University of Groningen Shigella spp. and entero-invasive Escherichia coli van den Beld, Maaike
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(2) Chapter 3 Multicenter evaluation of molecular and culture-dependent diagnostics for Shigella spp. and entero-invasive Escherichia coli in the Netherlands. Maaike J.C. van den Beld1,2, Alexander W. Friedrich2, Evert van Zanten3, Frans A.G. Reubsaet1, A.M.D. (Mirjam) Kooistra-Smid2,3#, John W.A. Rossen2# on behalf of participating Medical Microbiological Laboratories4 Infectious Disease Research, Diagnostics and laboratory Surveillance, Centre for Infectious Disease Control, National Institute for Public Health and the Environment, Bilthoven, The Netherlands 2 Department of Medical Microbiology and Infection Prevention, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands 3 Department of Medical Microbiology, Certe, Groningen, The Netherlands 4 Participating Medical Microbiological Laboratories are listed in the Acknowledgements. 1. #. last two authors contributed equally. Journal of Microbiological Methods 131 (2016) 10-15. Maaike van den Beld.
(3) PART I CHAPTER 3. MULTICENTER EVALUATION OF DIAGNOSTICS. Abstract. Introduction. An inter-laboratory collaborative trial for the evaluation of diagnostics for detection and identification of Shigella species and Entero-invasive Escherichia coli (EIEC) was performed. Sixteen Medical Microbiological Laboratories (MMLs) participated. MMLs were interviewed about their diagnostic methods and a sample panel, consisting of DNA-extracts and spiked stool samples with different concentrations of Shigella flexneri, was provided to each MML. The results of the trial showed an enormous variety in culture-dependent and molecular diagnostic techniques currently used among MMLs. Despite the various molecular procedures, 15 out of 16 MMLs were able to detect Shigella species or EIEC in all the samples provided, showing that the diversity of methods has no effect on the qualitative detection of Shigella flexneri. In contrast to semi quantitative analysis, the minimum and maximum values per sample differed by approximately five threshold cycles (Ct- value) between the MMLs included in the study. This indicates that defining a uniform Ct-value cut-off for notification to health authorities is not advisable.. Shigella species and entero-invasive Escherichia coli (EIEC) cause gastrointestinal diseases, ranging from mild diarrheal episodes to dysentery with complications. Although the incidence of diarrheal diseases is declining worldwide because of improved sanitary and hygienic conditions, 190 million cases of shigellosis are still reported worldwide each year. Shigellosis results in approximately 65,000 deaths per year, mostly in children under five years of age [1]. In the Netherlands, health authorities receive on average 450 notifications of confirmed shigellosis each year [2]. To calculate the real number of cases, a correction for under-estimation of incidence is applied, and the number of real cases is calculated to be 7561, with an annual disease-burden of 196 DALY (disability-adjusted life year) [2]. DALY is defined as the sum of the number of years lost due to premature mortality and the number of healthy years lost due to morbidity [2]. In contrast to shigellosis, in most countries there is no obligation to report EIEC infections to health authorities. Therefore, no global or regional information is available about the disease burden of EIEC. Shigella and EIEC both evolved on multiple occasions from ancestral E. coli, and they have acquired a large virulence plasmid (pINV) [3, 4]. The presence of multiple virulence genes on the invasion plasmid pINV and the subsequent pathoadaptation are the cause of the invasive nature of Shigella species and EIEC. Due to the close genetic relatedness of Shigella species and EIEC, extensive methods are required to distinguish them from each other and from noninvasive E. coli [5]. In the Netherlands, most Medical Microbiological Laboratories (MMLs) screen stool samples for Shigella and EIEC using molecular procedures, which target the antigen H (ipaH) genes, present on the pINV and the chromosome. Because these virulence genes are present in both Shigella species and EIEC, tests based on this target cannot distinguish Shigella species from EIEC [6]. Hence, the Dutch guidelines for control of infectious diseases require a confirmation of the molecular test results by culture before Shigella infections are notified to health authorities [7]. The genus Shigella comprises four species, Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei and classification is based on phenotypic and antigenic characteristics. To distinguish between the species and serotypes of Shigella and EIEC, culturing on selective media followed by extensive phenotypical testing and serotyping is required. However, this approach has several drawbacks: (i) the selection of Shigella species in culture is insensitive; (ii) other E. coli bacteria naturally present in stools hinder the selection of EIEC from stool samples; (iii) extensive phenotypical testing and serotyping are laborintensive and costly and are, therefore, not routinely performed by MMLs. Because of these drawbacks, some MMLs in the Netherlands report shigellosis infections to health authorities based on a cut-off in threshold cycle (Ct) value obtained from the molecular screening.. 62. 63. 3.
