 40.00 (10/25) 100.00 (2/2) nv 100.00 (20/20) 96.33 (105/109) 91.67 (22/24)
100.00 (0/230) 99.18 (2/244) 100.00 (0/225) nv 100.00 (0/242) 98.36 (4//244) 96.05 (9/228) 99.55 (1/220) 99.59 (1/243) nv 99.11 (2/225) 96.32 (5/136) 99.10 (2/221)
nv = not valuable.
cross-reaction in antigen detection (the declared specificity for the immunochromatographic assay is 94.6%). Rotavirus type A was the most commonly detected pathogen in our stool samples, with 91/245 concordant positive results. Three samples that were positive for rotavirus only by immunochromatographic assay were confirmed to be negative. The Rota-Strip test claims 100% specificity; therefore, we can only speculate that the RNA extraction procedure was not successful. Five stool samples that were positive for rotavirus only by multiplex PCR were confirmed to be negative. As these samples had a high intensity value (i.v. N90), we hypothesize that the RNA in the samples was degraded. Concerning the 16 stool samples discordant for adenovirus, homebrew PCR did not confirm 2/4 samples that were positive for adenovirus by immunochromatographic assay, perhaps resulting from cross-reactivity in antigen detection (the Adeno-Strip test claims 99.5% specificity). The 5/12 discordant unconfirmed samples that were positive by multiplex PCR may be a result of nonspecific amplification, given their i.v. b20. In our experience, we found that the numerical value ascribed by the ScreenTape system to a positive signal gave a reliable result only when N20; in fact, the majority of discordant positive results not confirmed by home brew PCR showed an i.v. ≤20. For the detection of viral enteropathogens, our data are in agreement with a recent report (Higgins et al., 2011), but with the exception of the sensitivity for norovirus, sensitivity and specificity are lower than 100%. Coupland et al. (2012) extended the evaluation to bacterial pathogens, reporting a high specificity and a sensitivity of 100% for all pathogens, excluding rotavirus, C. difficile toxin B, and Salmonella spp., in concordance with our data. The Seeplex® Diarrhea ACE detection assay can replace multiple approaches for the investigation of enteropathogens with just 1 procedure. It provides several advantages in a clinical laboratory routine, including reduction in time and costs. On the other hand, molecular detection of DNA or RNA can also be a hindrance, since it can reveal low and clinically insignificant pathogen levels too and does not provide any information about viability of infectious particles. The limitations of the current study are related to the low number or total absence of some pathogens in stool samples and to the impossibility to compare the assays for Vibrio spp. and astrovirus. Moreover, the panel of pathogens investigated by multiplex assay does not include parasites and emerging viruses, resulting in a possible underestimation of pathogens really present in stool samples. One more limitation of this study was that the limit of sensitivity for the detection of each pathogen by home-brew PCR was not evaluated. In conclusion, the Seeplex® Diarrhea ACE Detection assay was shown to be a rapid, sensitive, specific, and reliable diagnostic tool to directly detect the most common enteropathogens in stool samples
but should be optimized for recovery of Salmonella spp. and toxigenic C. difficile. Acknowledgments Preliminary results shown in this paper have been presented as poster format, at the European Congress of Clinical Microbiology and Infectious Diseases, Milano, Italia, May 7–10, 2011. This manuscript has been edited by native English-speaking experts of BioMed Proofreading. We would like to thank Dr Franco Ruggeri, Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Rome, Italy, for kindly providing primers for rotavirus and norovirus used in this study. We are also grateful to Dr Carlo Concato for his invaluable technical assistance and his precious suggestions. References Bessède E, Delcamp A, Sifré E, Buissonnière A, Mégraud F. New methods for detection of campylobacters in stool samples in comparison to culture. J Clin Microbiol 2011;49: 941–4. Colomba C, De Grazia S, Giammanco GM, Saporito L, Scarlata F, Titone L, et al. Viral gastroenteritis in children hospitalised in Sicily, Italy. Eur J Clin Microbiol Infect Dis 2006;25:570–5. Coupland LJ, McElarney I, Meader E, Cowley K, Alcock L, Naunton J, et al. Simultaneous detection of viral and bacterial enteric pathogens using the Seeplex® Diarrhoea ACE detection system. Epidemiol Infect 2012;5:1–11. Cunningham SA, Sloan LM, Nyre LM, Vetter EA, Mandrekar J, Patel R. Three-hour molecular detection of Campylobacter, Salmonella, Yersinia, and Shigella species in feces with accuracy as high as that of culture. J Clin Microbiol 2010;48:2929–33. de Boer RF, Ott A, Kesztyüs B, Kooistra-Smid AM. Improved detection of five major gastrointestinal pathogens by use of a molecular screening approach. J Clin Microbiol 2010;48:4140–6. Di Bartolo I, Monini M, Losio MN, Pavoni E, Lavazza A, Ruggeri FM. Molecular characterization of noroviruses and rotaviruses involved in a large outbreak of gastroenteritis in Northern Italy. Appl Environ Microbiol 2011;77:5545–8. Dionisi AM, Filetici E, Ocwzarek S, Arena