Helicobacter pylori (Hp) -infection and atrophic gastritis (AG) are the most important risk conditions preceding gastric cancer (GC). Following extensive research and development, a Finnish biotechnology company, Biohit Oyj, has launched the GastroPanel test, a panel of four stomach-specific biomarkers that give accurate information on both the structure and function of gastric mucosa.

by Dr Kari Syrjänen

Since the risk of GC and peptic ulcer disease among individuals with healthy stomach is very low, it is essential to distinguish between subjects with healthy stomach and those with gastric disorders. With GastroPanel – a  simple blood test – it is now possible to detect the patients who are at high risk for GC because they harbour either Hp -infection, AG or both in their stomach mucosa. Hp -infection alone increases the risk of GC several-fold, and this risk is over 90-fold among patients with Hp -related severe AG of both the corpus and antrum (pangastritis)^{[1, 3]}.

Another area of use for the GP test are the dyspeptic complaints, which in western countries appear in 20-40% of the population.  According to most current medical practices, the assessment of these complaints should invariably include a gastroscopic examination for which the existing resources are clearly insufficient and  which is actually not really necessary. The same applies to the costly and risky  “test” medications with proton-pump inhibitors (PPIs), since it is now possible to screen the patients at true risk and for whom gastroscopy is indicated by using the GastroPanel test. With this approach, approximately 40-70% of the limited and expensive endoscopy capacity can be released for colonoscopies, i.e. for screening and early detection of colorectal cancer. Because of the fact that, particularly among the elderly, dyspeptic complaints are frequently of large intestinal origin, it is cost-effective to supplement the examinations of these dyspeptic patients with colorectal screening methods like the ColonView test, a stool based detection of Hb and Hb/Hp complex, or colonoscopy.

The safe and cost-effective GastroPanel test enables early detection of many different disorders, and thus helps avoiding the majority of subsequent health problems. Besides gastric and esophageal cancer, undetected AG of the corpus (acid-free stomach)  can eventually also lead to malabsorption of vitamin B12, iron, magnesium, calcium, and some drugs. AG of the antrum, in turn, increases the risk of peptic ulcer disease and GC.

Concomitant AG of the antrum and corpus (pangastritis) is the single most important risk condition for GC. A minority of GCs can develop directly from HP-induced gastritis, without recognizable stages of mucosal atrophy. It is well known that vitamin B12 deficiency can lead to pernicious anemia (PA), dementia, depression, and injuries of the peripheral nervous system. Calcium deficiency, in turn, leads to osteoporosis. The absorption of many drugs is impaired in acid-free stomach. The risk of serious intestinal infections (giardiasis, malaria, Clostridium difficile and E. coli EHEC) can be increased particularly among senior citizens with AG.

Within public healthcare, it is possible to achieve substantial cost savings by replacing the systematic use of gastroscopy with a simple and inexpensive first-line diagnostic tool like the GastroPanel test for all patients with dyspeptic symptoms.

References
1. Suovaniemi O. GastroPanel-tutkimus osaksi dyspepsian hoitokäytäntöä. Yleislääkäri 2007; 4:104-106.
2. Malfertheiner P, Mégraud F, O’Morain C ym. Current concepts in the management of Helicobacter pylori infection: the Maastricht III Consensus Report. Gut 2007; 56:772-781.
3. Agreus L, Kuipers EJ, Kupcinskas L, Malfertheiner P, Di Mario F, Leja M, Mahachai V, Yaron N, van Oijen M, Perez Perez G, Rugge M, Ronkainen J, Salaspuro M, Sipponen P, Sugano K, Sung J. Rationale in diagnosis and screening of atrophic gastritis with stomach-specific plasma biomarkers. Scand J Gastroenterol 2012; 47:136-147.