(4) PART I CHAPTER 3. A national multicenter study, “Invasive Bacteria E.coli-Shigella Study” (IBESS), has been established to gain further insight into diagnostics, prevalence, clinical relevance and the risks of Shigella species and EIEC infections to public health. As this study is highly dependent on diagnostics performed by the participating MMLs, better understanding of the diagnostic methods used by the MMLs is required. For this reason, a collaborative trial was initiated, to evaluate the diagnostic methods used and to compare sample panel results from the different MMLs. The results of this collaborative trial are reported in this paper.. Material and Methods Evaluation of culture dependent diagnostic methods Two digital surveys, which comprised questions about the culture-dependent and molecular methods used to detect, identify and test the antimicrobial susceptibility of isolated Shigella species and EIEC from stool samples (Supplementary Files 1 and 2), were sent to 16 Dutch MMLs. For the comparison of the culture and identification techniques used, pie charts were created using GraphPad Prism 6.05 (La Jolla, USA). Molecular diagnostics and inter-laboratory evaluation A test panel was sent to the participating MMLs consisting of ten standardized and blinded samples; five DNA-extracts and five spiked stool samples. For DNA-extracts, type strain Shigella flexneri 2a, CIP 82.48 (Collection of Institute Pasteur, Paris, France) was used to prepare a ten-fold dilution series, ranging from 1.295*105fg DNA/µl to 1.295*101 fg DNA/ µl. For the preparation of the spiked stool samples, twenty stool samples were pooled to obtain enough volume to prepare the samples for the collaborative trial. All stool samples were confirmed negative for Salmonella enterica, Campylobacter jejuni, Giardia lamblia, Cryptosporidium, Dientamoebe fragilis, Entamoeba histolytica, Norovirus type I and II, Adenovirus type 40 and 41, Rotavirus, and Stx1- and Stx2 genes using real-time PCR assays in the laboratory of Certe, Groningen. The pooled stool samples were liquefied by adding 50% weight/volume NucliSENS easyMag Lysis buffer (BioMérieux, Boxtel, the Netherlands) and homogenized by vigorous mixing. Remaining debris was sedimented by gravity and the resulting supernatant was used to prepare the spiked samples. The stool samples were spiked with a ten-fold dilution series of a suspension of a clinical S. flexneri 2a strain, resulting in final concentrations ranging from 1.32*105 CFU/ml to 1.32*101 CFU/ml stool. All samples in the test panel were labeled with a non-identifiable code. 50 µl of the DNA-extracts and 500 µl of the spiked stool samples were provided to each participating MML. Homogeneity and stability were assessed by intra-run and inter-run comparison and this was conducted at one of the sixteen MMLs (Certe, Groningen). Inter-run comparison was performed over a period of four weeks, to assess the stability of the samples until the last results were received from the participating MMLs. Variance coefficients (Cv) were calculated from the intra-run and inter-run results.. 64. MULTICENTER EVALUATION OF DIAGNOSTICS. Each MML was coded with a letter to ensure anonymity. The MMLs were instructed to perform a DNA extraction on the stool samples, using their diagnostic extraction protocol. Subsequently, they screened both their prepared and the received DNA extracts for the presence of Shigella species using their diagnostic amplification protocols. Details of the extraction and amplification protocols for each of the MMLs are shown in Table 1. For comparison of the semi qualitative results of the molecular procedures, the mean, standard deviation, minimum and maximum of Ct-values per sample and R-squared correlation coefficients for each sample series were calculated using Excel and R Studio (Boston, USA). Within a ten-fold dilution series, a linear correlation between dilutions and Ct-values was expected, as every 10 fold dilution theoretically increases the Ct by 3.3 [8]. Linearity of the dilution series was determined with line plots, made in R Studio using the graphics package, version 3.2.0 (Boston, USA).. Results Evaluation of culture dependent diagnostic methods All MMLs (n=16) responded to the surveys and participated in the collaborative trial. The culture and identification techniques used by the MMLs are shown in Figure 1. Although only a few enrichment broths and selective agar plates for selection of Shigella spp. are available, a large diversity was observed in the combination of broths, plates, and protocols used by the MMLs (Figure 1A, 1B, 1C, 1D). Similar observations were made for identification and serotyping techniques (Figure 1E, 1F, 1H, 1I, 1J, 1K). Each MML used a unique combination of protocols for selection from stool samples, and subsequent identification and serotyping of Shigella species. However, for susceptibility testing, only a few techniques and protocols were used (Figure 1G). A limited number of MMLs performed culturing of EIEC from stool samples, and although some of them used the same plates as for selection of Shigella species, different protocols were used (Figure 1B). Molecular diagnostics and inter-laboratory evaluation For molecular methods, 14 out of 16 MMLs used a real-time PCR method targeting the ipaHgene for diagnosing Shigella species and EIEC. However, different extraction methods, amplification platforms and PCR master mixes for the real-time PCR were used. Each MML used a unique combination of techniques (Table 1). Two MMLs used a commercially purchased kit based on real-time PCR procedures with undisclosed targets. Variance coefficients from the intra-run and inter-run tests are displayed in Supplementary Table S1.. 65. 3.