S, Benedetti I, Lucarelli C, et al. (2011) Enternet international surveillance network. Report 2007–2009. Not Ist Super Sanitá 24: 3. El Sayed Zaki M, El-Adrosy H. Diagnosis of Shiga toxin producing Escherichia coli infection, contribution of genetic amplification technique. Microbes Infect 2007;9: 200–3. Gómez-Duarte OG, Bai J, Newell E. Detection of Escherichia coli, Salmonella spp., Shigella spp., Yersinia enterocolitica, Vibrio cholerae, and Campylobacter spp. enteropathogens by 3-reaction multiplex polymerase chain reaction. Diagn Microbiol Infect Dis 2009;63:1–9. Heikinheimo A, Lindström M, Granum PE, Korkeala H. Humans as reservoir for enterotoxin gene-carrying Clostridium perfringens type A. Emerg Infect Dis 2006;12:1724–9. Higgins RR, Beniprashad M, Cardona M, Masney S, Low DE, Gubbay JB. Evaluation and verification of the Seeplex Diarrhoea-V ACE assay for simultaneous detection of adenovirus, rotavirus, and norovirus genogroups I and II in clinical stool specimens. J Clin Microbiol 2011;49:3154–62. Iturriza Gómara M, Wong C, Blome S, Desselberger U, Gray J. Molecular characterization of VP6 genes of human rotavirus isolates: correlation of genogroups with subgroups and evidence of independent segregation. J Virol 2002;76:6596–601. Khamrin P, Okame M, Thongprachum A, Nantachit N, Nishimura S, Okitsu S, et al. A single-tube multiplex PCR for rapid detection in feces of 10 viruses causing diarrhoea. J Virol Methods 2011;173:390–3.
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Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Evaluation of a multiplex PCR assay for simultaneous detection of bacterial and viral enteropathogens in stool samples of paediatric patients☆ Manuela Onori a,⁎, Luana Coltella a, Livia Mancinelli a, Marta Argentieri b, Donato Menichella c, Alberto Villani d, Annalisa Grandin d, Diletta Valentini d, Massimiliano Raponi c, Cristina Russo a a
Department of Laboratory Medicine, Virology Unit, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy Department of Laboratory Medicine, Bacteriology Unit, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy c Medical Direction, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy d Paediatric and Infectious Disease Unit, Bambino Gesù Children's Hospital, IRCCS, Rome, Italy b
a r t i c l e
i n f o
Article history: Received 9 October 2013 Received in revised form 4 February 2014 Accepted 5 February 2014 Available online 22 February 2014 Keywords: Multiplex PCR Enteric pathogens Paediatric patients
a b s t r a c t We evaluated a multiplex PCR assay, the Seeplex Diarrhoea ACE detection, that simultaneously detects 15 enteric pathogens, including Salmonella spp., Shigella spp., Vibrio spp., toxin B producer Clostridium difficile, Campylobacter spp., Clostridium perfringens, Yersinia enterocolitica, Aeromonas spp., Escherichia coli O157:H7, verocytotoxin-producing Escherichia coli, adenovirus, Group A rotavirus, norovirus GI and GII, and astrovirus. We compared this assay with clinical methods routinely used in our laboratory, for detecting enteropathogens in stool samples collected from 245 paediatric patients with suspected infectious gastroenteritis. We recovered 61 bacterial pathogens and 121 enteric viruses with our laboratory assays, while we detected 78 bacteria and 167 viruses with the molecular assay. We calculated specificity and sensitivity for both methods after analysis of discordant results and demonstrated greater sensitivity for multiplex PCR than for our routine methods, with the exception of Salmonella spp. and toxigenic C. difficile detection. The multiplex PCR assay proved to be a reliable tool to directly detect the most common enteropathogens in stool samples but with some limitations. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Acute gastroenteritis is one of the most common causes of death amongst infants and children in developing countries (Thapar and Sanderson, 2004). In Europe, most cases are mild to moderately severe, and fatal outcomes are rare. About 40% of acute diarrhoeal episodes in the first 5 years of life are caused by rotaviruses, while 30% are caused by other viruses, primarily noroviruses and adenoviruses (Van Damme et al., 2007). A bacterial pathogen can be identified in stool in about 20% of children with gastroenteric infections, while parasites are detected in less than 5% of cases (Koletzko and Osterrieder, 2009). Currently, a national surveillance system that is able to determine the incidence of different etiologic agent of gastroenteritis is not in place in Italy. However, the surveillance of Acute Gastroenteritis is included in the official surveillance program of infectious disease (Italian National Surveillance System of Infectious Diseases) and in the laboratory-based surveillance network for enteric pathogens (as Enter-net Italia and RotaNet-Italia). Rotavirus represents the principal etiologic agent in ☆ Transparency declaration: All authors report that they have no potential conflicts of interest to declare. ⁎ Corresponding author. Tel.: +39-06-68592206; fax: +39-06-68592218. E-mail address: [email protected] (M. Onori). http://dx.doi.org/10.1016/j.diagmicrobio.2014.02.004 0732-8893/© 2014 Elsevier Inc. All rights reserved.
