Multiplex Diagnostic Test For Enteric Pathogens Biology Essay

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Diarrhoeal disease accounts for an estimated 2 billion episodes of illness and 1.5 million infant deaths globally each year (World Health Organisation, 2009). Such an evident impact of this illness on human mortality and morbidity requires the determination of optimal methods for overall management of enteropathogenic illness worldwide.

Classed as the passing of three or more abnormally liquid or unformed stools per day, diarrhoea is most commonly a symptom of gastrointestinal infection and the pathogens involved are generally spread through the oral-faecal

route (Guerrant et al, 2001). Unsafe water, inadequate sanitation or insufficient hygiene account for 80% of all diarrhoeal episodes (Prüss-Üstün et al, 2001) and with a 2004 report from the world health organisation and Unicef joint monitoring board declaring that up to 1.1 billion people globally were without improved sources of drinking water (World Health Organisation, 2004), there is great opportunity for these infections to occur and in high numbers.

4 main epidemiological settings have been identified as being associated with diarrhoeal illness, covering community-acquired, hospital-acquired, travel-acquired and persistent diarrhoea (Pawlowski et al, 2009) but within each, treatment of diarrhoeal episodes is either usually systematic due to its self-limited nature, consisting of oral rehydration therapy and administration of antibiotics in certain cases. Early diagnosis of diarrhoeal episodes can lead to interventions that alleviate sufferer's symptoms and prevent secondary transmission, allowing public control and surveillance of outbreaks (Guerrant et al, 2001). Acute episodes may require hospitalisation, can result in extreme squelae such as Guillain-Barre syndrome or haemolytic uraemic syndrome, and the most serious cases can result in death (Fischer Walker et al, 2010).

Despite a call to reverse the trend of illness due to insufficient hygiene in the UK, the incidence of foodborne illness causing gastroenteritis remains at an unacceptably high level since its peak in the 1990's, showing an increase in comparison with 2008 figures to above 70000 cases in 2009 (Food Standards Agency, 2011).

A wide diversity of organism types have been identified in cases of acute diarrhoeal episodes and infections are mainly attributable to viruses and bacteria, with parasites identified less commonly as the cause (Pawlowski et al, 2009).

Escherichia coli, Salmonella, Shigella, Campylobacter, Clostridium difficile and Yersinia entercolitica are all common bacterial types involved in the manifestation of diarrhoea (World Health Organisation, 2008) and, coupled with the addition of enteric viruses, there are a sizeable array of causative microbes. The number of viral agents associated with diarrhoeal disease has increased since the initial identification of noroviruses (NoVs) as gastroenteritis causative agents and subsequently, sapoviruses (SaVs), astroviruses (HaVs), and enteric adenoviruses have all been highlighted as important etiological causes of the disease (Roman et al, 2003).

Enteric Bacteria

Healthy adults are most likely to present with bacterial foodborne zoonotic infections than viruses, with 90% of reported cases having either Salmonella or Campylobacter as causative agents (Bloomfield et al, 2009). Campylobacter in particular is associated with polymicrobial enteric infection in developing countries (Isokpehi et al, 2002) and a recent report from the European Food Safety Authority and the European Centre for Disease Prevention and Control reported that infection due to due to this organism was the most commonly reported zoonosis in the European Union (2009 data) with 198,252 cases, representing a 4% increase from 2008 (European Food Standards Agency, 2009).

Numerous types of diarrhoea-causing strains of Escherichia coli have been identified worldwide and enterotoxigenic E. coli (ETEC) is the leading cause of enteric infection in travellers and of diarrhoeal disease in military populations (World Health Organisation, 2009) In addition to this, enteropathogenic E. coli (EPEC) is one of the most important agents involved in persistent diarrhoea, an infection-induced illness resulting from multiple consecutive infections due to an unresolved infection, secondary malabsorption or post-gastroenteritis syndrome. Despite improvements in treating acute diarrhoeal disease in children through replenishment of fluids and electrolytes, there has ultimately been an observed increase in deaths caused by EPEC. The E. coli pathogens produce enterotoxins or adhere to the brush border causing effacement of cells (Ochoa et al, 2008).

The most frequent causative infectious agent involved in nosocomial (hospital-acquired) diarrhoeal episodes is Clostridium difficile (Weis and Grimm, 2011), which is closely related to antibiotic usage. This pathogen is therefore of great importance in a health-care setting with symptoms ranging in severity from mild diarrhoea through to severe disease in the form of pseudomembranous colitis and toxic megacolon, both of which can lead to death. (Health Protection Agency, 2011). C. difficile is also an important agent in community-acquired infection (Pawlowski et al, 2009).