(5) 66. BioMérieux, Boxtel, the Netherland. b Applied Biosystems, Bleiswijk, the Netherlands .c Roche Diagnostics, Almere, the Netherlands.d Serosep, Limerick, Ireland. e Unknown, because of the use of a commercially available kit or system. f QIAGEN, Venlo, the Netherlands. g Agilent Technologies, Amstelveen, the Netherlands. h Isogen Life Science, De Meern, the Netherlands. i Invitrogen, Bleiswijk, the Netherlands. j TIB Molbiol, Berlin, Germany. a. Unknown e 20 Bacterial Gastroenteritis Lightmix j Unknown e Probes master c MagNA pure compact system c P. LightCycler 480 c. 64 25 [9] IpaH gene TaqMan Fast Advanced Master Mix b NucliSENS® EasyMag a O. 7500 Fast Real-Time PCR b. 64 20 [9] IpaH gene Platinum Quantitative PCR SuperMix-UDG i Arrow h N. Rotor-Gene Q f. 63 30 [9] IpaH gene TaqMan Fast Advanced Master Mix b NucliSENS® EasyMag a M. LightCycler 480 c MagNA pure compact system c L. 7500 Fast Real-Time PCR MagNA pure compact system K. 7500 Fast Real-Time PCR b. 64 15 [9] IpaH gene Probes master c. 64. 63 15. 20 In-house design. In-house design IpaH gene. TaqMan Fast Virus 1-Step Master IpaH gene Mix b. c. Probes master. b. LightCycler 480 c MagNA pure compact system c J. c. 64 20 [9] IpaH gene TaqPath 1-step RT-qPCR Master Mix b NucliSENS® EasyMag a I. 7500 Fast Real-Time PCR b. 64 30 [9] IpaH gene Brilliant III Ultra-Fast QPCR Master Mix g QIAsymphony f H. Rotor-Gene Q f. 64 25 [9] IpaH gene TaqMan Fast Universal PCR Master Mix b MagNA pure compact system c G. 7500 Fast Real-Time PCR b. 64 25 [9] IpaH gene TaqMan Fast Universal PCR Master Mix b NucliSENS® EasyMag a F. 7500 Fast Real-Time PCR b. 64. Unknown e 25. 25 [9]. Unknown e Unknown e. IpaH gene QuantiTect Multiplex PCR NoROX kit f. EntericBio d. QIAsymphony f E. LightCycler 480 c EntericBio d D. Rotor-Gene Q f. 64 30 [9] IpaH gene c. Probes master NucliSENS® EasyMag a C. LightCycler 480 c. 101 30 [10] IpaH gene TaqMan Fast Advanced Master Mix b MagNA pure compact system c B. 7500 Fast Real-Time PCR b. 64 25 [9] IpaH gene TaqMan Universal PCR Master Mix b NucliSENS® EasyMag a A. 7500 Fast Real-Time PCR b. Mastermix DNA extraction platform. PCR platform. MULTICENTER EVALUATION OF DIAGNOSTICS. MML. Table 1 Overview of the equipment, methods and primers used by the participating MMLs. Target. Primers from/based on reference. Reaction vol PCR (µl). Amplicon size (bp). PART I CHAPTER 3. A.. Enrichment broths. B.. Protocols for culturing of EIEC. None. No protocol. Selenite broth. With Shigella plates. Shigella broth. Other protocol T o ta l= 1 6. T o ta l= 1 6. C.. Combinations of agar plates. D.. Picked colonies from selective agar plates Shigella-lik e All Other protocol. only SS-agar only Hektoen only chromSS only one other. Total=16. 3. Hektoen, SS-agar Hektoen, SS-agar,ChromSS Hektoen, SS -agar, XLD XLD, other T o ta l= 1 6. E.. Combinations of identification techniques. F.. Combinations of tests in phenotyping. Only Bruker MS. Only API 20E. Bruker MS, phenotyping. TSI, urea. Bruker MS, Vitek 2/XL. TSI, urea, lysine. Bruker MS, Vitek 2/XL, phenotyping. TSI, urea, lysine,. Only Vitek MS. motility, indole, gas. Vitek MS, phenotyping. TSI, urea, lysine,. Vitek 2/XL, phenotyping. motility , indole, ONPG. T o ta l= 1 6. G.. Susceptibility testing. T o t a l= 7. H.. Serotyping used for Shigella species. Vitek 2/XL. Only Shigella serotyping. Agar disc diffussion. Shigella and E. coli serotyping. BD Phoenix. None Unknown, contracted out. T o ta l= 1 6. T o ta l= 1 6. I.. Serotyping used for EIEC None Shigella and E. coli O-typing E. coli O-typing T o ta l= 1 6. J.. Shigella antisera Polyvalent A, B, C, D. Polyvalent B,D Polyvalent A,B,C,D, monovalent S. flex, S. boyd Unknown, contracted out. T o ta l= 1 4. K.. E. coli O-typing antisera O157* STAT_EHEC* EIEC polyvalent 7, 8 O26, O91, O103, O111*. T o t a l= 6 * S T E C a s s o c ia t e d O - t y p e s , n o t E I E C a s s o c ia t e d O - t y p e s. Figure 1 Used culture and identification techniques, displayed as fractions from totals SS-agar = Salmonella-Shigella agar; Hektoen = Hektoen Enteric