The authors
Prof Kari Syrjänen,* MD, PhD, FIAC,
Chief Medical Director, Biohit Oyj.
Lea Paloheimo PhD
Director of Business Development and Quality, EurClinChem,

 *Corresponding author
E-mail: kari.syrjanen@biohit.fi

An adequate initial antibiotic therapy is a key determinant of therapeutic success in Pseudomonas aeruginosa – infected patients. Antibiotic efflux is an underestimated resistance mechanism because it may occur in strains categorized as susceptible. It is rarely or not at all diagnosed in routine laboratories and often masked by high-level resistance mechanisms.

by Dr Laetitia Avrain, Dr Pascal Mertens and Professor Françoise Van Bambeke

P. aeruginosa: state of the art of antibiotic susceptibility
P. aeruginosa is a Gram-negative bacterium recognized as a major cause of infections in hospitalized patients or in patients with impaired defences as observed in burn wounds or cystic fibrosis. In spite of improved hygiene measures, the risk of infection by P. aeruginosa in ICU remains high (infection incidence > 30/100 patients hospitalized in ICU). P. aeruginosa infections are associated with mortality rates as high as 30 % to 50 % in bacteremia [1] and up to 70% in patients with nosocomial pneumonia [2].

Yet, empirical selection of antibiotics is made difficult by the continuously evolving resistance of P. aeruginosa to antibiotics, notably due to the emergence of Multi Drug Resistance (MDR) phenotype (R ≥ 3 antibiotic classes). The MDR status of the strain as well as an initial inappropriate treatment negatively influence patient outcome [3].

Acquired high level resistance is due to the acquisition of genes coding for aminoglycoside-modifying enzymes or beta-lactamases, or to mutations in fluoroquinolone targets. Intrinsic antibiotic resistance is due to low outer membrane permeability mediated either by under production of the oprD porin, or by the expression of multidrug resistance efflux pumps. The genome of P. aeruginosa codes for numerous efflux pumps, among which MexAB-OprM and MexXY-oprM are of first clinical importance due to their large prevalence in clinical strains and their ability to expel several classes of chemically-unrelated antibiotics.

RND efflux pumps in P. aeruginosa
The main efflux pumps in P. aeruginosa belong to the Resistance-Nodulation-Division (RND) superfamily, which uses proton motive force as energy source. They constitute a tri-partite system, composed of an integral cytoplasmic membrane drug-proton transporter, an outer membrane channel and a periplasmic fusion protein linking the two other proteins. This assembly allows expelling the substrate from the inner membrane directly to the extracellular medium [Fig. 1, reproduced from [4]].

Ten efflux systems have been characterized in P. aeruginosa, among which MexAB-OprM and MeXY-OprM are constitutively expressed at a basal level in wild-type strains (expression of MeXY-OprM being however much lower than that of MexAB-OprM). Both systems are inducible when exposed to antibiotic substrates. The other systems (MexCD-OprJ, MexEF-OprN, MexJK, MexGHI-OpmD, MexVW, MexPQ-OpmE, MexMN, and TriABC are not expressed in wild type strains but may contribute to antibiotic or biocide resistance when expressed in resistant strains [5].

Antipseudomonal antibiotics released by P. aeruginosa multidrug efflux systems
RND efflux systems release multiple antimicrobials components including first-line antipseudomonal antibiotics such as β-lactams and β-lactamase inhibitors, fluoroquinolones, aminoglycosides [Table 1]. More specifically MexAB-OprM transports β-lactams fluoroquinolones, macrolides, tetracyclines, trimethoprim, sulfamides and chloramphenicol; MexXY-OprM, aminoglycosides, fluoroquinolones, macrolides, and tetracyclines; MexCD-oprJ, some β-lactams, fluoroquinolones, macrolides, tetracyclines, trimethoprim and chloramphenicol, and MexEF-OprN, fluoroquinolones, trimethoprim and chloramphenicol.  The latter is also involved in resistance to meropenem and doripenem, but this may rather result from the fact that the OrpD porin is downregulated in strains expressing this efflux system.

Colistin, the last resort drug for MDR P. aeruginosa, is not substrate for these efflux pumps. Thus, efflux is responsible for multidrug resistance, a single pump being able to transport several classes of drugs while at the same time some redundancy exists among transporters, fluoroquinolones for example being universal substrates for the main efflux systems. Moreover, the subsequent reduction in antibiotic concentration inside the bacteria may help selecting high level resistance mechanisms, in particular target mutations [6].