hospitalized children affected by acute diarrhea with an age b5 years (Palumbo et al., 2010). Norovirus is the major responsible virus of outbreak (Colomba et al., 2006; Di Bartolo et al., 2011), and with regard to sporadic enteritis, the few studies performed have reported prevalence rates from 2.1% to 18.6% (Medici et al., 2006). For bacteria, Salmonella is the most frequently isolated bacteria in enteric infections, while verotoxin-producing Escherichia coli (VTEC) is only sporadically reported (Dionisi et al., 2011). The annual rate of acute gastrointestinal illness observed in Italy falls within the range of incidence reported in other industrialized countries (Scavia et al., 2012); however, estimates of the incidence of enteric infections normally suffer from large underreporting (Rimoldi et al., 2011). The clinical presentation of patients with acute gastroenteritis symptoms is generally not indicative of a specific pathogen, and laboratory diagnosis is often difficult due to the long list of potential enteropathogens. Moreover, conventional diagnostic procedures for bacteria detection require time, expert microbiologists, and vital bacteria because based on culture procedures, the immunochromatographic detection of enteric viral antigens has limited sensitivity. Molecular techniques, such as PCR, are widely used for diagnosing infectious disease. However, singleplex PCR is unsuitable for this type of investigation, due to the wide variety of pathogens that can
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potentially be associated with diarrhoea. Multiplex PCR systems, which allow the simultaneous amplification of several targets, are promising diagnostic tools for detection of enteropathogens (Platts-Mills et al., 2012). Several papers describe molecular approaches for the simultaneous detection of multiple gastrointestinal bacteria (Cunningham et al., 2010; de Boer et al., 2010; Liu et al., 2012) and/or viruses (Khamrin et al., 2011; Liu et al., 2011; Wolffs et al., 2011). In this study, we compared the performance of a multiplex PCR assay (Seeplex® Diarrhea ACE Detection; Seegene, Seoul, Korea), which simultaneously detects 10 bacterial species and 5 viruses, with our routine clinical methods for investigating the aetiology of gastrointestinal infections in children with diarrhoea. 2. Materials and methods 2.1. Specimens A total of 245 liquid stools samples were collected from 245 paediatric patients, aged between 1 month and 16 years, admitted for presumptive infectious diarrhoea to the Paediatric and Infectious Disease Unit of Bambino Gesù Children's Hospital in Rome, Italy, between April 2010 and August 2011. Stool samples were immediately processed for routine detection of bacteria and viruses and for multiplex PCR. None of the children enrolled in this study were vaccinated for rotavirus. Informed consent was obtained from patients enrolled in the study. This study was conducted with the approval of the Ethics Committee of Bambino Gesù Children's Hospital. 2.2. Routine assays for detection of bacteria Stool samples were spread on selective media shown in Table 1. Species identification was carried out using the systems listed in Table 1. Evaluation of Clostridium difficile toxin B was performed by real-time PCR (GeneXpert C. difficile; Cepheid, Sunnyvale, CA, USA), employing a laboratory-validated procedure: a 10-μL loop of isolated colonies grown on selective media was suspended directly in 2 mL of sample reagent. 2.3. Routine assays for detection of viruses Immunochromatographic assays were used for direct detection of viral antigens in faecal samples. Antigens of group A rotavirus, adenovirus serotypes 40 and 41, and norovirus genotypes I and II were detected, respectively, using Rota-Strip C1001 (Coris BioConcept, Gembloux, Belgium), Adeno-Strip C1002 (Coris BioConcept) and RIDA®QUICK Norovirus (R-Biopharm AG, Darmstadt, Germany). In our routine testing, no diagnostic tool was available for recovery of astrovirus.
