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Molecular diagnostic testing for common stool pathogens
Journal of Hospital Infection 90 (2015) 196e198 Available online at www.sciencedirect.com
Journal of Hospital Infection journal homepage: www.elsevierhealth.com/journals/jhin
Commentary
Molecular diagnostic testing for common stool pathogens G. Trafford a, N. Ratnaraja b, N. Wickramasinghe b, * a b
Department of Infectious Diseases, Heart of England Foundation Trust, Bordesley Green East, Birmingham, UK Department of Microbiology, Sandwell and West Birmingham Hospitals NHS Trust, City Hospital, Birmingham, UK
A R T I C L E
I N F O
Article history: Received 19 January 2015 Accepted 21 January 2015 Available online 21 February 2015
Introduction Traditional culture methods for identifying enteric pathogens have long been the mainstay of investigation of patients with acute diarrhoea. These methods have been refined to provide the best possible yield within the time and resource constraints of a typical laboratory. The benefit of obtaining culture of a causative organism is not to be underestimated as it allows sensitivity testing and strain typing, but sensitivity is variable and the process is time-consuming and not without cost. Although the majority of diarrhoeal illness is self-limiting, the impact on the health of populations as a whole is a further strong justification for diagnosing the cause of individual cases and managing outbreaks. The paper by Buchanan et al.1 in this issue outlines how molecular methods could change infection prevention and control practice, based on responses to questionnaires administered to hospitals across England. Rapid diagnostic approaches with high sensitivity and capacity for automation are an attractive prospect due to the potential for increasing
DOI of original article: http://dx.doi.org/10.1016/j.jhin.2014.12.021.
* Corresponding author. Address: Department of Microbiology, Sandwell and West Birmingham Hospitals NHS Trust, City Hospital, Dudley Road, Birmingham B18 7QH, UK. Tel.: + Tel: +44 1215076486. E-mail address: [email protected] (N. Wickramasinghe).
the diagnostic yield while reducing both turnaround time and laboratory running costs. Developers of molecular methods have tended to favour polymerase chain reaction (PCR)-based techniques, and the technology has progressed rapidly to allow real-time detection of multiple targets. This, in turn, may ultimately be supplanted by genome sequencing or some other gene detection method. There are, however, numerous pitfalls and important considerations when moving from familiar organism isolation to gene detection, some of which may not be immediately apparent.
Potential benefits of pathogen detection by PCR PCR has potential to improve many aspects of gastrointestinal pathogen detection, and to contribute, directly or indirectly, to better infection prevention and control. There are many PCR tests for gastrointestinal pathogens in current use, but usually as single target detection systems for specific scenarios, such as Clostridium difficile or norovirus. However, multi-target assays that detect a large number of pathogens either by multiplex or array systems are becoming more widely available, and have the potential to be employed either for routine samples or for targeted patient groups such as paediatrics, returned travellers or the immunocompromised. In terms of sensitivity, PCR has been shown to increase the detection of many common and important organisms with the
http://dx.doi.org/10.1016/j.jhin.2015.01.019 0195-6701/ª 2015 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.
G. Trafford et al. / Journal of Hospital Infection 90 (2015) 196e198 notable exception of salmonellae,2e7 where selenite enrichment still has higher sensitivity, particularly when a low organism burden is expected, such as in chronic carriage. Campylobacter and Shigella spp. are notoriously difficult to culture, and the current method for identifying verocytotoxigenic Escherichia coli (VTEC) is limited in scope to the 0157 serotype. Culture for all of these pathogens requires several different culture media, takes a minimum of 24 h to produce preliminary negative results,8 and may be susceptible to methodological and interpretative difficulties or errors at any stage of processing from specimen transport to reading of cultures. Obtaining a rapid diagnosis of the cause of acute diarrhoea could result in more appropriate management that might indirectly benefit infection prevention and control. For example, cessation of unnecessary antibiotic treatment may be facilitated, or the elimination of diagnostic uncertainty may mean that invasive investigations or even surgery may be avoided. In some cases, an early positive diagnosis may give clinicians the confidence to discharge patients earlier, although this will also depend on clinical parameters such as the need for ongoing intravenous hydration or electrolyte management. A negative PCR result could also be helpful to exclude common enteric pathogens in cases of non-infectious diarrhoea, such as inflammatory colitis, where immunosuppression is being considered. For infection prevention and control or public health practitioners, identifying the cause of acute diarrhoea early on in the clinical course could have a profound impact on diagnosing, terminating or limiting an outbreak. Identifying linked cases at an early stage could allow earlier interventions such as ward or bay closures. This benefit is likely to have the most significance in the community setting, as many infection prevention and control decisions in hospital are taken based on patients’ symptoms, rather than accurate knowledge of a causative organism.9 For many micro-organisms, the PCR method is more sensitive, and increasing detection of foodborne disease has an obvious benefit for public health, both for the more common Campylobacter cases and the infrequent but more serious outbreaks of organisms carrying the verocytotoxin gene. Compared with the alternative of waiting 48 h for a negative culture, a negative PCR received in less than 24 h using a highly sensitive assay could also be of use when deciding to de-isolate patients in high-dependency areas such as critical care units, where resources are stretched and non-infective causes of diarrhoea are not infrequent. For the laboratory, being able to screen out negative results in a timely, automated fashion can dramatically reduce the time and cost of processing and reading culture plates. This area is perhaps the most attractive to those responsible for making the decision to move to gene detection, as the financial benefits are easier to model and can be considerable. For example, although they require a higher degree of scientific oversight, most PCR systems can be operated and interpreted by non-scientific staff, freeing up more experienced staff for other tasks.
