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
Mass spectrometry is poised for a new era, as clinical labs and researchers, hospital managers and industry prepare themselves for expansion in its use. Fuelling growth are trends towards personalized healthcare, the identification of novel biomarkers for translational medicine, large-scale epidemiological screenings as well as everyday clinical chemistry tests beyond just toxicology and endocrinology. There is room for such growth. At present, clinical lab applications of mass spectrometers account for only about 5% of the market.
Superior sensitivity and specificity, samples reusable
Mass spectrometry identifies a molecule by its unique mass-to-charge ratio, and is both highly sensitive and specific. In spite of concerns about cost and steep learning curves, the superiority of mass spectrometry versus immunoassays has never been disputed. Indeed, a study by the US National Cancer Institute (NCI) in 2008 focused on using mass spectrometry to distinguish between breath samples from patients with ovarian epithelial cancer versus those with polycystic ovarian syndrome or endometriosis.
Another advantage of mass spectrometry is its ability to use the same serum for multiple analyte profiling. This makes it useful in large-scale clinical studies, where samples have often been archived. Another NCI study, for instance, used mass spectrometry to identify biomarkers in blood from patients with acute myeloid leukemia; some of the samples were almost 10 years old. Dated samples have also been used for a range of other biomarkers, including malignant melanoma, soft tissue sarcomas and non-small cell lung cancer.
Gas chromatography and liquid chromatography
As a technology, mass spectrometry is not new in a lab setting. Gas chromatograph MS (GC-MS) has been used for ages in the diagnosis of organic metabolic disorders. More recently, liquid chromatograph mass spectrometry (LC-MS) has become a recommended resource for screening newborns.
The longer use of GC-MS means a bigger user base, as well as a more extensive legacy database, richer software libraries and advanced algorithms. Although GC-MS requires more complex processes for sample preparation (discussed below), it is relatively inexpensive compared to LC-MS systems, and has been considered effective enough for the bulk of applications.
The challenge of standardization
However, there is still some way to go before mass spectrometry attains wider use. One key barrier is a lack of standardization, above all in the preparation of samples. Clinical labs have different approaches to this issue, especially in terms of purification. This leads to sometimes-significant differences in results. Confounding the problem are continuing changes in the methods used for sample preparation, over time even within individual laboratories.
In the US, the Clinical and Laboratory Standards Institute has published two sets of recommendations on the use of MS. However, these leave quite a bit of room for interpretation and are considered no more than broad guidelines.
Preparation of samples for mass spectrometry
Typically, two steps are involved in preparing a sample: the concentrating of analytes, followed by ionization. The sample itself consists of two parts: the analytes of interest, and other components which are collectively known as the sample matrix. Sample preparation is considered the most difficult when whole blood or fractions are involved, given a relatively low density of analytes. Urine lies at the the other end of the spectrum, since the kidneys have already done most of the job of concentrating analytes.
Techniques for preparing samples include solid-phase extraction (SPE), immunoextraction (or immunoaffinity purification) and so-called ‘dilute-and- shoot’. In SPE, analytes and other matrix compounds are separated on the basis of their physical and chemical properties, among them charge and polarity. SPE systems consist of a liquid, mobile phase and a solid stationary phase (usually disposable cartridge-based). The liquid phase uses two different solvents, one for binding and washing, and another for elution.
Immunoextraction separates antibodies bound to the analytes from ‘free’ matrix components, by immobilizing them to a chromatographic column or polystyrene beads. After incubation with an immobilized antibody, unwanted components are washed away, and the enriched analyte is then eluted; another method is to concentrate the sample by drying, followed by re-suspension and injection into the chromatography system.
The third mechanism for preparing MS samples, dilute-and-shoot, is generally used in samples with a relatively high concentrations of analytes (e.g. urine). Here, dilution is usually effective enough to reduce matrix components to a
manageable level.
Successful ionization essential
The process of analysis relies wholly on successful ionization, as mass spectrometers can only detect charged analytes in a gaseous phase. Ionization can be either positive (cationic) or negative (anionic). The most common techniques for ionization in a clinical lab consist of chemical ionization and electrospray.
Chemical ionization generates ions by combining heat and plasma (produced by high-voltage electricity), at atmospheric pressure. While high temperatures vaporize the sample, the plasma (also known as a corona discharge) ionizes the evaporated solvent. Following this, mechanical interaction of the sample components (including analytes of interest) leads to the formation of negative or positive ions.
