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Featured Articles
Monitoring kidney function in aminoglycoside therapy
Aminoglycosides are antibiotics largely used at the hospital. Their nephrotoxicity imposes therapeutic drug monitoring as well as kidney function monitoring. Creatinine is the most widely used biochemical marker; however, new biomarkers such as neutrophil gelatinase associated lipocalin (NGAL), cystatin C (Cys C) or kidney injury molecule-1 (KIM-1) can allow the detection of acute kidney injury more quickly.
by F. Fraissinet, E. Sacchetto and Dr E. Bigot-Corbel
Background
Aminoglycosides are bactericidal antibiotics used for the treatment of Gram-negative or endocarditis infections. The most important adverse effects of aminoglycosides are nephrotoxicity and ototoxicity. The prevalence of aminoglycoside-associated nephrotoxicity is estimated at 10 to 20 %, although it also depends on the patient’s clinical condition and exposure to nephrotoxic drugs such as cyclosporine, anti-inflammatory or iodinated drugs. In intensive care units, nephrotoxicity is most frequent (60% of patients) and associated with a high rate of mortality [1, 2]. Nephrotoxicity mainly results in nonoliguric acute kidney injury which occurs following 7 to 10 days. This toxicity may manifest as a decrease in the glomerular filtration rate with glycosuria, hypokalemia and hypocalcemia. Aminoglycosides are often administered by intravenous drip and are freely eliminated by glomerular filtration and reabsorbed by the proximal tubule. Of the injected dose, 5% is retained by epithelial cells of the proximal tubule. After endocytosis in tubular cells, these molecules accumulate in lysosomes and induce phospholipidosis, alteration of key cellular components and apoptosis. Aminoglycosides also have glomerular effects: gentamicin stimulates mesangial proliferation, produces mesangial contraction and induces neutralization of negative charges of the glomerulus [3]. Gentamicin also induces a reduction in renal blood flow with an increased renal vascular resistance. These factors contribute to decrease the glomerular filtration rate (GFR). Additional risk factors for nephrotoxicity induced by aminoglycoside have been identified as sepsis, prolonged therapy, renal or liver dysfunction, hypokalemia or hypomagnesemia. Nephrotoxicity is less frequent when aminoglycosides are administered once daily compared with 12 h [4].
Methods of detection of acute kidney injury induced by aminoglycoside therapy
Classic markers: creatinine and creatinine clearance
Creatinine is the most widely used marker in the diagnosis of the acute renal insufficiency. For defining AKI, the Risk Injury Failure Loss End stage kidney disease (RIFLE) classification is based on increase of serum creatinine concentration and decrease of glomerular filtration rate. The introduction of the RIFLE classification has increased the conceptual understanding of AKI syndrome, and this classification has been successfully tested in a number of clinical studies [5].
In spite of an easily accessible dosage, creatinine as a marker of AKI has some drawbacks. Creatinine is filtered by the glomerulus and is not bound to plasma proteins. In standard physiological conditions, the daily rate of creatinine production is constant; however, the rate of creatinine production is affected by conditions of muscular pathology or muscular loss (as occurs in intensive care and cirrhosis). In these patients, AKI does not result in an increase of serum creatinine levels. Other factors, such as age, sex, ethnic group and diet, also influence serum concentrations of creatinine. Creatinine is not the ideal marker to estimate GFR, because it is secreted by renal tubule, which artefacutally increases glomerular filtration rate. At low serum creating concentrations, creatinine is lacks sensitivity to estimate GFR. Large changes in GFR may be associated with relatively small changes in serum creatinine (See Figure 2 in Delanaye et al. [6]). The rise of the creatinine is late (occurring after 3–5 days) and is not specific for nephrotoxicity induced by aminoglycosides, and an increase of creatinine in AKI is a function of the initial concentration of creatinine [7].
To estimate GFR, formulas that use creatinine plasma concentration, such as the Modified Diet in Renal Disease formula (MDRD), Chronic Kidney Disease Epidemiology collaboration formula (CKD-EPI) or estimation of clearance creatinine by Cockroft–Gault (CG) equation, were derived in subjects with chronic, not acute, kidney disease. A limitation of the MDRD equation was an underestimation of GFR in the high range. The CKD-EPI equation performs better at high GFR levels (GFR >60 mL/min/1.73 m²). Use of serum creatinine concentration to estimate GFR supposes a steady-state between creatinine production and excretion [8]. In spite of the use of correction factors, it is more difficult to estimate GFR in Asian or African populations as well as in elderly or obese patients [9].
New biomarkers
Cystatin C
Cystatin C (CysC), a 13-kDa endogenous cysteine proteinase inhibitor, plays an important role in intracellular catabolism of various peptides and proteins. CysC is considered to be a good biomarker of decreased kidney function because it is produced at a relatively constant rate and released into plasma, and is filtered by glomeruli without tubular secretion. The influence of muscular mass is less than for creatinine, and CysC allows diagnosis of AKI 48 h before serum creatinine [10]. Equations with serum CysC concentration can also estimate GFR. If GFR is great than 60 mL/min/m², CysC measurement is more powerful than the MDRD equation. CysC is a useful biomarker for early detection of AKI in the pediatric population and for patients in the intensive care unit, as CysC determination can be performed in serum and/or in urine. In spite of efforts to standardize the procedure, there is no reference method. Production of CysC also depends on hormonal factors, so CysC cannot be used in cases of thyroid dysfunction.