Potential pitfalls of pathogen detection by PCR Assays that are commercially available, or are in development, for PCR detection vary in their scopes, costs and
197
processing times. Regardless, the most likely role of any of these methods is as a screening step, with positive test results subject to confirmation and further testing, usually using culture-based methodologies. As such, a high negative predictive value is essential, and this also applies if a negative test result is going to be used by infection prevention and control practitioners to lift isolation restrictions. A high negative predictive value may be achieved at the expense of reduced specificity. Most assays target either conserved regions of microbial DNA to detect a broad range of strains or species within a particular pathogen group, or detect a gene known to be associated with pathogenicity. The former may be prone to overdiagnosis if it identifies nonpathogenic strains within the group. From the point of view of infection prevention and control practice, this is usually preferable to the risk of underascertainment of cases if the target is narrowed to improve specificity at the expense of poorer sensitivity. Targeting specific genes has the advantage of detecting only those organisms within a group carrying a pathogenic gene, and hence capable of causing disease. The diagnostic accuracy of tests using such targets relies on the presence of the gene correlating with pathogenicity; detection of genes that are not expressed may lead to overdiagnosis. Of course, PCR is not flexible in the face of new pathogens, or established pathogens that are not included within the test repertoire. Attention needs to be given to the possibility that a patient with a negative screening result has an unexpected pathogen before lifting infection prevention and control restrictions. A number of studies have shown that PCR is more likely to result in the detection of more than one species per sample, and elucidating the clinical relevance of each can be difficult. The previously mentioned higher sensitivity of culture for Salmonella spp. in certain scenarios means that PCR tests should not be used as standalone tests of cure, investigating chronic carriage or when symptoms have been present for some time. Rarer bacterial (e.g. Aeromonas spp.), viral, parasitic and protozoal pathogens are included in some assays, which can cause difficulties. For example, there may be little benefit in detecting pathogens that are self-limiting (such as viral gastroenteritis), not unequivocal pathogens (such as Aeromonas spp.), or rare for most laboratories (such as Cryptosporidium spp.). The interpretation of results that are not confirmed by culture is also difficult; there is insufficient experience, to date, to determine the infection prevention and control management of patients with discordant PCR and culture results.10 PCR does not always allow for antimicrobial susceptibility testing or strain typing. If these are required, culture must usually be performed following gene detection. At this early stage in the development of PCR tests, clinicians may not have confidence in the results, meaning that the intended benefits are not achieved. In particular, without careful training and education, they may lose confidence in a screening test that produces positive results that are not confirmed by further testing. There is also the potential for unconfirmed positive results to cause unnecessary work or anxiety for what, in many cases, may be a self-limited illness with very little threat to the wider public health. Quantitative PCR methods have been employed to distinguish between infection and incidental colonization in rotavirus and norovirus infection, so there appears to be correlation between lower
198
G. Trafford et al. / Journal of Hospital Infection 90 (2015) 196e198
pathogen burden and milder disease.11e13 The vast majority of these ‘PCR positive, culture negative’ results for bacterial pathogens will remain unresolved, as reference laboratory services to investigate these further are unlikely to be available, with the possible exception of verocytotoxin-positive results. Understanding the meaning of a positive result in any particular assay, and deciding how and when to communicate this to other clinical teams is of great importance and should ideally be agreed in advance. Finally, and importantly, there are implications of these tests for laboratory services. Moving to automated detection will result in a deskilled laboratory workforce, such that when traditional methods are required to resolve difficult cases, this ability could have been lost. Also, adequate provision needs to be made in the event of equipment downtime.
