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Comparison of an immunochromatographic rapid test with enzyme-linked immunosorbent assay and polymerase chain reaction for the detection of Shiga toxins from human stool samples
Diagnostic Microbiology and Infectious Disease 59 (2007) 97 – 99 www.elsevier.com/locate/diagmicrobio
Comparison of an immunochromatographic rapid test with enzyme-linked immunosorbent assay and polymerase chain reaction for the detection of Shiga toxins from human stool samples Katharina Grif ⁎, Dorothea Orth, Manfred P. Dierich, Reinhard Würzner Department of Hygiene, Microbiology and Social Medicine, Innsbruck Medical University and Austrian Reference Centre for Enterohemorrhagic Escherichia coli, A-6020 Innsbruck, Austria Received 24 November 2006; accepted 26 March 2007
Abstract Rapid detection of enterohemorrhagic Escherichia coli is important for its successful treatment. We have evaluated the immunochromatographic Duopath Verotoxin-test™ for detection of Shiga toxins, in comparison with enzyme-linked immunosorbent assay and polymerase chain reaction, on 240 clinical human stool samples. The Duopath-test showed a lower sensitivity and specificity. © 2007 Elsevier Inc. All rights reserved. Keywords: EHEC; Duopath Verotoxin-test™; ELISA; PCR
Infections with enterohemorrhagic Escherichia coli (EHEC) are the major cause of hemolytic–uremic syndrome (HUS), the most common cause of acute renal failure in childhood (Karch et al., 2005). Detection of Shiga toxins (Stx) by means of enzymelinked immunosorbent assay (ELISA) and determination of stx genes by molecular methods are commonly implemented for the diagnosis of EHEC. Because these methods are very time consuming, a quicker “screening test” is required. There are some rapid screening tests available (Aldus et al., 2003; Capps et al., 2004); however, most of them are designed for testing food samples or bacterial cultures. Awareness of the limitations of the presently available tests has prompted the development of tests designed for screening human stool samples to diagnose patients suspected of suffering from HUS. The Duopath Verotoxin (DV)-test™ (Merck, Darmstadt, Germany) is a newly developed immunochromatographic rapid test for the qualitative identification of Stx1 and 2 using monoclonal antibodies. The DV-test™ was previously only
⁎ Corresponding author. Tel.: +43-512-900370780; fax: +43-512900373750. E-mail address: [email protected] (K. Grif). 0732-8893/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2007.03.022
evaluated for detection of Stx1 and 2 from cultures of human stool samples (Park et al., 2003). The aim of this study was to evaluate the DV-test™ with regard to the identification of the presence of Stx in direct routine screening. To this purpose, 240 clinical human stool samples were tested using 3 different methods: DV-test™, Premier EHECELISA, and polymerase chain reaction (PCR) (as reference method). Before performing the different assays, all stool specimens were suspended in 5 mL of enrichment broth (EHEC Direct Medium; Heipha, Heidelberg, Germany) and incubated at 37 °C for 18 to 24 h. Of the 240 human stool specimens tested with the DVtest™, 145 samples were tested using the standard protocol according to the manufacturer's instructions (Merck) and 95 samples using a modified protocol. For those samples tested according to the standard protocol, we included an enrichment step in the sample preparation. In the modified protocol, 0.25 mL of liquid or one pea size of nonliquid stool specimen was applied to 5 mL of saline and incubated for 5 min at room temperature. Stool–saline solution (0.1 mL) was transferred to 5 mL of enrichment broth and incubated at 37 °C overnight for 18 to 24 h. Enriched samples were processed according to the manufacturer's instructions and tested for toxins by means of EHEC-ELISA (Premier EHEC;
98
K. Grif et al. / Diagnostic Microbiology and Infectious Disease 59 (2007) 97–99
Meridian, Milan, Italy). PCR was used for detection of stx1 and stx2 genes (Friedrich et al., 2002). All EHEC isolates were serotyped (Ørskov and Ørskov, 1984) using diagnostic antisera from Statens Serum Institute, SSI, Copenhagen, Denmark. Twenty-two of the EHEC isolates (33%) belonged to the serogroup O157, the remaining 44 isolates were non-O157. This, however, did not influence the comparison, as expected (data not shown). Using PCR, 66 stool samples were found to be stx positive: 21 were positive for only stx1, 30 were positive for only stx2, and 15 were positive for both stx1 and stx2. Of the 145 human stool samples tested using the DVtest™ according to the standard protocol, 30 samples were found to be positive for only Stx1, 22 were positive for only Stx2 (Table 1), 4 of which