Enteric Viruses

Rotaviruses have been highlighted as the leading cause of severe diarrhoeal disease and dehydration in infants and children under the age of 5 worldwide, accounting for up to 40% of cases and the pathogen is cited as the most common cause of diarrhoeal deaths in developing countries (Guandalini and Vaziri, 2011). The viruses infect the small intestine villi tip enterocytes, causing atrophy of the epithelium and repopulation by immature secretor cells. There is a loss of intestinal macromolecule permeability, a decrease in intestinal disaccharidase and an induction of intestinal electrolyte and water secretion by stimulation of the enteric nervous system caused by these viruses (Roman et al, 2003).

Human astroviruses (HaVs) are one of the most important causes of acute paediatric gastroenteritis, together with RoVs and both calciviruses (the virus family encompassing NoVs and SaVs) (Guix et al, 2005). Viral replication of HaVs occurs in intestinal tissue but the detailed pathogenesis of diarrhoeal disease caused by astrovirus is not well understood.

The exact mechanism by which calciviruses cause diarrhoea is also unknown to some extent, however the effect is thought to be contributed to by an expansion of the proximal small intestine villi and a shortening of the microvilli, with an accompanying delay in gastric emptying (Roman et al, 2003). With a low infectious dose, inanimate surface stability and conventional cleaning agent resistance, norovirus is the leading cause of foodborne disease worldwide and is second only to rotaviruses as the cause of severe gastroenteritis in children globally under 5 years of age (Koo, 2010).

It has been observed that adenoviruses produce lesions in the enterocytes of the intestinal villi, leading to atropy of the villi, triggering crypt hyperplasia and essentially leading to loss of fluids (Roman et al, 2009).

Organism-specific Diagnosis

The need for organism specific diagnosis of pathogens involved in gastroenteritis is many-fold due to the varied number of causative microbes that present with the same symptoms. This means that lack of testing for the pathogen involved in the disease can lead to empirical and often inappropriate treatment of the general patient population on an individual as well as a group level.

Lack of or incorrect antibiotic use can be counterproductive in gastroenteritis treatment and indiscriminate antibiotic use has resulted in an observed increase in drug resistant pathogens that are easily spread throughout the populace, rendering therapies less useful (Bloomfield et al, 2009).

Specifically, antibiotic use is contraindicated in nontyphoidal Salmonella infections since their use prolongs shedding, increases the likelihood of a carrier state and promotes emergence of resistant strains while in comparison antibiotics are always recommended for public health reasons in Shigella infection in order to give a reduced period of excretion and reduced diarrheal duration. C. difficile is recommended as a suspected causative agent in anyone who develops diarrhoea following antibiotic therapy and, if possible, the offending medication should be discontinued and antidiarrhoeals avoided (Guandalini and Vaziri, 2011).

Antibiotic use, when required, is often infection-specific and takes into account current observed drug resistance of certain pathogen types. For example, the recommended treatment to decrease, when appropriate, symptoms of E. coli infection, is with Fluoroquinolones (FQ), trimethoprim-sulfamethoxazole (TMP-SMX), azithromycin, or rifaximin used in conjunction with antidiarrhoeals. FQs are suitable for use in patients with Campylobacter infections with symptoms for 1 week or more or in severe cases but ciprofloxacin-resistant strains of the bacterium are now becoming more prevalent and macrolides antibiotics are therefore a more suitable choice (Guandalini and Vaziri, 2011). Knowledge of the pathogen type in these cases therefore, allows for appropriate treatment and the reduction in the development and prevelance of drug-resistant organisms.

Further to this, unnecessary treatments or procedures for diseases with similar symptoms to gastroenteritis such as invasive endoscopies for Irritable Bowel Syndrome (IBS) are avoided as in the case of Yersinia infections which present with symptoms similar to IBS, Crohn's disease or appendicitis (Guandalini and Vaziri, 2011), diseases which have very different different treatment requirements. A recent case study highlights the issue of incorrect diagnosis leading to adverse effects where a presentation of diarrhoea and sickness was treated as cholera but subsequently diagnosed as Salmonella, delaying treatment (Saidi, 2010).