Cost implications The cost of setting up a PCR screening test can be considerable, incorporating the upfront equipment cost and any ongoing maintenance and consumable costs. In addition, a validation phase and a formal training programme for staff involved in running the assay both divert resources from routine laboratory tasks, although this is for a limited time period. These direct costs are relatively easy to quantify, but many factors are much less straightforward (e.g. calculating laboratory time saved or cost savings to the organization as a whole). Similarly, the time spent troubleshooting the new methods, devising automatic comments for reports, integrating them into the existing information technology systems and producing information for stakeholders is not insignificant but is difficult to quantify. The potential for generating a higher number of positive PCR results could lead to a greatly increased workload in the realm of public health. A clear understanding of the meaning of a positive PCR result and the limitations around subsequent culture methods are needed in order to prevent unnecessarily stringent public health measures. Clear and regular communication between the laboratory, clinical staff and public health is vital in order to achieve this.
Conclusions Molecular diagnostic techniques can play a valuable role in identification of enteric pathogens, with potential for a significant impact on infection prevention and control. However, one must be wary of the costs and limitations of these techniques, and be aware that the current standard of culture will still play a major role in the investigation of patients with diarrhoeal disease.
Conflict of interest statement None declared. Funding sources None.
References 1. Buchanan J, Wordsworth S, O’Connor L, et al. Management of patients with suspected infectious diarrhoea in hospitals in England. J Hosp Infect 2015;90:199e207. 2. Koziel M, Kiely R, Blake L, et al. Improved detection of bacterial pathogens in patients presenting with gastroenteritis using the EntericBio Real-Time Gastro Panel I assay. J Clin Microbiol 2013;51:2679. 3. Cunningham S, Sloan LM, Nyre LM, et al. 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;48:2929e2933. 4. O’Leary J, Corcoran D, Lucey B. Comparison of the EntericBio multiplex PCR system with routine culture for detection of bacterial enteric pathogens. J Clin Microbiol 2009;47:3449e3453. 5. Wessels E, Rusman LG, van Bussel MJ, Claas EC. Added value of multiplex Luminex Gastrointestinal Pathogen Panel (xTAG GPP) testing in the diagnosis of infectious gastroenteritis. Clin Microbiol Infect 2014;20:O182eO187. 6. Lee HM, Lee S, Lee BI, et al. Clinical significance of fecal lactoferrin and multiplex polymerase chain reaction in patients with acute diarrhea. Gut Liver 2014. http://dx.doi.org/10.5009/ gnl14106 [Epub ahead of print]. 7. Abubakar I, Irvine L, Aldus CF, et al. A systematic review of the clinical, public health and cost-effectiveness of rapid diagnostic tests for the detection and identification of bacterial intestinal pathogens in faeces and food. Health Technol Assess 2007;11(36). 8. Public Health England. UK Standards for microbiology investigations ID22i3. Identification of Escherichia coli O157. London: Public Health England; 2014. Available at: https://www.gov. uk/government/publications/smi-id-22-identification-ofescherichia-coli-o157. Last accessed 19th January 2015. 9. Pankhurst L, Macfarlane-Smith L, Buchanan J, et al. Can rapid integrated polymerase chain reaction-based diagnostics for gastrointestinal pathogens improve routine hospital infection control practice? A diagnostic study. Health Technol Assess 2014;18(53). 10. Khare R, Espy MJ, Cebelinski E, et al. Comparative evaluation of two commercial multiplex panels for detection of gastrointestinal pathogens by use of clinical stool specimens. J Clin Microbiol 2014;52:3667e3673. 11. Kang G, Iturizza-Gomara M, Wheeler JG, et al. Quantitation of group A rotavirus by real-time reverse-transcription-polymerase chain reaction: correlation with clinical severity in children in South India. J Med Virol 2004;73:118e122. 12. Phillips G, Lopman B, Tam CC, Iturizza-Gomara M, Brown D, Gray J. Diagnosing norovirus-associated infectious intestinal disease using viral load. BMC Infect Dis 2009;9:63. 13. Platts-Mills JA, Liu J, Houpt ER. New concepts in diagnostics for infectious diarrhea. Mucosal Immunol 2013;6:876e885.