Over-expression of efflux pumps: impact on antimicrobial susceptibility
A study published in 2010 examined the impact of antibiotic treatment on the susceptibility of P. aeruginosa, by collecting successive isolates from ICU patients at the time of diagnosis of infection and during treatment [7]. Globally, mean minimum inhibitory concentration (MIC) values increased after exposure to antibiotics, with statistically significant effects being observed for amikacin, ciprofloxacin, cefepime, meropenem and piperacillin/tazobactam, bringing mean MICs to values higher than the EUCAST susceptibility breakpoints. Three quarters of the isolates showed a moderate elevation of the MIC (≤16X initial MIC), suggesting the involvement of low to moderate levels resistance mechanisms as those affecting membrane permeability [Fig 2, reproduced from [7]].

The analysis of the expression of efflux pumps in this collection revealed that a high proportion of the strains (34 %) did overexpress MexAB-OprM and MexXY-OprM in the initial isolate, but that this proportion further increased during the antibiotic treatment, with about two third of the strain overexpressing at least one of these efflux systems [Fig.3, adapted from [8]].
 
Diagnosis of efflux in clinical laboratory
Because efflux in P. aeruginosa almost always co-operates with other mechanisms of resistance, differential diagnosis by phenotypic antimicrobial analysis is complex, high levels resistance mechanisms masking the effect of the expression of efflux systems on MICs. Moreover, efflux pumps can be overexpressed during treatment, which may explain therapeutic failures with antibiotics that are considered active based on the original determination of the susceptibility profile.

Resistance by efflux can be detected using Efflux Pumps Inhibitors (EPI), which revert MICs to those strains that do not express efflux systems. Among them MC-207,110 (phenylalanine arginyl beta-naphthylamide) is a broad spectrum inhibitor that has been widely used in vitro to investigate the impact of efflux on susceptibility to antibiotics of P. aeruginosa. Inhibitors specific of a given transporter are also under investigation. Yet, in MDR strains with additional resistance mechanisms, EPI do not allow restoring antibiotic activity, which may lead to false-negative results [9].

In this context, molecular methods appear as the only way to evidence the expression of efflux pumps in clinical isolates. Immunoblotting methods were developed first but were rapidly replaced by Reverse Transcriptase quantitative PCR (RT-qPCR) due to its higher specificity and rapidity. RT-qPCRs were thus developed to detect and quantify the expression of the genes coding for the different proteins of a given RND pump. This method remains applicable whatever the other resistance mechanisms present in the clinical strain and can thus be applied in clinical laboratories. Typically, a 2-fold increase in the expression of mexA and mexB genes causes a decrease in antibiotic susceptibility, while overexpression of mexX needs to be higher (≥ 5-fold) to increase MIC values. This low level of overexpression implies that all the steps for RT-qPCR should be carefully standardized [10]. The commercial mex Q-TesT kit includes two housekeeping genes to standardize the RT-qPCR and facilitates the interpretation of mexA and mexX genes expression of clinical Pseudomonas aeruginosa strains in comparison to wild type strain PAO1.

Conclusion
Resistance by efflux has now well been characterized in specialized laboratories but is still rarely or not at all diagnosed in routine laboratories. The complexity of resistance in P. aeruginosa with MDR phenotypes and the lack of diagnostic tools are probably the main reasons why this mechanism is neglected. Because this resistance mechanism can also contribute to therapeutic failures, accurate diagnosis is of prime importance for selecting adequate therapy.