Certain population groups that are at higher risk of complication from gastroenteritis require specific, proper and timely treatment and it is reported that often the very worst symptoms are observed in the elderly, the very young and the immunocompromised (Pawlowski et al, 2009). The last thirty years have seen the era of the HIV/AIDS pandemic, with the overall level of newly diagnosed infections remaining high and the number of people living with HIV/AIDS increasing due to improved antiretroviral therapy, leading to direct reductions in mortality (United Nations, 2010). Gastrointestinal (GI) disorders are among the most common and most debilitating conditions that affect individuals with HIV/AIDS and several viruses have been implicated as common causative agents of these enteric disorders in this vulnerable patient population, often in a polymicrobial fashion (Silva, 2010). Also, patients with neutropenia (linked to the advent and rising use of chemotherapeutic agents), recipients of bone marrow or solid organ transplant patients, as well as those with underlying chronic pulmonary disease also come under this patient section and require organism-specific diagnosis for astute medical care (Echavarria, 2008).

Current Detection Methods

Traditional culture methods based on phenotype have been used for many years in the identification of enteric bacteria. The conventional microbiological method (following National Standard Methods) for bacterial growth and detection is to use a selective agar and these are widely available for the growth and detection of many enteric bacteria including Salmonella, Shigella, Vibrio, Yersinia, Campylobacter and E. coli, amongst others (Pawlowski et al, 2009). Most of these agents are easily cultivated but isolation and subsequent final identification of pathogen(s) is time consuming, resulting in a 2 to 3 day time period for correct assessment of a clinical isolate. Patients are left without diagnosis for this period, are left untreated and pose a risk of secondary transmission (Leland and Ginnocchio, 2007).

Viruses are difficult in comparison with bacteria to grow in cell culture and low particle numbers make this pathogen type difficult to identify. This has resulted in past traditional electron-microscopy based visualisation techniques for virus identification being superseded by less tedious antigen-based and molecular diagnostic methods (Pawlowski et al, 2009).

More sensitive antigen detection techniques that have been developed for pathogen detection are based on enzyme-immunoassay (EIA), latex particle agglutination (LA) and, more recently, immunochromatography (ICG) (Leland and Ginnocchio, 2007). Enteric viruses, such as sapovirus (Farkas et al, 2006) and norovirus are detectable by enzyme-immunoassay and EIA methods have been described for the detection of Campylobacter (Constantini et al, 2010).

Molecular Detection Methods

Molecular methods involving DNA amplification that have been applied to diagnostics in the last 25 years offer a faster, more sensitive and highly specific alternative to EIA and traditional culture techniques (Lauri et al, 2009) and despite the fact that this technique was once confined to research laboratories, amplification of both bacterial and viral DNA and RNA by Polymerase Chain Reaction (PCR) methods are now becoming more widely used in diagnostic laboratories. (Speers, 2006)

Viruses as well as bacteria can be detected in clinical samples using many different molecular-based methods, including PCR, both traditional and real-time, Reverse Transcription (RT)-PCR, Nucleic Acid Sequence Based Amplification (NASBA), strand displacement amplification and transcription-mediated amplification (Yang and Rothman, 2004).

All these methods are advantageous in that lengthy incubation periods for pathogen isolation in culture is not required (results are generally available in 2.5 to 3 hours), less technical expertise is usually needed and there is no issue with non-proliferation of viruses due to the culture independent nature of the technique. (Leland and Ginnocchio, 2007).

The clinical applicability, however, of these PCR-based methods are detracted from due to the fact that detection and analysis of products obtained through these techniques has often been achieved either using laborious and time consuming gel-electrophoresis (and subsequent sequencing) or limited fluorescence spectroscopy in the case of RT-PCR methods (Yang and Rothman, 2004). Agarose gels are low in precision, sensitivity, have a short dynamic range, low resolution and allow for size-based discrimination only. Gels cover only a short dynamic range (<2 log) where results are not displayed as numbers (Dorak, 2010). In addition to this, the broad spectral overlap obtained from more than one fluorophore excited with a single light source results in a limited detection space for simultaneous identification of DNA sequences using this technique (Yang and Rothman, 2004).

Multiplex PCR-based assays have been reported for detection of enteric viruses (Liu et al, 2011) and RT- PCR has been demonstrated as a more or comparatively sensitive method for detecting norovirus, sapovirus, astrovirus (Logan et al, 2007), adenovirus (Heim et al, 2003) and rotavirus (Zheng et al, 2009) as well as the enteric bacteria in comparison with traditional PCR and culture techniques. However, despite this advantage, RT-PCR is a contamination prone, complex, multi-step procedure and is not recommended as a high throughput multiplex assay format. (Yang and Rothman, 2004).