Journal of Hospital Infection journal homepage: www.elsevierhealth.com/journals/jhin
Commentary
Molecular diagnostic testing for common stool pathogens G. Trafford a, N. Ratnaraja b, N. Wickramasinghe b, * a b
Department of Infectious Diseases, Heart of England Foundation Trust, Bordesley Green East, Birmingham, UK Department of Microbiology, Sandwell and West Birmingham Hospitals NHS Trust, City Hospital, Birmingham, UK
A R T I C L E
I N F O
Article history: Received 19 January 2015 Accepted 21 January 2015 Available online 21 February 2015
Introduction Traditional culture methods for identifying enteric pathogens have long been the mainstay of investigation of patients with acute diarrhoea. These methods have been refined to provide the best possible yield within the time and resource constraints of a typical laboratory. The benefit of obtaining culture of a causative organism is not to be underestimated as it allows sensitivity testing and strain typing, but sensitivity is variable and the process is time-consuming and not without cost. Although the majority of diarrhoeal illness is self-limiting, the impact on the health of populations as a whole is a further strong justification for diagnosing the cause of individual cases and managing outbreaks. The paper by Buchanan et al.1 in this issue outlines how molecular methods could change infection prevention and control practice, based on responses to questionnaires administered to hospitals across England. Rapid diagnostic approaches with high sensitivity and capacity for automation are an attractive prospect due to the potential for increasing
DOI of original article: http://dx.doi.org/10.1016/j.jhin.2014.12.021.
* Corresponding author. Address: Department of Microbiology, Sandwell and West Birmingham Hospitals NHS Trust, City Hospital, Dudley Road, Birmingham B18 7QH, UK. Tel.: + Tel: +44 1215076486. E-mail address: [email protected] (N. Wickramasinghe).
the diagnostic yield while reducing both turnaround time and laboratory running costs. Developers of molecular methods have tended to favour polymerase chain reaction (PCR)-based techniques, and the technology has progressed rapidly to allow real-time detection of multiple targets. This, in turn, may ultimately be supplanted by genome sequencing or some other gene detection method. There are, however, numerous pitfalls and important considerations when moving from familiar organism isolation to gene detection, some of which may not be immediately apparent.
Potential benefits of pathogen detection by PCR PCR has potential to improve many aspects of gastrointestinal pathogen detection, and to contribute, directly or indirectly, to better infection prevention and control. There are many PCR tests for gastrointestinal pathogens in current use, but usually as single target detection systems for specific scenarios, such as Clostridium difficile or norovirus. However, multi-target assays that detect a large number of pathogens either by multiplex or array systems are becoming more widely available, and have the potential to be employed either for routine samples or for targeted patient groups such as paediatrics, returned travellers or the immunocompromised. In terms of sensitivity, PCR has been shown to increase the detection of many common and important organisms with the
http://dx.doi.org/10.1016/j.jhin.2015.01.019 0195-6701/ª 2015 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.
G. Trafford et al. / Journal of Hospital Infection 90 (2015) 196e198 notable exception of salmonellae,2e7 where selenite enrichment still has higher sensitivity, particularly when a low organism burden is expected, such as in chronic carriage. Campylobacter and Shigella spp. are notoriously difficult to culture, and the current method for identifying verocytotoxigenic Escherichia coli (VTEC) is limited in scope to the 0157 serotype. Culture for all of these pathogens requires several different culture media, takes a minimum of 24 h to produce preliminary negative results,8 and may be susceptible to methodological and interpretative difficulties or errors at any stage of processing from specimen transport to reading of cultures. Obtaining a rapid diagnosis of the cause of acute diarrhoea could result in more appropriate management that might indirectly benefit infection prevention and control. For example, cessation of unnecessary antibiotic treatment may be facilitated, or the elimination of diagnostic uncertainty may mean that invasive investigations or even surgery may be avoided. In some cases, an early positive diagnosis may give clinicians the confidence to discharge patients earlier, although this will also depend on clinical parameters such as the need for ongoing intravenous hydration or electrolyte management. A negative PCR result could also be helpful to exclude common enteric pathogens in cases of non-infectious diarrhoea, such as inflammatory colitis, where immunosuppression is being considered. For infection prevention and control or public health practitioners, identifying the cause of acute diarrhoea early on in the clinical course could have a profound impact on diagnosing, terminating or limiting an outbreak. Identifying linked cases at an early stage could allow earlier interventions such as ward or bay closures. This benefit is likely to have the most significance in the community setting, as many infection prevention and control decisions in hospital are taken based on patients’ symptoms, rather than accurate knowledge of a causative organism.9 For many micro-organisms, the PCR method is more sensitive, and increasing detection of foodborne disease has an obvious benefit for public health, both for the more common Campylobacter cases and the infrequent but more serious outbreaks of organisms carrying the verocytotoxin gene. Compared with the alternative of waiting 48 h for a negative culture, a negative PCR received in less than 24 h using a highly sensitive assay could also be of use when deciding to de-isolate patients in high-dependency areas such as critical care units, where resources are stretched and non-infective causes of diarrhoea are not infrequent. For the laboratory, being able to screen out negative results in a timely, automated fashion can dramatically reduce the time and cost of processing and reading culture plates. This area is perhaps the most attractive to those responsible for making the decision to move to gene detection, as the financial benefits are easier to model and can be considerable. For example, although they require a higher degree of scientific oversight, most PCR systems can be operated and interpreted by non-scientific staff, freeing up more experienced staff for other tasks.