Agar; chromSS = chromogenic Salmonella-Shigella agar; XLD = xylose lysine deoxycholate agar; Bruker MS = Matrix-assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectronomy performed using an Microflex™ apparatus with the MALDI Biotyper software, Bruker Daltonik GmbH, Bremen, Germany; Vitek 2/XL = VITEK 2 or VITEK2 XL in combination with GN cards, BioMérieux, Boxtel, the Netherlands; Vitek MS = MALDI-TOF mass spectronomy performed using a VITEK MS with the IVD-CE marked database, BioMérieux, Boxtel, the Netherlands; API 20 E, BioMérieux, Boxtel, the Netherlands; TSI = Triple Sugar Iron agar; BD Phoenix = BD Phoenix™ Automated Microbiology System, Becton, Dickinson and Company, New Jersey, USA; STAT_EHEC = ImmunoCard STAT! EHEC, Meridian Bioscience Inc., Veghel, the Netherlands.. 67.
(6) PART I CHAPTER 3. MULTICENTER EVALUATION OF DIAGNOSTICS. Ct−value. 35. 30. 45. Results feces samples lab A−H. MML. 45. I. A. B. J. B. C. 40. D. K. 40. L. E. F. N. F. 35. O P. H. mean. mean. mean. 2SD. 30. 2SD. 30. J 40. 35. 30. 25. 25. 20. 20. 20. 20. 15. 15. 15. 15. 1.3*10^3 fg/µl DNA. 1.3*10^2. 1.3*10^1. 1.3*10^5. 1.3*10^4. 1.3*10^3. 1.3*10^2. 1.3*10^1. fg/µl DNA. Figure 2 Results of DNA extracts in line plots, plotted per MML in relation to the mean and plus or minus 2 standard deviation Results of MMLs that were not linear were considered as outliers and were excluded from the calculations of mean, standard deviation, and minimum and maximum Ct-values (Supplementary Table S2). This was the case for the results of the DNAextracts of MML D and the results of stool samples of MML A, D, and N.. 1.3*10^5. N O mean. 2SD. 25. 1.3*10^4. L. P. 25. 1.3*10^5. K M. G. H. MML I. D. M 35. 45. C. E G. Results feces samples lab I−P. MML. A. Ct−value. 40. MML. Ct−value. 45. Results DNA extracts lab I−P. Ct−value. Results DNA extracts lab A−H. 1.3*10^4. 1.3*10^3. 1.3*10^2. 1.3*10^1. CFU/ml spiked stool. 3. 2SD. 1.3*10^5. 1.3*10^4. 1.3*10^3. 1.3*10^2. 1.3*10^1. CFU/ml spiked stool. Figure 3 Results of stool samples in line plots, plotted per MML in relation to the mean and plus or minus 2 standard deviation. Discussion and conclusions All MMLs, except MML D, detected Shigella species or EIEC in all provided DNA-extracts and stool samples (Supplementary tables S3 and S4). The individual results in Ct-values of each MML in relation to the mean and two standard deviations are presented in Figure 2 and Figure 3. The results of all MMLs, except for MML D, demonstrated a high correlation between Ct-values and concentration of the DNA-extracts (Figure 2) and the R-squared correlation coefficients were 0.99 (Supplementary Table S3). For the spiked stool samples, the expected 3.3 Ct difference between the mean measured concentrations could only be observed for the three highest concentrations. However, for all MMLs the two lowest concentrations of the spiked stool samples did not display linearity, as the mean differences in Ct-values were 5.21 and 2.93 (Figure 3). Obviously, this influenced the R-squared correlation coefficients, which were lower than the correlation coefficients of the DNA-extracts (Supplementary Table S4).. 68. There is considerable variety in culture-dependent diagnostic protocols; in this study, each MML used a unique combination of techniques. Performance testing of the culturedependent methods was not within the scope of this study. All MMLs used a real-time PCR technique for molecular detection of Shigella species and EIEC in stool samples, but each MML used a different combination of extraction and amplification platforms, PCR master mixes, primers and probes, and reaction volumes. Nevertheless, the qualitative results were similar. Every MML, except MML D, detected Shigella species or EIEC in all provided DNA-extracts and stool samples. MML D showed a negative result for the lowest concentration of the spiked stool sample, indicating that their technique has a lower sensitivity. Furthermore, the Ct-values of MML D, for both the DNA-extracts and the spiked stool samples, showed less correlation with concentration than the results of other MMLs. Because the tests were not repeated, it is not clear if sample handling, human errors, or factors related to the equipment or assay were the cause for this observation. MML D used the EntericBio assay, which does not include a. 69.