2.4. Multiplex PCR assay Stool samples were pre-treated by dissolving 1 mL of faeces in 5 mL of phosphate-buffered saline, followed by centrifugation at 4000 × g for 30 min. The supernatant (500 μL) was used for extraction of DNA/RNA using the NucliSENS® easyMAG® System (bioMérieux, Marcy l'Etoile, France). Extracted nucleic acids were eluted in a final volume of 100 μL. Reverse transcription was performed using the GeneAmp® RNA PCR kit (Applied Biosystems, Carlsbad, CA, USA), according to the manufacturer's instructions. Thermal cycling parameters were: 25 °C for 10 min, 42 °C for 60 min, and 95 °C for 5 min. The Seeplex® Diarrhea ACE Detection assay is composed of 3 separate primer mixtures (B1, B2, and V) in 3 reaction tubes and is able to identify Salmonella spp. (Salmonella bongori and Salmonella enterica), Shigella spp. (Shigella flexneri, Shigella boydii, Shigella sonnei, and Shigella dysenteriae), Vibrio spp. (Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus), C. difficile toxin B, Campylobacter spp. (Campylobacter jejuni and Campylobacter coli), Clostridium perfringens toxin, Yersinia enterocolitica, Aeromonas spp. (Aeromonas salmonicida, Aeromonas sobria, Aeromonas bivalvum, and Aeromonas hydrophila), E. coli O157:H7, VTEC, group A rotavirus, adenovirus serotypes 40 and 41, astrovirus, and norovirus GI and GII. PCR inhibitory effects were assessed by co-amplification of an internal control included in the primer mixtures. Multiplex PCR was performed according to the manufacturer's instructions. The detection limit declared by the manufacturer is 100 copies/reaction. PCR products were detected by the ScreenTape system (Lab901, Loanhead, UK), according to the manufacturer's instructions. The numerical value ascribed by the ScreenTape system to a positive signal in the detection step represents the intensity value (i.v.) relative to the marker. Manufacturer's instructions do not indicate a range or a breakpoint value to assign a positive result.
2.5. Confirmatory tests All extracts for which discordant results were obtained by multiplex PCR and our routine methods were retested using a home-brew singleplex PCR (Table 2). The primers employed for the home-brew PCR were selected according to the literature, as the multiplex assay does not specify the target region for each pathogen. Primers and probes were synthesized by Sigma Aldrich (Saint Louis, MO, USA) and Applied Biosystems, respectively. Reaction mixtures for Salmonella spp., Shigella spp., Yersinia enterocolitica, Aeromonas spp., VTEC, C. difficile, and C. perfringens (final volume 25 μL) contained: 10.75 μL of sterile water, 2.5 μL of 10× buffer solution, 1.5 μL MgCl2, 1.25 μL of DMSO, 2 μL of dNTP mix (10 mmol/L), 2 μL each of forward and reverse primers (10 μmol/L), 0.5 μL of Taq DNA polymerase, and 2.5 μL of template DNA. Reaction
Table 1 Media, growth conditions, and identification systems used for the detection of bacterial pathogens from stool samples by routine methods. Pathogen
Media
Growth conditions
Identification systems
Time of detection
Salmonella spp.
Hektoen enteric agar (bioMérieux) CHROMagar Salmonella (BD, Franklin Lakes, NJ, USA) after 24 h of enrichment in selenite broth (Copan Italia, Brescia, Italy) Hektoen enteric agar (bioMérieux) Campylosel agar (bioMérieux) Thiosulfate Citrate Bile Salts Sucrose agar (Biolife, Milan, Italy) MacConkey agar (bioMérieux) Cefsulodina-irgasan-novobiocina agar (bioMérieux) Aeromonas Yersinia agar (BD) Sulphite polimixin sulphadiazine agar (Liofilchem, TE, Italy) CLO agar (bioMérieux) CHROMagar O157 (BD) Sorbitol MacConkey (Biolife)
35–37 °C aerobic
VITEK® 2 system (bioMérieux)
24–48 h
35–37 °C aerobic 42 °C microaerophilic 35–37 °C aerobic
VITEK® 2 system API-Campy system (bioMérieux) VITEK® 2 system
24 h 48–72 h 24–48 h
30 °C aerobic 30 °C aerobic 35–37 °C anaerobic 35–37 °C anaerobic 35–37 °C aerobic
VITEK® 2 system VITEK® 2 system RapID™ System (Remel, Lenexa, KS, USA) RapID™ System ImmunoCard STAT!® EHEC (Meridian Bioscience Inc.)
24 h 24 h 7d 7d 24 h
Shigella spp. Campylobacter spp. Vibrio spp. Y. enterocolitica Aeromonas spp. C. perfringens C. difficile E. coli O157:H7
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Table 2 Primers and probes used for analysis of discordant results. Target
PCR method
Primers sequence
Target gene
Amplicon size
Reference
Salmonella spp.