Potential pitfalls of pathogen detection by PCR Assays that are commercially available, or are in development, for PCR detection vary in their scopes, costs and
197
processing times. Regardless, the most likely role of any of these methods is as a screening step, with positive test results subject to confirmation and further testing, usually using culture-based methodologies. As such, a high negative predictive value is essential, and this also applies if a negative test result is going to be used by infection prevention and control practitioners to lift isolation restrictions. A high negative predictive value may be achieved at the expense of reduced specificity. Most assays target either conserved regions of microbial DNA to detect a broad range of strains or species within a particular pathogen group, or detect a gene known to be associated with pathogenicity. The former may be prone to overdiagnosis if it identifies nonpathogenic strains within the group. From the point of view of infection prevention and control practice, this is usually preferable to the risk of underascertainment of cases if the target is narrowed to improve specificity at the expense of poorer sensitivity. Targeting specific genes has the advantage of detecting only those organisms within a group carrying a pathogenic gene, and hence capable of causing disease. The diagnostic accuracy of tests using such targets relies on the presence of the gene correlating with pathogenicity; detection of genes that are not expressed may lead to overdiagnosis. Of course, PCR is not flexible in the face of new pathogens, or established pathogens that are not included within the test repertoire. Attention needs to be given to the possibility that a patient with a negative screening result has an unexpected pathogen before lifting infection prevention and control restrictions. A number of studies have shown that PCR is more likely to result in the detection of more than one species per sample, and elucidating the clinical relevance of each can be difficult. The previously mentioned higher sensitivity of culture for Salmonella spp. in certain scenarios means that PCR tests should not be used as standalone tests of cure, investigating chronic carriage or when symptoms have been present for some time. Rarer bacterial (e.g. Aeromonas spp.), viral, parasitic and protozoal pathogens are included in some assays, which can cause difficulties. For example, there may be little benefit in detecting pathogens that are self-limiting (such as viral gastroenteritis), not unequivocal pathogens (such as Aeromonas spp.), or rare for most laboratories (such as Cryptosporidium spp.). The interpretation of results that are not confirmed by culture is also difficult; there is insufficient experience, to date, to determine the infection prevention and control management of patients with discordant PCR and culture results.10 PCR does not always allow for antimicrobial susceptibility testing or strain typing. If these are required, culture must usually be performed following gene detection. At this early stage in the development of PCR tests, clinicians may not have confidence in the results, meaning that the intended benefits are not achieved. In particular, without careful training and education, they may lose confidence in a screening test that produces positive results that are not confirmed by further testing. There is also the potential for unconfirmed positive results to cause unnecessary work or anxiety for what, in many cases, may be a self-limited illness with very little threat to the wider public health. Quantitative PCR methods have been employed to distinguish between infection and incidental colonization in rotavirus and norovirus infection, so there appears to be correlation between lower
198
G. Trafford et al. / Journal of Hospital Infection 90 (2015) 196e198
pathogen burden and milder disease.11e13 The vast majority of these ‘PCR positive, culture negative’ results for bacterial pathogens will remain unresolved, as reference laboratory services to investigate these further are unlikely to be available, with the possible exception of verocytotoxin-positive results. Understanding the meaning of a positive result in any particular assay, and deciding how and when to communicate this to other clinical teams is of great importance and should ideally be agreed in advance. Finally, and importantly, there are implications of these tests for laboratory services. Moving to automated detection will result in a deskilled laboratory workforce, such that when traditional methods are required to resolve difficult cases, this ability could have been lost. Also, adequate provision needs to be made in the event of equipment downtime.