On its part, electrospray ionization uses electricity, heat and air to successively reduce the size of droplets that elute off the chromatographic column and sharply increase their charge. Ions (above all, proteins) desorb from the liquid droplet surface into a gas phase and then enter the mass spectrometer.
Challenges for vendors
Until recently, industry has focused on process improvements, while researchers have concentrated on improving the specificity and sensitivity of mass spectroscopy. Innovations from vendors have aimed at increasing the efficiency of ionization and of ion transfer, and accelerating discovery of biomarkers by combining size exclusion and affinity capture to enrich low molecular weight proteins, and more quickly separate diseased from clear samples. Some companies have also coupled reference databases of micro-organisms to their mass spectroscopy systems.
The greatest challenge for industry, however, has been to increase user acceptability. Research scientists rather than clinical lab technologists have been the traditional target for mass spectrometry manufacturers. The former, typically, have more interest in top-of-the-line technical specifications and performance than user-friendliness. The potential demand from clinical labs is forcing vendors to change approach. As a result, several are now beginning to package equipment sales with training and support.
Industry is also paying attention to systems integration, to bundle sample preparation instrumentation into a mass spectrometry suite and control its findings. Indeed, software has so far proved to be one of the biggest impediments to the growth of mass spectrometry, once again given the delicate balance between enabling new users to operate a system on the one side, while permitting complex adjustment of performance parameters on the other. OEMs have sought to plug this gap with bespoke add-ons but, as all IT systems designers know, this adds to system cost.
Researchers aim for more precision, ease of use
On the R&D side, a potentially promising area consists of so-called time-of-flight (TOF) mass spectrometers. TOF provides accuracy of 1 part per million by accelerating gas phase ions toward a detector via an electric field. Other initiatives are focused on robotic assistance, turbulent-flow chromatography and ion mobility – with considerable potential seen in linear ion traps. Scientists are also exploring the use of nanospray interfaces as well as microfluidics, though most successes to date have been at bench scale. In the future, such improvements will permit a reduction of detection thresholds, along with greater precision, ease of use and efficiency.
Some trade-offs inevitable
For both researchers and industry, the Holy Grail is to devise adequate user-configurability for trade-offs between high throughput on one side (required, for example, in epidemiological studies or newborn screening), and sensitivity and specificity on the other. Even now, detection of steroids such as cortisol, estradiol and testosterone remain a challenge at the lower end of their reference range, but require high precision in certain categories of patients, for example elderly female patients.
Lab use of mass spectrometry still minor, room for growth
No one doubts that the market for mass spectrometry is potentially huge. Globally, sales have been rising briskly, after falling due to the recession. A study from Los Angeles-based Strategic Directions International estimates the 2011 mass spectrometer market at USD 3.9 billion, with projections of USD 4.8 billion by 2014. The US and Canada hold the largest share of the market (38%) followed by Europe (31%) and Japan (13%), with other countries accounting for the remainder. Leaders in the mass spectrometer market include AB Sciex, Thermo Fisher Scientific, Waters and Agilent Technologies (all from the US), along with Hitachi and Shimadzu. European companies have a smaller presence, and include Germany’s GSG, Spectromat and Thermolinear.
As mentioned before, the clinical lab segment accounts for a very small share of total sales. The biggest users are pharmaceutical companies (a share of 20% of sales, with mass spectrometers increasingly used for metabolomic screening and drug discovery). Government follows closely (with an 18.5% share), universities (12.6%) and environmental/general testing services (9.4%). Electronics, the food and chemical industries also buy more mass spectrometers than clinical laboratories or hospitals.
However, the hope is that continuing growth in this entrenched base of other users will drive down unit costs of mass spectrometers, just as clinical labs get ready to increase their own requirements.
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
1 Coris BioConcept, Gembloux, Belgium
2 Molecular and cellular pharmacology,
Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium
*Corresponding author
E-mail: laetitia.avrain@corisbio.com
February | March 2025
Beukenlaan 137
5616 VD Eindhoven
The Netherlands
+31 85064 55 82
info@clinlabint.com
PanGlobal Media is not responsible for any error or omission that might occur in the electronic display of product or company data.