End-point PCR
ITS
312 bp
(Park et al., 2006)
Shigella spp. (chromosome and plasmid)
End-point PCR
5′-TATAGCCCCATCGTGTAGTCAGAAC-3′ F 5′-TGCGGCTGGATCACCTCCTT-3′ R 5′-AGCTCAGGCAATGAAACTTTGAC-3′ F 5′-TGGGCTTGATATTCCGATAAGTC-3′ R 5′-CTCGGCACGTTTTAATAGTCTGG-3′ F 5′-GTGGAGAGCTGAAGTTTCTCTGC-3′ R 5′-CACGTGTCACAATGGCATAT-3′ F 5′-GGCTTCATGCTCTCGAGTT-3′ R 5′-VIC-AGACGCAATACCGC-TAMRA-3′ Probe 5′-GTTAATGCTGTCTTCATTTGGAGC-3′ F 5′-GACATCCCAATCACTACTGACTTC-3′ R 5′-CTACTTTTGCCGGCGAGCGG-3′ F 5′-TGATTCCCGAAGGCACTCCC-3′ R 5′-ACAGGTACCTTTAGCCAATC-3′ F 5′-AATCTTTCTGTAGCAGCAGC-3′ R 5′-GGATTTGGAATAACTATAGG-3′ F 5′-CTGCAGATGTTTTACTAAGC-3′ R 5′-GGAAAAGAGAATGGTTTTATTAA-3′ F 5′-ATCTTTAGTTATAACTTTGACATCTTT-3′ R 5′-ACCCTGTAACGAAGTTTGAC-3′ F 5′-ATCTCATGCGACTCTTGAC-3′ R 5′-TTAACCACACCCACGGCAGT-3′ F 5′-GCTCTGGATGCATCTCTGGT-3′ R 5′-ATACCACTATGATGCAGATTA-3′ F 5′-TCATCATCACCATAGAAAGAG-3′ R 5′-GACGGVGCRACTACATGGT-3′ F 5′-GTCCAATTCATNCCTGGTGG-3′ R 5′-AACTGTGCCTTGGAATCATC-3′ F 5′-TAAGGTTAAAGCCCCGTTT-3′ R
virF
628 bp
(Vidal et al., 2005)
ipaH
933 bp
16S rRNA
~ 115 bp
(Leblanc-Maridor et al., 2011)
YST
145 bp
(Gómez-Duarte et al., 2009)
16S rRNA
~935 bp
(Lee et al., 2002)
cpe
199 bp
(Miwa et al., 1998)
tcdB
160 bp
(Lemee et al., 2004)
VT1
135 bp
(El Sayed and El-Adrosy, 2007)
VT2
346 bp
RNA pol
310 bp
(Vinjé and Koopmans, 1996)
VP6
382 bp
(Iturriza Gómara et al., 2002)
Fiber protein
499 bp
(La Rosa et al., 2011)
Campylobacter spp.
Real-time PCR
Y. enterocolitica
End-point PCR
Aeromonas spp.
End-point PCR
C. perfringens
Nested-PCR
C. difficile toxin B
End-point PCR
E. coli Shiga toxin 1-2
End-point PCR
Norovirus
End-point PCR
Rotavirus
End-point PCR
Adenovirus
End-point PCR
ITS = Salmonella internal transcribed spacer region of 16S-23S rRNA; virF = Shigella virulence regolatory gene; ipaH = Shigella invasion plasmid antigen gene; 16S rRNA = Campylobacter conserved regions of 16S rDNA gene sequences; YST = Yersinia heat-stable enterotoxin gene; 16S rRNA = Aeromonas conserved regions of 16S rDNA gene sequences; cpe = C. perfringens enterotoxin gene; tcdB = internal fragment of the C. difficile toxin B gene; VT1/VT2 = E. coli verotoxin gene; RNA pol = norovirus RNA polymerase gene; VP6 = rotavirus middle-layer protein; fiber protein of adenovirus.