(7) PART I CHAPTER 3. DNA extraction and purification process; PCR is performed directly on chemical and heattreated fecal suspensions. However, the observed discrepancy in Ct values cannot solely be explained by the absence of a DNA extraction process, because the Ct-values of MML D for the DNA-extract samples also showed less correlation with concentration than the Ct-values of the other MMLs . The lowest concentration of S. flexneri in the spiked stool samples did not result in the highest Ct -value in the test results of MMLs A and N. Sample-handling errors of the two stool samples with the lowest concentration may provide an explanation for this observation. The molecular procedures of most MMLs displayed, as expected, a high correlation between Ct-values and concentration. However, for all MMLs, the two stool samples with the lowest concentrations of S. flexneri did not result in the expected 3.3 Ct difference, despite the approximate 3.3 Ct difference between every dilution was being observed in an initial test panel, tested by Certe. In order to obtain enough volume for the collaborative trial these spiked stool samples were remade and scaled-up to larger volumes. As all MMLs show the same discrepancies in Ct -values at the two lowest concentrations tested in the collaborative trial, the cause for this phenomenon may have arisen from the volume increase required for the collaborative trial sample preparation. An alternative explanation may be that PCR is a stochastic process, especially at low concentrations of the target [11]. The large differences in minimum and maximum Ct-values obtained for the test panel (Supplementary Table S2) show that an inter-laboratory comparison of absolute bacteria quantities in samples based on Ct-values only, is not possible. Therefore, defining a uniform cut-off based on Ct-value for reporting the presence of EIEC or Shigella species is not advisable. If a cut-off needs to be defined it should be expressed in real quantitative, international units, or alternatively in CFU or ng DNA per ml.. MULTICENTER EVALUATION OF DIAGNOSTICS. Acknowledgements The following MMLs participated in this pilot study. Listing is in random order and includes a named contact for each MML: - L. E. S. Bruijnesteijn van Coppenraet, Isala, Zwolle; - A. P. van Dam, OLVG General Hospital, Amsterdam; - I. Linde, Amsterdam Health Service, Amsterdam; - L. C. Smeets, Reinier de Graaf Group, Delft; - J. J. Verweij, Elisabeth Tweesteden Hospital, Tilburg; - L. J. M. van Mook, Amphia, Breda; - A. A. Demeulemeester, SHL-Group, Etten-Leur; - J. H. B. van de Bovenkamp, PAMM Laboratory for Medical Microbiology, Veldhoven; - K. Waar, Izore, Leeuwarden; - A. M. C. Bergmans, Laboratory of Medical Microbiology Roosendaal - Bergen op Zoom, Roosendaal; - D. L. J. Hess, LabMicTA, Laboratory for Medical Microbiology and Public Health, Hengelo; - E. Reinders, St. Antonius Hospital Nieuwegein, Nieuwegein; - S. Svraka-Latifovic, Tergooi Hospital, Hilversum; - C. F. M. Linssen, Zuyderland Medical Centre, Heerlen We would like to thank Rose Maase from the Centre for Infectious Disease Research, Diagnostics and Screening of the National Institute for Public Health and the Environment for editing the manuscript.. In conclusion, currently used diagnostics for detection and identification of Shigella species and EIEC in the Netherlands are very diverse. Each MML enrolled in the study, uses a unique combination of culture-dependent and molecular procedures. These different combinations of molecular techniques have no consequence for semi qualitative detection of Shigella species and EIEC. However, due to differences in absolute Ct-values between MMLs, defining a uniform cut-off based on Ct-values for reporting to health authorities is not recommended.. 70. 71. 3.