for both. Thirteen and 3 samples gave borderline results for Stx1 and 2, respectively, yielding in both cases a high percentage of equivocal results and a low sensitivity, in particular, for Stx1 positivity, but also for Stx2positive stool samples. Using the modified protocol, the sensitivity for both Stx was markedly increased and even reached 100% for Stx2. The specificity, however, was lower using the modified protocol when compared with the results obtained using the standard protocol (Table 2). Compared with PCR results, the Premier EHEC-ELISA test yielded a sensitivity of 95.4%, a specificity of 97.2%, and positive or negative predictive values of 0.93 and 0.98, respectively (Tables 1 and 2). The ELISA test displayed a negative result for 2 samples, where the PCR detected stx genes. The phenomenon that Stx-positive cultures do not produce Stx and are thus negative in the ELISA has already been described (Zhang et al., 2005). On the other hand, 5 samples were positive in the Stx ELISA but found to be negative in the PCR analysis, which may be due to a cross-reaction. To date, there is only a single study describing an evaluation of the DV-test™ (Park et al., 2003). In the latter study, 41 known Stx-positive stool samples (31 EHEC O157 and 10 EHEC non-O157) were retrospectively tested using the standard protocol. The authors used colony sweeps from
Table 1 Stx-positive strains detected using the standard protocol of the DV-test™ and the EHEC-ELISA in comparison with PCR
Correct positive False positive Correct negative False negative Sensitivity (%) Specificity (%) Positive predictive value Negative predictive value
DV-test™ a
EHEC-ELISA
PCR
Stx1
Stx2
Stx
stx1
stx2
6 24 95 7 46.15 79.8 0.20 0.93
21 1 113 7 75.0 99.1 0.95 0.94
35 4 104 2 94.6 96.3 0.90 0.98
16 0 129 0 100 100 1 1
29 0 116 0 100 100 1 1
a The borderline results (13 for Stx1 and 3 for Stx2) are not shown in the table.
Table 2 Stx-positive strains detected using the modified protocol of the DV-test™ and the EHEC-ELISA in comparison with PCR
Correct positive False positive Correct negative False negative Sensitivity (%) Specificity (%) Positive predictive value Negative predictive value
DV-test™
EHEC-ELISA
PCR
Stx1
Stx2
Stx
stx1
stx2
16 14 61 4 80.0 81.3 0.53 0.94
16 30 49 0 100 62.0 0.35 1
28 1 65 1 96.6 98.5 0.97 0.98
20 0 75 0 100 100 1 1
16 0 79 0 100 100 1 1
the Sorbitol MacConkey (SMAC) agar plates as inoculum for the DV-test™, without any enrichment of the stool samples before application onto the SMAC plates. However, it was not reported as to whether the positive stool samples were artificially contaminated or whether they originated from patients (Park et al., 2003): there is a marked difference in the amount of inoculum in artificially contaminated stool samples compared with that in patient stool samples. This point is of particular significance when considered in the light of the known fact that the few EHEC bacteria in patient samples are sufficient to cause HUS, but that they are unable to grow on SMAC plates without enrichment (Karmali et al., 1985). All 41 Stx-positive stool specimens tested by Park et al. were found to be positive using the DV-test™. These results demonstrate that the DV-test™, applied using the standard protocol, works very well on EHEC-positive bacterial cultures. In their prospective study, Park et al. also investigated 250 stool samples without enrichment. They found only 2 EHEC-positive bacterial cultures on SMAC plates, which is not predictive. Furthermore, it is not clear as to whether or not the author used a reference method such as PCR or hybridization for the evaluation of the DV-test™. In our study, we used 2 different protocols for the DVtest™. However, the modified protocol of the DV-test™ did not result in a time benefit when compared with the EHECELISA analysis. In addition, the DV-test™ does not give any information regarding the intensity of the positive result. In contrast, the Premier EHEC-ELISA analysis gives a quantitative result and indicates weakly positive samples, which may prove to be negative. Epidemiologic studies have shown that EHEC expressing Stx2 are more prone to cause HUS than those producing Stx1 alone (Friedrich et al., 2002; Tarr et al., 2005). Thus, the advantage of the DV-test™ is the differentiation between Stx1 and 2, which can give important information regarding the pathogenicity of this EHEC strain. In conclusion, we found no advantage in the use of the DVtest™ standard protocol for routine screening of human stool samples, with a low sensitivity and specificity and a quite high percentage of equivocal results being observed. The modified protocol of the DV-test™ produced more reliable results for direct screening of clinical human stool samples.