mixtures for rotavirus, adenovirus, and norovirus (final volume 25 μL) contained: 12.7 μL of sterile water, 2.5 μL of 10× buffer solution, 2 μL MgCl2, 0.5 μL of dNTP mix (10 mmol/L), 2 μL each of forward and reverse primers (10 μmol/L), 0.3 μL of Taq DNA polymerase, and 3 μL of template DNA. The PCR products were analysed by electrophoresis on 2.2% agarose gel (Flash gel; Lonza, Basel Switzerland). The real-time PCR for Campylobacter spp. was performed in an ABI PRISM® 7300 Sequence Detection System (Applied Biosystems). Reaction mixtures (final volume 50 μL) contained: 25 μL of 2× Taqman Universal PCR Mastermix (Applied Biosystems), containing AmpliTaq Gold™ DNA polymerase, dNTPs, Passive reference (ROX), and optimized buffer components including 5 mmol/L MgCl2, 17.55 μL of sterile water, 0.5 μL of 20 μmol/L Camp Probe, 0.45 μL of 100 μmol/L Camp Forward primer, 1.5 μL of 10 μmol/L Camp Reverse primer, and 5 μL of template DNA. In each reaction was included a concordant positive and a concordant negative sample to confirm home-brew reliability. The PCR methods were performed according to published protocols (El Sayed and El-Adrosy, 2007; Gómez-Duarte et al., 2009; Iturriza Gómara et al., 2002; La Rosa et al., 2011; Leblanc-Maridor et al., 2011; Lee et al., 2002; Lemee et al., 2004; Miwa et al., 1998; Park et al., 2006; Vidal et al., 2005; Vinjé and Koopmans, 1996). No limit of detection was calculated for each singleplex PCR assay. 2.6. Sensitivity and specificity After the analysis of discordant results, the sensitivity and specificity of both the Seeplex® Diarrhea ACE Detection assay and of our routine methods were determined. The sensitivity was calculated as the number of positive results obtained by either multiplex PCR or routine methods, divided by the number of total positive results (concordant positive results plus positive results by
confirmatory testing). The specificity was calculated as the number of negative results obtained by either multiplex PCR or by routine methods divided by the number of total negative results (concordant negative results plus negative results by confirmatory test). 3. Results Out of 245 stool samples processed, our routine methods detected 61 bacteria (16 Salmonella spp., 5 Campylobacter spp., 13 C. perfringens, 25 toxigenic C. difficile, and 2 VTEC, 1 of which was E. coli O157:H7) and 121 viruses (7 norovirus, 98 rotavirus, and 16 adenovirus). These methods allowed for the diagnosis of bacterial diarrhoea in 43/245 (17.6%) cases, viral diarrhoea in 106/245 (43.2%) cases, and bacterial-viral co-infections in 14/245 (5.7%) cases. Therefore, at least 1 pathogen responsible for infectious gastroenteritis was detected in 163/245 patients (66.5%). No aetiological agent was detected in 82/245 (33.5%) cases. Multiplex PCR detected 78 bacteria (9 Salmonella spp., 3 Shigella spp., 19 Campylobacter spp., 3 Y. enterocolitica, 5 Aeromonas spp., 25 C. perfringens, 11 toxigenic C. difficile, and 3 VTEC, 1 of which was E. coli O157:H7) and 167 viruses (11 astrovirus, 4 norovirus GI, 18 norovirus GII, 110 rotavirus, and 24 adenovirus). The molecular method allowed for the diagnosis of bacterial diarrhoea in 36/245 (14.7%) cases, viral diarrhoea in 121/245 (49.4%) cases, and bacterial-viral co-infections in 30/245 (12.2%) cases. Therefore, at least 1 pathogen responsible for infectious gastroenteritis was detected in 187/245 patients (76.3%). No aetiological agent was detected in 58/245 (23.7%) cases. Forty-four stool samples resulted concordant negative, positive results were obtained for each pathogen by both methods, and concordant and discordant results are shown in Table 3. All discordant samples were investigated further by confirmatory tests (Table 4) and the following results were confirmed positive: 6/7 Salmonella spp., 1/3 Shigella spp., 16/16 Campylobacter spp., 3/3 Y.
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Table 3 Summary of results obtained with routine methods and multiplex PCR assay. Pathogen
Routine methods
Multiplex PCR
Concordant results
Discordant results
Salmonella spp. Shigella spp. Campylobacter spp. Vibrio spp. Y. enterocolitica Aeromonas spp. C. perfringens C. difficile toxin B VTEC Astrovirus Norovirus Rotavirus Adenovirus
16 0 5 0 0 0 13 25 2 7 98 16
9 3 19 0 3 5 25 11 3 11 22 (4 GI; 18 GII) 110 24
9 4 7 7 2 6 91 12
7 3 16 3 5 24 22 1 17 26 16
enterocolitica, 1/5 Aeromonas spp., 16/24 C. perfringens enterotoxin (CPE) producers, 18/22 toxigenic C. difficile, 14/17 norovirus, 18/26 rotavirus, and 9/16 adenovirus. Among the not confirmed results, those resulted positive only by multiplex PCR, with the exception of VTEC and rotavirus, showed an i.v. b20, specifically: 2/3 Shigella spp. (i.v. 18 and 6), 4/5 Aeromonas spp. (i.v. 19, 18, 15, and 17), 1/4 C. difficile (i.v. 12), 2/16 norovirus (i.v. 9 and 13), and 5/12 adenovirus (i.v. 5, 5, 11, 9, and 17). After analysis of discordant results, multiplex PCR was found to have a higher level of sensitivity than our routine detection methods for common enteric pathogens, with the exception of Salmonella spp. and toxigenic C. difficile (Table 5). Evaluation of sensitivity is unreliable for Shigella spp., Y. enterocolitica, and Aeromonas spp., due to the lack of positive results by culture methods and the few or no positive results by multiplex PCR. The use of multiplex PCR did not always result in a significant improvement in specificity.