(8) PART I CHAPTER 3. References Pires, S.M., et al., Aetiology-specific estimates of the global and regional incidence and mortality of diarrhoeal diseases commonly transmitted through food. PLoS One, 2015. 10(12): p. e0142927. 2. van Lier, A., et al., Disease Burden of 32 Infectious Diseases in the Netherlands, 2007-2011. PLoS One, 2016. 11(4): p. e0153106. 3. Maurelli, A.T., Black holes, antivirulence genes, and gene inactivation in the evolution of bacterial pathogens. FEMS Microbiol Lett, 2007. 267(1): p. 1-8. 4. Lan, R., et al., Molecular evolutionary relationships of enteroinvasive Escherichia coli and Shigella spp. Infect Immun, 2004. 72(9): p. 5080-8. 5. van den Beld, M.J. and F.A. Reubsaet, Differentiation between Shigella, enteroinvasive Escherichia coli (EIEC) and noninvasive Escherichia coli. Eur J Clin Microbiol Infect Dis, 2012. 31(6): p. 899-904. 6. Hartman, A.B., et al., Sequence and molecular characterization of a multicopy invasion plasmid antigen gene, ipaH, of Shigella flexneri. J Bacteriol, 1990. 172(4): p. 1905-15. 7. RIVM. LCI-richtlijn Shigellose. 2011; Available from: http://www.rivm.nl/Documenten_en_publicaties/Professioneel_ Praktisch/Richtlijnen/Infectieziekten/LCI_richtlijnen/LCI_richtlijn_Shigellose. 8. Bustin, S.A. and T. Nolan, A-Z of Quantitative PCR, ed. S.A. Bustin. 2006, La Jolla, USA: International University Line. 882. 9. Vu, D.T., et al., Detection of Shigella by a PCR assay targeting the ipaH gene suggests increased prevalence of shigellosis in Nha Trang, Vietnam. J Clin Microbiol, 2004. 42(5): p. 2031-5. 10. de Boer, R.F., et al., Improved detection of five major gastrointestinal pathogens by use of a molecular screening approach. J Clin Microbiol, 2010. 48(11): p. 4140-6. 11. Weusten, J. and J. Herbergs, A stochastic model of the processes in PCR based amplification of STR DNA in forensic applications. Forensic Sci Int Genet, 2012. 6(1): p. 17-25.. MULTICENTER EVALUATION OF DIAGNOSTICS. Supplementary Material. 1.. Supplementary Table 1 Variance coefficients from intra-runs, inter-runs, homogeneity and stability Sample. Cv intra-run (homogeneity). Cv 4 inter-runs (stability). 1.3*105 fg/µl DNA. 0.4%. 1.3*104 fg/µl DNA. 0.3%. 0.3% 0.2%. 1.3*103 fg/µl DNA. 0.5%. 1.1%. 1.3*102 fg/µl DNA. 0.3%. 1.3*101 fg/µl DNA. 0.9%. 0.5% 0.3%. DNA extracts a. 3. Suspensions spiked in stool samples b. a. 1.3*105 CFU/ml stool. 0.2%. 1.3*104 CFU/ml stool. 0.03%. 1.3*103 CFU/ml stool. 0.4%. 1.3*102 CFU/ml stool. 0.3%. 1.3*101 CFU/ml stool. 1.4%. 1.5% 1.2% 0.9% 1.0% 0.9%. for intra-run, n=5. for intra-run, n=2. b. Supplementary Table 2 Calculation of mean, standard deviation, minimum, and maximum of Ct-values per sample Concentration. Mean. Standard deviation. Minimum. Maximum. 19.9 23.3 26.7 30.0 33.6. DNA extracts a 1.3*105 fg/µl DNA. 18.0. 1.6. 14.6. 1.3*104 fg/µl DNA. 21.5. 1.5. 18.5. 1.3*103 fg/µl DNA. 24.6. 1.6. 21.5. 1.3*102 fg/µl DNA. 28.1. 1.5. 25.1. 1.3*101 fg/µl DNA. 31.5. 1.4. 28.6. Suspensions spiked in stool samples b. a. 72. 1.3*105 CFU/ml stool. 16.9. 1.7. 14.5. 19.3. 1.3*104 CFU/ml stool. 20.2. 1.8. 17.2. 1.3*103 CFU/ml stool. 23.5. 1.6. 20.7. 1.3*102 CFU/ml stool. 28.7. 1.6. 25.9. 22.7 25.7 31.7. 1.3*101 CFU/ml stool. 31.6. 1.8. 28.5. 34.7. Ct-values of MML D excluded from calculations. b Ct-values of MMLs A, D and N excluded from calculations.. 73.