K. Grif et al. / Diagnostic Microbiology and Infectious Disease 59 (2007) 97–99
However, this method provides no time benefit when compared with the Premier EHEC-ELISA analysis, which has a higher sensitivity and specificity in general. Thus, a rapid screening of clinical human stool samples for the presence of Stx, in particular, Stx2, has still to be developed. Acknowledgment This work was supported by Merck, Darmstadt, Germany. The authors thank C. Ortner and A. Rief for excellent technical assistance. References Aldus CF, Van Amerongen A, Ariens RM, Peck MW, Wichers JH, Wyatt GM (2003) Principles of some novel rapid dipstick methods for detection and characterization of verotoxigenic Escherichia coli. J Appl Microbiol 95:380–389. Capps KL, McLaughlin EM, Murray AW, Aldus CF, Wyatt GM, Peck MW, Van Amerongen A, Ariens RM, Wichers JH, Baylis CL, Wareing DR,
99
Boltoln FJ (2004) Validation of three rapid screening methods for detection of verotoxin-producing Escherichia coli in foods: interlaboratory study. J AOAC Int 87:68–77. Friedrich AW, Bielaszewska M, Zhang WL, Pulz M, Kuczius T, Ammon A, Karch H (2002) Escherichia coli harboring Shiga toxin 2 gene variants: frequency and association with clinical symptoms. J Infect Dis 185:74–84. Karch H, Tarr PI, Bielaszewska M (2005) Enterohaemorrhagic Escherichia coli in human medicine. Int J Med Microbiol 295:405–418. Karmali MA, Petric M, Lim C, Cheung R, Arbus GS (1985) Sensitive method for detecting low numbers of verotoxin-producing Escherichia coli in mixed cultures by use of colony sweeps and polymyxin extraction of verotoxin. J Clin Microbiol 22:614–619. Ørskov F, Ørskov I (1984) Serotyping of Escherichia coli. Meth Microbiol 14:43–112. Park AH, Kim HJ, Hixon DL, Bubert A (2003) Evaluation of the duopath verotoxin test for detection of Shiga toxins in cultures of human stools. J Clin Microbiol 41:2650–2653. Tarr PI, Gordon CA, Chandler WL (2005) Shiga-toxin–producing Escherichia coli and haemolytic uraemic syndrome. Lancet 365:1073–1086. Zhang W, Bielaszewska M, Friedrich AW, Kuczius T, Karch H (2005) Transcriptional analysis of genes encoding Shiga toxin 2 and its variants in Escherichia coli. Appl Environ Microbiol 71:558–561.
Comparison of an immunochromatographic rapid test with enzyme-linked immunosorbent assay and polymerase chain reaction for the detection of Shiga toxins from human stool samples Katharina Grif ⁎, Dorothea Orth, Manfred P. Dierich, Reinhard Würzner Department of Hygiene, Microbiology and Social Medicine, Innsbruck Medical University and Austrian Reference Centre for Enterohemorrhagic Escherichia coli, A-6020 Innsbruck, Austria Received 24 November 2006; accepted 26 March 2007
Abstract Rapid detection of enterohemorrhagic Escherichia coli is important for its successful treatment. We have evaluated the immunochromatographic Duopath Verotoxin-test™ for detection of Shiga toxins, in comparison with enzyme-linked immunosorbent assay and polymerase chain reaction, on 240 clinical human stool samples. The Duopath-test showed a lower sensitivity and specificity. © 2007 Elsevier Inc. All rights reserved. Keywords: EHEC; Duopath Verotoxin-test™; ELISA; PCR
Infections with enterohemorrhagic Escherichia coli (EHEC) are the major cause of hemolytic–uremic syndrome (HUS), the most common cause of acute renal failure in childhood (Karch et al., 2005). Detection of Shiga toxins (Stx) by means of enzymelinked immunosorbent assay (ELISA) and determination of stx genes by molecular methods are commonly implemented for the diagnosis of EHEC. Because these methods are very time consuming, a quicker “screening test” is required. There are some rapid screening tests available (Aldus et al., 2003; Capps et al., 2004); however, most of them are designed for testing food samples or bacterial cultures. Awareness of the limitations of the presently available tests has prompted the development of tests designed for screening human stool samples to diagnose patients suspected of suffering from HUS. The Duopath Verotoxin (DV)-test™ (Merck, Darmstadt, Germany) is a newly developed immunochromatographic rapid test for the qualitative identification of Stx1 and 2 using monoclonal antibodies. The DV-test™ was previously only