4. Discussion We evaluated the Seeplex® Diarrhea ACE Detection assay for the detection of enteropathogens in stool samples from paediatric patients with diarrhoea, comparing results obtained versus our routine methods. Analyzing our data by patients, between the traditional and the molecular method, we obtained an overall concordance for 133 samples (89 positive and 44 negative), resulting in a 54.3% of patients that would have received the same diagnosis. For 45/245 patients (18.4%), the result was partially concordant and for 67/245 (27.3%), the result was completely discordant.
Table 4 Summary of analysis of discordant results. Pathogen (n)
Salmonella spp. (7) Shigella spp. (3) Campylobacter spp. (16) Y. enterocolitica (3) Aeromonas spp. (5) C. perfringens (24) C. difficile toxin B (22) VTEC (1) Norovirus (17) Rotavirus (26) Adenovirus (16)
Positive results Routine methods
Multiplex PCR
7 0 1 0 0 6 18 0 1 7 4
0 3 15 3 5 18 4 1 16 19 12
Confirmed positive results (home-made PCR) 6 1 16 (1/1 + 15/15)a 3 1 10 (1/6 + 9/18)a 18 (15/18 + 3/4)a 0 14 (0/1 + 14/16)a 18 (4/7 + 14/19)a 9 (2/4 + 7/12)a
a Numbers in parentheses are related to specimens confirmed positive by homebrew PCR, referred to routine methods and multiplex PCR, respectively.
If our laboratory were to use only the Seeplex Diarrhea ACE Detection, we would obtain a 10.6% (26/245) of positive patients more than if only routine methods were used, with a total of 76.3% versus 66.5% of patients with at least a positivity for a gastrointestinal pathogen. A causative agent was not found in 44/245 (18%) of the presumed infectious gastroenteritis cases; however, we did not evaluate parasitic enteric pathogens (Cryptosporidium spp., Giardia lamblia, Entamoeba histolytica, etc.) or emerging viruses related to gastroenteritis, such as Aichi virus, parechovirus, enterovirus, and human bocavirus (Pham et al., 2010; Van Damme et al., 2007). The analysis of discordant results revealed that Salmonella spp. was detected more frequently by routine culture methods, most likely due to the use of selective enrichment broth, which increases bacterial load and recovery. The unique unconfirmed sample may be the result of a bacterial load that was so low that it could not be detected even by singleplex PCR. Using routine culture methods, there were no positive results for Shigella spp., Y. enterocolitica, and Aeromonas spp. Singleplex PCR confirmed positive only those samples that were assigned a high intensity value by the detection system. Our results confirmed the limitations of detection by culture methods for Campylobacter spp. (Bessède et al., 2011), as 15/15 stool samples that were shown to be positive only by multiplex PCR were confirmed by real-time PCR. Since CPE-producing C. perfringens type A is 1 of the most common food poisoning agents and is implicated in 5–15% of cases of antimicrobial drug-associated diarrhoea and sporadic diarrhoea (Heikinheimo et al., 2006; Morris and Fernández-Miyakawa, 2009), the 24 discordant stool samples were tested by singleplex PCR targeting cpe enterotoxin. Culture methods detected 6/24 C. perfringens, with no information about toxin production and only 1 was revealed to be a CPE producer by singleplex PCR. On the other hand, toxin-producing C. perfringens were detected by multiplex PCR in 18/ 24 discordant sample, and of these, 9 were confirmed to be CPE producers. The target toxin is not specified in the multiplex PCR assay, but according to our results, we assume that this toxin is not necessarily CPE. For toxigenic C. difficile, as well as for Salmonella spp., the routine method proved to be the most sensitive. This is likely because the assay for the detection of toxin B was performed on isolated colonies grown on selective agar, resulting in a higher DNA load with no interference by the DNA from other pathogens or cells. In 3/18 stool samples that were positive only by routine methods, home-brew PCR did not confirm C. difficile toxin B, likely due to a low load of toxin B DNA in these samples. Similarly, 1/4 stool samples that were positive only by multiplex PCR were not confirmed by singleplex PCR, likely a result of a nonspecific amplification (i.v. 12) in the multiplex PCR. Among the 3/4 samples positive by multiplex PCR and confirmed by home-brew singleplex PCR, 2 were positive by culture for nontoxigenic C. difficile. Therefore, it is possible that toxigenic and nontoxigenic populations coexisted in the same culture and that the assay to detect toxin was performed on an isolated non-toxigenic strain. The third sample could have given a negative result by culture method because of low bacterial viability. The only 1 sample discordant for VTEC was confirmed to be negative by home-brew PCR. This result is likely due to crossreactivity in the multiplex PCR. We obtained 11/245 stool samples positive for astrovirus by multiplex PCR, but no concordance evaluation was possible. Norovirus was confirmed by home-brew PCR in 14/16 stool samples that were positive only by multiplex PCR, supporting claims of low sensitivity for the immunoassay currently in use (86%); the 2/16 stool samples, which resulted negative, were the only samples with a very low intensity value. The unique stool sample that was positive only by immunochromatographic assay and not confirmed was likely due to a