(9) PART I CHAPTER 3. MULTICENTER EVALUATION OF DIAGNOSTICS. Supplementary Table 3 Ct-values of provided DNA-extracts of each MML, including R-squared correlation coefficients MML. Supplementary File 1 Digital survey number 1 Supplementary File 1 Digital survey number 1. A1 Ct-value. A2 Ct-value. A3 Ct-value. A4 Ct-value. A5 Ct-value. R-squared correlation coefficient. A. 30.5. 20.4. 16.8. 27.2. 23.2. 0.9981. B. 32.8. 23.1. 19.4. 29.3. 26.2. 0.9988. This survey is an assessment of the used diagnostics for detection and identification of Shigella and Entero-invasive Escherichia coli (EIEC).. C D E F G H I J K L M N O P. 32.4. 22.1. 18.8. 28.9. The results of this survey are used as a starting point for IBESS (Invasive Bacteria E. coli-Shigella Study), which is a cross-sectinal studiy into Shigella and EIEC.. 26.8. 22.3. 40.5. 0.9370. It is necessary for the research group of IBESS to be able to reduce the results of this survey to individual laboratories. However, the results will be anonymized before oral or written publication.. 29.4. 18.5. 15.2. 25.6. 30.8. 20.6. 17.0. 27.1. 31.8. 21.6. 18.2. 28.6. 25.5 28.5 22.2 23.7 24.6. 0.9999. 45.0. 28.6. 18.5. 14.6. 25.1. 21.5. 0.9989. 32.6. 22.6. 19.3. 29.5. 22.8. 19.5. 29.6. 32.1. 21.5. 18.2. 28.1. 30.9. 22.7. 18.5. 28.7. 32.1. 22.1. 18.9. 28.8. 30.0. 20.0. 16.5. 26.7. 32.1. 22.2. 19.0. 29.1. 25.9 26.5 24.7 25.1 25.4 22.4 25.6 26.7. 0.9997. 32.5. 33.6. 23.3. 19.9. 30.0. Diagnostics of Shigella and EIEC for IBESS, version 2. '8 *hich colonies from the (s)elective agarplates for Shigella are followed up? Only colonies suspect for Shigella All colonies Other, specified below:. '' Do you have a protocol for the culturing of EIEC from fecal samples? Yes. 0.9996. Diagnostics of Shigella and EIEC for IBESS, version 2. 0.9994. Bo. ') Which selection method is used according your protocol for culturing of EIEC?. 0.9987. . ' Who is completing this survey? (please fill in name). 3. *use the same (s)elective agarplates as we use for the selection of Shigella species *use a different selection method, specified below:. ) Which laboratory (or laboratories) do you represent?. 0.9979 0.9981. + Which technique do you use for molecular diagnostics?