Cost implications The cost of setting up a PCR screening test can be considerable, incorporating the upfront equipment cost and any ongoing maintenance and consumable costs. In addition, a validation phase and a formal training programme for staff involved in running the assay both divert resources from routine laboratory tasks, although this is for a limited time period. These direct costs are relatively easy to quantify, but many factors are much less straightforward (e.g. calculating laboratory time saved or cost savings to the organization as a whole). Similarly, the time spent troubleshooting the new methods, devising automatic comments for reports, integrating them into the existing information technology systems and producing information for stakeholders is not insignificant but is difficult to quantify. The potential for generating a higher number of positive PCR results could lead to a greatly increased workload in the realm of public health. A clear understanding of the meaning of a positive PCR result and the limitations around subsequent culture methods are needed in order to prevent unnecessarily stringent public health measures. Clear and regular communication between the laboratory, clinical staff and public health is vital in order to achieve this.
Conclusions Molecular diagnostic techniques can play a valuable role in identification of enteric pathogens, with potential for a significant impact on infection prevention and control. However, one must be wary of the costs and limitations of these techniques, and be aware that the current standard of culture will still play a major role in the investigation of patients with diarrhoeal disease.
Conflict of interest statement None declared. Funding sources None.
References 1. Buchanan J, Wordsworth S, O’Connor L, et al. Management of patients with suspected infectious diarrhoea in hospitals in England. J Hosp Infect 2015;90:199e207. 2. Koziel M, Kiely R, Blake L, et al. Improved detection of bacterial pathogens in patients presenting with gastroenteritis using the EntericBio Real-Time Gastro Panel I assay. J Clin Microbiol 2013;51:2679. 3. Cunningham S, Sloan LM, Nyre LM, et al. 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;48:2929e2933. 4. O’Leary J, Corcoran D, Lucey B. Comparison of the EntericBio multiplex PCR system with routine culture for detection of bacterial enteric pathogens. J Clin Microbiol 2009;47:3449e3453. 5. Wessels E, Rusman LG, van Bussel MJ, Claas EC. Added value of multiplex Luminex Gastrointestinal Pathogen Panel (xTAG GPP) testing in the diagnosis of infectious gastroenteritis. Clin Microbiol Infect 2014;20:O182eO187. 6. Lee HM, Lee S, Lee BI, et al. Clinical significance of fecal lactoferrin and multiplex polymerase chain reaction in patients with acute diarrhea. Gut Liver 2014. http://dx.doi.org/10.5009/ gnl14106 [Epub ahead of print]. 7. Abubakar I, Irvine L, Aldus CF, et al. A systematic review of the clinical, public health and cost-effectiveness of rapid diagnostic tests for the detection and identification of bacterial intestinal pathogens in faeces and food. Health Technol Assess 2007;11(36). 8. Public Health England. UK Standards for microbiology investigations ID22i3. Identification of Escherichia coli O157. London: Public Health England; 2014. Available at: https://www.gov. uk/government/publications/smi-id-22-identification-ofescherichia-coli-o157. Last accessed 19th January 2015. 9. Pankhurst L, Macfarlane-Smith L, Buchanan J, et al. Can rapid integrated polymerase chain reaction-based diagnostics for gastrointestinal pathogens improve routine hospital infection control practice? A diagnostic study. Health Technol Assess 2014;18(53). 10. Khare R, Espy MJ, Cebelinski E, et al. Comparative evaluation of two commercial multiplex panels for detection of gastrointestinal pathogens by use of clinical stool specimens. J Clin Microbiol 2014;52:3667e3673. 11. Kang G, Iturizza-Gomara M, Wheeler JG, et al. Quantitation of group A rotavirus by real-time reverse-transcription-polymerase chain reaction: correlation with clinical severity in children in South India. J Med Virol 2004;73:118e122. 12. Phillips G, Lopman B, Tam CC, Iturizza-Gomara M, Brown D, Gray J. Diagnosing norovirus-associated infectious intestinal disease using viral load. BMC Infect Dis 2009;9:63. 13. Platts-Mills JA, Liu J, Houpt ER. New concepts in diagnostics for infectious diarrhea. Mucosal Immunol 2013;6:876e885.