References
1. Aliaga L, Mediavilla JD, et al.  A clinical index predicting mortality with Pseudomonas aeruginosa bacteraemia. J Med Microbiol 2002; 51(7): 615-619.
2. Alp E, Guven M, et al. Incidence, risk factors and mortality of nosocomial pneumonia in intensive care units: a prospective study. Ann Clin Microbiol Antimicrob 2004; 3: 17.
3. Hirsch EB, Tam VH. Impact of multidrug-resistant Pseudomonas aeruginosa infection on patient outcomes. Expert Rev Pharmacoecon Outcomes Res 2010; 10(4): 441-451.
4. Mesaros N, Van Bambeke F, et al. L’efflux actif des antibiotiques et la résistance bactérienne: état de la question et implications. La lettre de l’infectiologue 2005; (4): 117-126.
5. Lister PD, Wolter DJ, et al. Antibacterial-resistant Pseudomonas aeruginosa: clinical impact and complex regulation of chromosomally encoded resistance mechanisms. Clin Microbiol Rev 2009; 22(4): 582-610.
6. Zhanel GG, Hoban DJ, et al. Role of efflux mechanisms on fluoroquinolone resistance in Streptococcus pneumoniae and Pseudomonas aeruginosa. Int J Antimicrob Agents 2004; 24(6): 529-535.
7. Riou M, Carbonnelle S, et al. In vivo development of antimicrobial resistance in Pseudomonas aeruginosa strains isolated from the lower respiratory tract of Intensive Care Unit patients with nosocomial pneumonia and receiving antipseudomonal therapy. Int J Antimicrob Agents 2010; 36(6): 513-522.
8. Riou M, Avrain L, et al. Influence of antibiotic treatments on gene expression of RND efflux pumps in successive isolates of Pseudomonas aeruginosa collected from patients with nosocomial pneumonia hospitalized in Intensive Care Units from Belgian Teaching Hospitals. ECCMID, 10-13 April 2010, Vienna, Austria. P780.
9. Van Bambeke F, Pages JM, et al.  Inhibitors of bacterial efflux pumps as adjuvants in antibiotic treatments and diagnostic tools for detection of resistance by efflux. Recent Pat Antiinfect Drug Discov 2006; 1(2): 157-175.
10. Avrain L, Hocquet D, et al. Pre-Real-Time PCR steps standardization for appropriate interpretation of mexA and mexX gene expression by mex Q-Test in P. aeruginosa. ECCMID, 10-13 April 2010, Vienna, Austria. P590.

The authors
Laetitia Avrain PhD¹*, Pascal Mertens PhD¹ and Françoise Van Bambeke, Professor, Maître de Recherche FNRS, PhD²

*Corresponding author
E-mail: laetitia.avrain@corisbio.com

Current methods for the detection of gastroenteric pathogens are insensitive, slow, and laborious. Testing directed at specific organisms misses important infections. Systematically testing all fecal samples using multiplex PCR for common viral, bacterial and parasitic pathogens allows laboratories to increase diagnostic yield, improve workflow, reduce waste and turn-around times.

by Dr Gary McAuliffe

Introduction
Within a single diagnostic laboratory, multiple methods are used to detect gastroenteric pathogens. Selective agar plates differentiate bacterial pathogens, whereas immunoassays are used to detect viruses and parasites such as Giardia lamblia and Cryptosporidium spp.. Laboratories perform microscopy with special stains for the detection of Entamoeba histolytica and Dientamoeba fragilis. PCR has generally been restricted to the detection of norovirus, but many studies have demonstrated its potential for the detection of other enteric pathogens.

Multiplex PCR (M-PCR) panels have been shown to enhance detection of gastroenteric organisms. These panels combine several enteric pathogen targets in one or more PCR reaction vessel(s). O’ Leary et al. demonstrated 100% sensitivity compared with culture for the detection of four bacterial pathogens [1]. Wolffs et al. used a panel targeting seven viral pathogens and detected a pathogen in 97% of samples compared with 49% by conventional methods [2]. Stark et al. showed 100% sensitivity and specificity of a M-PCR panel targeting four parasites, which also reliably differentiates E. histolytica from non-pathogenic E. dispar and E. moshkovskii [3]. de Boer et al. successfully replaced bacterial culture at their institution with a molecular screening approach targeting four bacteria and G. lamblia [4].

Several commercial M-PCR fecal panels are available [Table 1]. These may be directed against parasites, viruses or bacteria, or contain targets from all three groups of organisms, allowing systematic testing of stool samples for all common gastrointestinal pathogens.   