(10) al, -. 0.9889. - &, -with. / !. 0.9999. - &, -with0-.!. 0.9937. A commercially available kit/system or another technique. Specify below. Diagnostics of Shigella and EIEC for IBESS, version 2. - &, -with& !. The questions below are about indentification of Shigella and/or EIEC strains that you have isolated from fecal samples.. 0.9996 4 Which platform do you use for your real-time PCR on Shigella and/or EIEC? (For example: Roche Lightcycler 480, Applied Biosystems 7500, Biorad CFX96 Touch). 0.9998. '+ When you have isolated a colony suspected for Shigella and/or EIEC, do you use molecular diagnostics directly on the isolate for (a part of the) identification ? Bo$we do not use molecular diagnostics for identification of colonies suspect for Shigella and/or EIEC. Yes$ do use molecular diagnostics directly on the isolate. For this, we use the same target(s) that we use for detection of Shigella/EIEC in fecal samples.. ; Which target(s) do you use for detection of Shigella and/or EIEC? (multiple answers are possible). Yes, we do use molecular diagnostics directly on the isolate. For this, we use other target(s) that we use for detection of Shigella/EIEC in fecal samples.. ! ?e. Supplementary Table 4 Ct-values of stool samples of each MML, including R-squared correlation coefficients. a. MML. B1 Ct-value. B2 Ct-value. B3 Ct-value. B4 Ct-value. B5 Ct-value. R-squared correlation coefficient. A. 19.0. 33.8. 37.0. 22.5. 0.8417. B C D. 21.3. 33.2. 29.9. 24.3. 19.8. 31.0. 28.4. 23.0. 15.8 17.8 17.5. 26.7. neg. 40.7. 29.5. 22.8. 0.9012. E. 19.3. 30.9. 27.8. 22.1. 14.8. 0.9907. F G H I J K L M N O P. 19.0. 30.4. 27.4. 22.5. 15.5 16.7 14.5 17.9 18.5 14.6 17.4 19.3 21.9 16.1 19.3. 0.9949. a. 19.9. 30.5. 27.7. 22.7. 17.3. 28.5. 26.4. 21.4. 21.8. 32.9. 30.3. 24.4. 21.7. 32.5. 29.5. 24.8. 17.3. 29.2. 25.9. 20.7. 20.2. 31.7. 28.8. 24.0. 22.7. 34.7. 30.2. 25.7. 24.7. 32.3. 33.7. 28.2. 20.4. 31.2. 28.7. 23.7. 22.4. 34.3. 31.7. 25.7. Missing value of negative sample excluded from calculation of R-squared correlation coefficient.. 0.9793. 0.9865 0.9851. '; When you have isolated a colony suspected for Shigella and/or EIEC, do you use one or more of the following systems for (a part of the) identification? &ultiple answers are possible > Which primers and probes (if applicable) do you use for detection of Shigella and/or EIEC?. "/96 .@0 % /. -
(11) ! &;A+A 5. % )2<6. ,';A+A 5. ", D. ,);A+A 5. a. '4 *hich do you use in your molecular diagnostic test(s) for identification directly on the isolate?. Other (specify below). 0 ! &;A+A 5. 0.9893. 0.9906. unknown, I use a commercially available kit/system. B" Your used primer and probe sequences will never made public. They are only used to elucidate possible discrepancies during the inclusion period of IBESS.. None of the systems above. '> When you have isolated a colony suspected for Shigella and/or EIEC, do you useconventional phenotypic tests for (a part of the) identification? Yes. Diagnostics of Shigella and EIEC for IBESS, version 2 The questions below are about culturing Shigella and/or EIEC from fecal samples.. Bo. ': Which conventional phenotypic test(s) do you use for identification of Shigella and/or EIEC? /ultiple answers are possible . orG . : Do you use an enrichment broth for culturing Shigella and/or EIEC(. Ha. Bo$ inoculate the fecal samples directly on the (s)elec
(12) agar! esf and2r . e. Yes$ use an enrichment broth. The (s)elective agarplates are only inoculated from this enrichment broth.. Motility. 0.9942. yes, we use an enrichment broth. The (s)elective agarplates are inoculated from this enrichment broth, and we inoculate the. 63 D3 6 . 0.9885. @ther, specified below:. fecal samples direclty on these plates.. 0.9922 0.9925. 7 *hich enrichment broth do you use? /ultiple answers are possible . 0.9012 0.9924 0.9836. Other specify below . '7 When you have isolated a colony suspected for Shigella and/or EIEC, do you use serotyping for (a part of the) identification? Yes Bo. = Which selective agarplates do you use for the culturing of Shigella species from fecal samples? (multiple answers are possible) ?9? 9 . Diagnostics of Shigella and EIEC for IBESS, version 2. & <6 <3 63 D3 & . PAY ATTENTIONIThe questions below are about used serotyping techniques when a Shigella strain is isolated, not an EIEC strain!. Other, specified below:. 74. 75.
(13) PART I CHAPTER 3. MULTICENTER EVALUATION OF DIAGNOSTICS. Supplementary File 2 Digital survey number 2 Supplementary File 2 Digital survey number 2. '= When you have isolated a colony suspect for Shigella species, which serotyping techiques do you use?. ); *hich Shigella sonnei antisera do you use for your classical Shigella serotyping? (Multiple answers are possible . *do not use serotyping techniques when a colony suspected for Shigella is isolated.. *do not use antisera against . Only Shigella serotyping.. ,3
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(105) Part II Optimizing diagnostics of Shigella spp. and EIEC.
(106)
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