⁎ Corresponding author. Tel.: +43-512-900370780; fax: +43-512900373750. E-mail address: [email protected] (K. Grif). 0732-8893/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2007.03.022
evaluated for detection of Stx1 and 2 from cultures of human stool samples (Park et al., 2003). The aim of this study was to evaluate the DV-test™ with regard to the identification of the presence of Stx in direct routine screening. To this purpose, 240 clinical human stool samples were tested using 3 different methods: DV-test™, Premier EHECELISA, and polymerase chain reaction (PCR) (as reference method). Before performing the different assays, all stool specimens were suspended in 5 mL of enrichment broth (EHEC Direct Medium; Heipha, Heidelberg, Germany) and incubated at 37 °C for 18 to 24 h. Of the 240 human stool specimens tested with the DVtest™, 145 samples were tested using the standard protocol according to the manufacturer's instructions (Merck) and 95 samples using a modified protocol. For those samples tested according to the standard protocol, we included an enrichment step in the sample preparation. In the modified protocol, 0.25 mL of liquid or one pea size of nonliquid stool specimen was applied to 5 mL of saline and incubated for 5 min at room temperature. Stool–saline solution (0.1 mL) was transferred to 5 mL of enrichment broth and incubated at 37 °C overnight for 18 to 24 h. Enriched samples were processed according to the manufacturer's instructions and tested for toxins by means of EHEC-ELISA (Premier EHEC;
98
K. Grif et al. / Diagnostic Microbiology and Infectious Disease 59 (2007) 97–99
Meridian, Milan, Italy). PCR was used for detection of stx1 and stx2 genes (Friedrich et al., 2002). All EHEC isolates were serotyped (Ørskov and Ørskov, 1984) using diagnostic antisera from Statens Serum Institute, SSI, Copenhagen, Denmark. Twenty-two of the EHEC isolates (33%) belonged to the serogroup O157, the remaining 44 isolates were non-O157. This, however, did not influence the comparison, as expected (data not shown). Using PCR, 66 stool samples were found to be stx positive: 21 were positive for only stx1, 30 were positive for only stx2, and 15 were positive for both stx1 and stx2. Of the 145 human stool samples tested using the DVtest™ according to the standard protocol, 30 samples were found to be positive for only Stx1, 22 were positive for only Stx2 (Table 1), 4 of which for both. Thirteen and 3 samples gave borderline results for Stx1 and 2, respectively, yielding in both cases a high percentage of equivocal results and a low sensitivity, in particular, for Stx1 positivity, but also for Stx2positive stool samples. Using the modified protocol, the sensitivity for both Stx was markedly increased and even reached 100% for Stx2. The specificity, however, was lower using the modified protocol when compared with the results obtained using the standard protocol (Table 2). Compared with PCR results, the Premier EHEC-ELISA test yielded a sensitivity of 95.4%, a specificity of 97.2%, and positive or negative predictive values of 0.93 and 0.98, respectively (Tables 1 and 2). The ELISA test displayed a negative result for 2 samples, where the PCR detected stx genes. The phenomenon that Stx-positive cultures do not produce Stx and are thus negative in the ELISA has already been described (Zhang et al., 2005). On the other hand, 5 samples were positive in the Stx ELISA but found to be negative in the PCR analysis, which may be due to a cross-reaction. To date, there is only a single study describing an evaluation of the DV-test™ (Park et al., 2003). In the latter study, 41 known Stx-positive stool samples (31 EHEC O157 and 10 EHEC non-O157) were retrospectively tested using the standard protocol. The authors used colony sweeps from
Table 1 Stx-positive strains detected using the standard protocol of the DV-test™ and the EHEC-ELISA in comparison with PCR
Correct positive False positive Correct negative False negative Sensitivity (%) Specificity (%) Positive predictive value Negative predictive value
DV-test™ a
EHEC-ELISA
PCR
Stx1
Stx2
Stx
stx1
stx2
6 24 95 7 46.15 79.8 0.20 0.93
21 1 113 7 75.0 99.1 0.95 0.94
35 4 104 2 94.6 96.3 0.90 0.98
16 0 129 0 100 100 1 1
29 0 116 0 100 100 1 1
a The borderline results (13 for Stx1 and 3 for Stx2) are not shown in the table.