In the author’s study, 1758 samples from community and hospital patients were tested using the Fast-Track Diagnostics bacterial, viral and parasite M-PCR panels. Pathogens were detected in 30% of samples by this systematic M-PCR approach compared with 18% using conventional testing as directed by clinician request [5][Table 2].

Advantages of a systematic M-PCR approach
Studies have demonstrated enhanced detection of entero-hemorrhagic E. coli (EHEC), Clostridium difficile, G. lamblia, E. histolytica, norovirus, adenovirus and rotavirus by PCR compared with immunoassays and other conventional assays [2–4, 6, 7]. Bacterial PCR has generally performed comparably with culture, with the exception of Salmonella spp., where reduced detection by PCR has been demonstrated in several, but not all, studies [4, 5, 8]. An advantage of increased sensitivity is that smaller amounts of feces are required for testing, and multiple samples are not required for the detection of common parasites. Clinicians should be aware that organisms such as adenovirus and norovirus can be shed in feces for several weeks following infection and that PCR detects low levels of these organisms which may not be clinically relevant. Selective bacterial media lack specificity, requiring time and further testing to discount commensal organisms. E. histolytica cysts cannot be reliably differentiated from other members of the Entamoeba complex by microscopy and staining. M-PCR generally overcomes these issues, though specificity depends on the target sequence chosen. For adenovirus, some panels target the hexon gene which does not differentiate between enteric and non-enteric serotypes, whereas others target sequences specific to enteric sub-types.

Currently laboratories restrict their testing to a limited range of pathogens for which they have sensitive and affordable assays available. A number of organisms that laboratories do not commonly test for, such as astrovirus, sapovirus, Vibrio spp., and non-O157 EHEC, can be missed. M-PCR allows a wider range of organisms to be targeted. In the author’s study astrovirus was found to be the second most common cause of gastroenteritis, and non-O157 EHECs were detected in sixteen samples by targeting the stx genes. Our laboratories did not have assays for these organisms prior to the study. Laboratories may design their own panels to reflect locally and internationally important pathogens, or buy commercial panels which reflect these requirements. Rare or imported infections not targeted by the panels will not be detected, and laboratories need to decide when additional tests are required. Up to five targets may be tested in a single real-time PCR reaction vessel; therefore increasing the number of targets reduces the number of samples on a given PCR amplification run. The xTAG system (Luminex Corp.) overcomes this limitation by post-amplification analysis using microspheres with up to 100 differing spectra. Eleven targets are currently included in the xTAG gastroenteritis panel within a single PCR reaction vessel [9]. TaqMan array cards (Life technologies) also overcome this restriction by allowing simultaneous real-time PCR in 384 PCR reaction wells. This platform allows up to eight samples to be run in parallel [10].

Fecal samples are usually tested for a limited range of viruses, bacteria, or parasites dependent upon clinician request and laboratory algorithms. Studies have shown that important pathogens such as G. lamblia and EHEC are missed using this approach. Testing for enteric viruses is not widely employed outside hospitals despite their prevalence. The M-PCR approach tests every sample for every target in the panels. It is less reliant on clinicians’ knowledge of the organisms that the patient has likely been exposed to, or those that may be causing the patients clinical syndrome. This systematic approach accounted for the majority of the increased diagnostic yield in the author’s study.

In our laboratory it can take 3–4 days for the identification of Salmonella spp. by culture. Time to generation of results is significantly reduced with the M-PCR approach.  In the author’s study, samples collected were batch tested the following day, giving results for eleven pathogens simultaneously within 24 hours of collection. Hands-on time is also reduced. The preparation and reading of a trichrome stain can take up to 40 minutes by an experienced operator, whereas the hands-on time for testing a sample by PCR is a quarter of this.

The workflow created by the systematic M-PCR approach fits well with current laboratory systems; testing is performed in a single pathway rather than by several laboratory departments. Conventional fecal testing employs multiple diagnostic kits and selective media, some of which come with significant cost, short expiry times, and extensive quality control requirements. In our laboratory stool samples for bacterial culture are inoculated onto seven agar plates which need to be stored, processed, and incubated, generating a significant amount of waste.  O’ Leary et al. reported that M-PCR significantly reduced this wastage [1]. M-PCR does not obviate the need for bacterial culture as it does not offer an antibiogram, but it allows focused testing of samples found to be positive for these bacterial targets.