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Table 5 Sensitivity and specificity of Seeplex® Diarrhea Ace Detection multiplex PCR results for each target, after analysis of discordant results. Pathogens
Salmonella spp. Shigella spp. Campylobacter spp. Vibrio spp. Y. enterocolitica Aeromonas spp. C. perfringens C. difficile toxin B VTEC Astrovirus Norovirus Rotavirus Adenovirus
Routine methods
Multiplex PCR
Sensitivity %
Specificity %
Sensitivity %
Specificity %
100.00 (15/15) 0 (0/1) 25.00 (5/20) nv 0 (0/3) 0 (0/1) 47.06 (8/17) 88.00 (22/25) 100.00 (2/2) nv 30.00 (6/20) 87.16 (95/109) 58.33 (14/24)
99.57 (1/230) 100.00 (0/244) 100.00 (0/225) nv 100.00 (0/242) 100.00 (0/244) 97.81 (5/228) 98.64 (3/220) 100.00 (0/243) nv 99.56 (1/225) 97.79 (3/136) 99.10 (2/221)
60.00 (9/15) 100.00 (1/1) 95.00 (19/20) nv 100.00 (3/3) 100.00 (1/1) 94.12 (16/17) 40.00 (10/25) 100.00 (2/2) nv 100.00 (20/20) 96.33 (105/109) 91.67 (22/24)
100.00 (0/230) 99.18 (2/244) 100.00 (0/225) nv 100.00 (0/242) 98.36 (4//244) 96.05 (9/228) 99.55 (1/220) 99.59 (1/243) nv 99.11 (2/225) 96.32 (5/136) 99.10 (2/221)
nv = not valuable.
cross-reaction in antigen detection (the declared specificity for the immunochromatographic assay is 94.6%). Rotavirus type A was the most commonly detected pathogen in our stool samples, with 91/245 concordant positive results. Three samples that were positive for rotavirus only by immunochromatographic assay were confirmed to be negative. The Rota-Strip test claims 100% specificity; therefore, we can only speculate that the RNA extraction procedure was not successful. Five stool samples that were positive for rotavirus only by multiplex PCR were confirmed to be negative. As these samples had a high intensity value (i.v. N90), we hypothesize that the RNA in the samples was degraded. Concerning the 16 stool samples discordant for adenovirus, homebrew PCR did not confirm 2/4 samples that were positive for adenovirus by immunochromatographic assay, perhaps resulting from cross-reactivity in antigen detection (the Adeno-Strip test claims 99.5% specificity). The 5/12 discordant unconfirmed samples that were positive by multiplex PCR may be a result of nonspecific amplification, given their i.v. b20. In our experience, we found that the numerical value ascribed by the ScreenTape system to a positive signal gave a reliable result only when N20; in fact, the majority of discordant positive results not confirmed by home brew PCR showed an i.v. ≤20. For the detection of viral enteropathogens, our data are in agreement with a recent report (Higgins et al., 2011), but with the exception of the sensitivity for norovirus, sensitivity and specificity are lower than 100%. Coupland et al. (2012) extended the evaluation to bacterial pathogens, reporting a high specificity and a sensitivity of 100% for all pathogens, excluding rotavirus, C. difficile toxin B, and Salmonella spp., in concordance with our data. The Seeplex® Diarrhea ACE detection assay can replace multiple approaches for the investigation of enteropathogens with just 1 procedure. It provides several advantages in a clinical laboratory routine, including reduction in time and costs. On the other hand, molecular detection of DNA or RNA can also be a hindrance, since it can reveal low and clinically insignificant pathogen levels too and does not provide any information about viability of infectious particles. The limitations of the current study are related to the low number or total absence of some pathogens in stool samples and to the impossibility to compare the assays for Vibrio spp. and astrovirus. Moreover, the panel of pathogens investigated by multiplex assay does not include parasites and emerging viruses, resulting in a possible underestimation of pathogens really present in stool samples. One more limitation of this study was that the limit of sensitivity for the detection of each pathogen by home-brew PCR was not evaluated. In conclusion, the Seeplex® Diarrhea ACE Detection assay was shown to be a rapid, sensitive, specific, and reliable diagnostic tool to directly detect the most common enteropathogens in stool samples
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