Table 2 Stx-positive strains detected using the modified protocol of the DV-test™ and the EHEC-ELISA in comparison with PCR
Correct positive False positive Correct negative False negative Sensitivity (%) Specificity (%) Positive predictive value Negative predictive value
DV-test™
EHEC-ELISA
PCR
Stx1
Stx2
Stx
stx1
stx2
16 14 61 4 80.0 81.3 0.53 0.94
16 30 49 0 100 62.0 0.35 1
28 1 65 1 96.6 98.5 0.97 0.98
20 0 75 0 100 100 1 1
16 0 79 0 100 100 1 1
the Sorbitol MacConkey (SMAC) agar plates as inoculum for the DV-test™, without any enrichment of the stool samples before application onto the SMAC plates. However, it was not reported as to whether the positive stool samples were artificially contaminated or whether they originated from patients (Park et al., 2003): there is a marked difference in the amount of inoculum in artificially contaminated stool samples compared with that in patient stool samples. This point is of particular significance when considered in the light of the known fact that the few EHEC bacteria in patient samples are sufficient to cause HUS, but that they are unable to grow on SMAC plates without enrichment (Karmali et al., 1985). All 41 Stx-positive stool specimens tested by Park et al. were found to be positive using the DV-test™. These results demonstrate that the DV-test™, applied using the standard protocol, works very well on EHEC-positive bacterial cultures. In their prospective study, Park et al. also investigated 250 stool samples without enrichment. They found only 2 EHEC-positive bacterial cultures on SMAC plates, which is not predictive. Furthermore, it is not clear as to whether or not the author used a reference method such as PCR or hybridization for the evaluation of the DV-test™. In our study, we used 2 different protocols for the DVtest™. However, the modified protocol of the DV-test™ did not result in a time benefit when compared with the EHECELISA analysis. In addition, the DV-test™ does not give any information regarding the intensity of the positive result. In contrast, the Premier EHEC-ELISA analysis gives a quantitative result and indicates weakly positive samples, which may prove to be negative. Epidemiologic studies have shown that EHEC expressing Stx2 are more prone to cause HUS than those producing Stx1 alone (Friedrich et al., 2002; Tarr et al., 2005). Thus, the advantage of the DV-test™ is the differentiation between Stx1 and 2, which can give important information regarding the pathogenicity of this EHEC strain. In conclusion, we found no advantage in the use of the DVtest™ standard protocol for routine screening of human stool samples, with a low sensitivity and specificity and a quite high percentage of equivocal results being observed. The modified protocol of the DV-test™ produced more reliable results for direct screening of clinical human stool samples.
K. Grif et al. / Diagnostic Microbiology and Infectious Disease 59 (2007) 97–99
However, this method provides no time benefit when compared with the Premier EHEC-ELISA analysis, which has a higher sensitivity and specificity in general. Thus, a rapid screening of clinical human stool samples for the presence of Stx, in particular, Stx2, has still to be developed. Acknowledgment This work was supported by Merck, Darmstadt, Germany. The authors thank C. Ortner and A. Rief for excellent technical assistance. References Aldus CF, Van Amerongen A, Ariens RM, Peck MW, Wichers JH, Wyatt GM (2003) Principles of some novel rapid dipstick methods for detection and characterization of verotoxigenic Escherichia coli. J Appl Microbiol 95:380–389. Capps KL, McLaughlin EM, Murray AW, Aldus CF, Wyatt GM, Peck MW, Van Amerongen A, Ariens RM, Wichers JH, Baylis CL, Wareing DR,
99
Boltoln FJ (2004) Validation of three rapid screening methods for detection of verotoxin-producing Escherichia coli in foods: interlaboratory study. J AOAC Int 87:68–77. Friedrich AW, Bielaszewska M, Zhang WL, Pulz M, Kuczius T, Ammon A, Karch H (2002) Escherichia coli harboring Shiga toxin 2 gene variants: frequency and association with clinical symptoms. J Infect Dis 185:74–84. Karch H, Tarr PI, Bielaszewska M (2005) Enterohaemorrhagic Escherichia coli in human medicine. Int J Med Microbiol 295:405–418. Karmali MA, Petric M, Lim C, Cheung R, Arbus GS (1985) Sensitive method for detecting low numbers of verotoxin-producing Escherichia coli in mixed cultures by use of colony sweeps and polymyxin extraction of verotoxin. J Clin Microbiol 22:614–619. Ørskov F, Ørskov I (1984) Serotyping of Escherichia coli. Meth Microbiol 14:43–112. Park AH, Kim HJ, Hixon DL, Bubert A (2003) Evaluation of the duopath verotoxin test for detection of Shiga toxins in cultures of human stools. J Clin Microbiol 41:2650–2653. Tarr PI, Gordon CA, Chandler WL (2005) Shiga-toxin–producing Escherichia coli and haemolytic uraemic syndrome. Lancet 365:1073–1086. Zhang W, Bielaszewska M, Friedrich AW, Kuczius T, Karch H (2005) Transcriptional analysis of genes encoding Shiga toxin 2 and its variants in Escherichia coli. Appl Environ Microbiol 71:558–561.