Raman scattering is the term used to describe the process by which electron cloud distortion, caused by photon-molecule interaction, is coupled with nuclear motion to give scattering of that photon with different energy (Smith and Dent, 2005). Further to this, Surface-Enhanced Raman Scattering (SERS), an effect first described by Fleishmann et al (1975), is the spectroscopic effect whereby this phenomenon of inelastic scattering of light is enhanced by a factor of up to 106 (in comparison with normal Raman scattering), through adsorption of an analyte onto an appropriately roughened metal surface. The increase in the intensity of this scattering of light is not fully understood but it is thought to be contributed to by both electromagnetic and charge transfer enhancements (Smith and Dent, 2010).

Use of a chromophore or fluorescent label to tag the analyte under investigation results in a further resonance contribution between the label and the exciting source, provided that the excitation source used matches or is near to the electronic transition of the label. This technique, termed Surface-Enhanced Resonance Raman Scattering (SERRS), gives a further enhancement in the sensitivity and selectivity of Raman signals, and signal enhancements of up to 1014 have been reported using this technique (Smith, 2008).

Solid substrates with randomly rough textures and metal colloidal suspensions are the two main classes of enhancing media that have dominated the field of SERS and SERRS measurements, with the primary advantage being the degree of control that can be exercised over these materials (Bell and Sirimuthu, 2007). The detection of SERRS from several dyes adsorbed on colloidal gold and silver nanoparticles was first described by Lee and Meisel (1982) and was the first demonstration of the high efficiency of signal that can be obtained by this method.

These enhancements in scattering efficiency obtained through SERRS of labelled oglionuleotides (up to 1014 compared to standard Raman scattering) gives a sensitivity that rivals or surpasses that achievable with fluorescence (Faulds et al, 2004). The sharp fingerprint spectra that are obtained through surface-enhanced resonance Raman scattering also make for a spectroscopic technique that is ideal for the simultaneous detection of a higher number of molecules than is achievable with PCR-based fluorescent techniques (Faulds et al, 2007). Additionally, quenching of fluorophore signal allows for detection of these fluorescent molecule types through SERRS and identification of particular analytes is often much more certain with this technique due to the more distinct molecular fingerprint in comparison with fluorescent spectroscopy (Faulds and Graham, 2009).

Both SERS and SERRS have been demonstrated to have a wide and versatile range of application possibilities, from the qualitative detection of organic colourants in works of art (Leona, 2009) and the investigation of drug molecule-target interactions (Yan et al, 1997) to the quantification of glucose or anthrax in biosensor units. (Haynes et al, 2007)

The standard approach to detect DNA using SERRS is to use dye-labelled DNA probes which will recognise the complimentary strand of the target DNA under investigation, whereupon it is the dye 'marker' that is detected in the analytical procedure (Smith and Dent, 2005).

A Surface Enhanced Resonance Raman Spectroscopy-Based Diagnostic Assay

The development of these DNA probes, coupled with the development of Raman spectroscopy has now provided a way forward for the simultaneous detection of multiple bacterial or viral biological targets in low concentrations from patient samples. Presented here is a diagnostic tool based on that technique in the form of a SERRS assay for the sensitive multiplex detection of enteric pathogens.

The method involves traditional PCR amplification of nucleic acid targets which are subsequently biotinylated at their 5' end. SERRS-active dye-labelled DNA probes with organism-specific sequences are then allowed to hybridise to the target organism DNA, before the specific probes are captured by binding their biotin moiety to magnetic streptavidin coated beads. A wash removes unbound nucleic acid targets and probes leaving behind only probes that have interacted with their target. These probes of interest are eluted from their bound state and are then combined with an aggregating agent and silver nanoparticulate colloid for analysis by SERRS. A unique fingerprint spectrum which is specific for the dye molecule is produced which can be related back to its associated probe for identification of the bacterial or viral biological target.

The Medical Device Industry Requirement For Proficiency Testing

Proficiency testing (PT) is an essential quality control and quality guarantee method within the medical device industry. As a complementary but independent external audit to on-site laboratory analyses, it provides invaluable evidence on the effectiveness of newly developed assay formats and the trueness/accuracy of results gained through these (Duarte et al, 2007). The essential information gained through interlaboratory comparison also allows for business improvement, ranging from product to personnel, identifying, preventing or remedying issues of impedance to successful validation of a medical device and its position in the marketplace. From an end-user perspective, proficiency testing gives customer confidence and reassurance in a final product offering, which is fundamental to commercial success for medical device companies.


Considering the need for PT, we discuss here the development of a method for the preparation, storage and transport of proficiency panels which are to be used for validation purposes of SERRS assay kits during pre-clinical and clinical evaluations by various collaborators. These pre-clinical and clinical evaluations are required to ensure that the SERRS assay kits can detect the enteric pathogen target species under investigation at various copy number concentrations of plasmid DNA and RNA template (53).