Pathogens such as Shigella spp, G. lamblia and D. fragilis are labile in stool. Delays may occur in transportation, inoculation or examination which can compromise yield. For M-PCR less initial processing is required. Specimens are inoculated into tubes containing Stool Transport and Recovery buffer (STAR) (Roche Diagnostics) which binds inhibitors, and stabilizes nucleic acids for later processing. PCR is also able to detect organisms rendered non-viable by inadequate transport, or the use of antibiotics. Feces contains high levels of bilirubin and bile salts, which can lead to inhibition of PCR amplification. Inoculating samples into STAR buffer on arrival, and using extraction methods such as the EasyMag platform (Biomerieux) can help overcome this issue. In the author’s study 1.7% of samples exhibited inhibition, but all gave adequate internal control amplification following dilution and repeat testing.

Cost is a major factor preventing systematic testing of fecal samples by conventional techniques in diagnostic laboratories. The costs of PCR are reducing relative to conventional tests, and batch testing of samples using the systematic M-PCR approach is becoming a viable option. In the author’s study testing a sample for bacteria, viruses and parasites was significantly cheaper by M-PCR than using equivalent conventional tests [NZ$152 (£75) versus NZ$280 (£140)]. Laboratories have successfully reported replacing their conventional methods with a molecular screening approach for bacteria [1, 4] or viruses [2]. Where the cost of replacing all traditional diagnostics with systematic testing may currently remain restrictive, laboratories may choose to replace testing for organism groups, e.g. viruses, and institute systematic testing at a later date.

Conclusion
Current conventional methods for the detection of enteric pathogens are labour intensive, insensitive, slow, and applied piecemeal to submitted fecal samples. Testing stool samples using multiplex PCR panels which simultaneously detect all common bacteria, viruses and parasites increases the detection of gastroenteric pathogens. This approach improves turn-around time, workflow, reduces labour and waste. Costs are reducing, making systematic M-PCR testing an attractive alternative to currently used techniques.

References
1. O’Leary J, et al. Comparison of the EntericBio multiplex PCR system with routine culture for detection of bacterial enteric pathogens. J Clin Microbiol. 2009; 47: 3449–3453.
2. Wolffs PF, et al. Replacing traditional diagnostics of fecal viral pathogens by a comprehensive panel of real-time PCRs. J Clin Microbiol. 2011; 49: 1926–1931.
3. Stark D, et al. Evaluation of multiplex tandem real-time PCR for detection of Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis in clinical stool samples. J Clin Microbiol. 2011; 49: 257–262.
4. de Boer RF, et al. Improved detection of five major gastrointestinal pathogens by use of a molecular screening approach. J Clin Microbiol. 2010; 48: 4140–4146.
5. McAuliffe GN, et al. Systematic application of multiplex PCR enhances the detection of bacteria, parasites, and viruses in stool samples. J Infect. 2013; 67(2): 122–129.
6. Costantini V, et al. Diagnostic accuracy and analytical sensitivity of IDEIA norovirus assay for routine screening of human norovirus. J Clin Microbiol. 2010; 48: 2770–2778.
7. Luna RA, et al. Rapid stool-based diagnosis of Clostridium difficile infection by real-time PCR in a children’s hospital. J Clin Microbiol. 2011; 49: 851–857.
8. Cunningham SA, 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; 48:2929–2933.
9. Coste JF, et al. Microbiological diagnosis of severe diarrhea in kidney transplant recipients by use of multiplex PCR assays. J Clin Microbiol. 2013; 51(6): 1841–1849.
10. Liu J, et al. A laboratory developed TaqMan array card for simultaneous detection of nineteen enteropathogens. J Clin Microbiol. 2013; 51(2): 472–480.

The author
Gary McAuliffe MBBS
Microbiology Department, LabPlus Laboratory, Auckland, New Zealand
E-mail: GMcAuliffe@adhb.govt.nz