Neutrophil gelatinase associated lipocalin
Neutrophil gelatinase associated lipocalin (NGAL) is a protein of 25 kDa protein of the lipocalin family and is covalently bound to matrix metalloproteinase-9. NGAL is expressed early in ischemic kidney impairment in animal models. During AKI, NGAL expression is induced in distal nephron epithelia resulting in elevated plasma and urinary levels of NGAL (Fig. 1) [2]. NGAL determination can be performed on serum and/or urine by immunoturbimetric or immunofluorimetric assays. There is a general agreement on a cut-off value of >150 ng/mL, but a clear cut-off NGAL concentration for AKI has not been reported. Several studies show the importance of NGAL in cardiac surgery or critically ill patients for predicting AKI. NGAL is also useful for the detection of nephrotoxicity induced by contrast agents and has prognostic value for mortality or initiation of renal replacement therapy. Plasma NGAL measurements may be influenced by a number of coexisting variables as chronic hypertension, systemic infections, inflammatory conditions or hypoxia. Changes in NGAL values are potentially associated with septic state or aminoglycoside therapy [11].
Kidney injury molecule-1
Kidney injury molecule-1 (KIM-1) is a glycoprotein localized in the apical membrane of the proximal tubule of kidney, and KIM-1 expression can be induced by nephrotoxic drugs. Urine KIM-1 is a promising biomarker of proximal tubular injury. As with NGAL, urinary KIM-1 levels predicted adverse clinical outcomes such as dialysis requirement and mortality. In a previous study, urinary KIM-1 is correlated with AKI severity in non-critically ill children treated by aminoglycosides [12].
Conclusion
Patients treated with aminoglycosides must be carefully monitored for nephrotoxicity. Creatinine has been the most used biochemical marker of AKI, but new biomarkers, such as NGAL and KIM-1, have been developed in recent years.
References
1. Oliveira JF, Silva CA, Barbieri CD, Oliveira GM, Zanetta DM, Burdmann EA. Antimicrob Agents Chemother. 2009; 53(7): 2887-2891.
2. Schmidt-Ott KM. Nephrol Dial Transplant. 2011; 26(3): 762-764.
3. Lopez-Novoa JM, Quiros Y, Vicente L, Morales AI, Lopez-Hernandez FJ. Kidney Int. 2011; 79(1): 33-45.
4. Rybak MJ, Abate BJ, Kang SL, Ruffing MJ, Lerner SA, Drusano GL. Antimicrob Agents Chemother. 1999; 43(7): 1549-1555.
5. Ricci Z, Cruz DN, Ronco C. Nat Rev Nephrol. 2011; 7(4) :201-208.
6. Delanaye P, Cavalier E, Maillard N, Krzesinski JM, Mariat C, Cristol JP, et al. [Creatinine: past and present]. Annales de Biologie Clinique 2010; 68(5): 531-543 (in French).
7. Waikar SS, Bonventre JV. J Am Soc Nephrol. 2009; 20(3): 672-679.
8. Nguyen MT, Maynard SE, Kimmel PL. Clin J Am Soc Nephrol. 2009; 4(3): 528-34.
9. Delanaye P, Cavalier E, Mariat C, Krzesinski JM, Rule AD. Kidney Int. 2011; 80(5): 439-440.
10. Herget-Rosenthal S, Marggraf G, Husing J, Goring F, Pietruck F, Janssen O, et al. Kidney Int. 2004; 66(3): 1115-1122.
11. Devarajan P. Nephrology (Carlton) 2010; 15(4): 419-428.
12. McWilliam SJ, Antoine DJ, Sabbisetti V, Turner MA, Farragher T, Bonventre JV, et al. PLoS One 2012; 7(8): e43809.
The authors
François Fraissinet1 BSc, Emilie Sacchetto2 and Edith Bigot-Corbel2* PhD
1Laboratoire de Biochimie, 86021 Poitiers, France
2Laboratoire de Biochimie, CHU de Nantes, Hôpital G et R Laënnec, 44800 Saint-Herblain, France
*Corresponding author
E-mail: edith.bigot@chu-nantes.fr
A new approach for diagnosis of Clostridium difficile based on evolution of a unique volatile organic compound identifier
Clostridium difficile is a major cause of nosocomial infections and rapid diagnosis of the disease is essential for infection control. Several methods for C. difficile detection are employed in clinical laboratories; each method has its advantages and disadvantages. A novel method has recently been developed that allows differentiation between C. difficile-positive and -negative stool samples based on volatile organic compound evolution and their detection by headspace solid-phase microextraction gas chromatography–mass spectrometry.
by Dr Emma Tait, Prof. Stephen P. Stanforth, Prof John D. Perry and Prof. John R. Dean
Introduction
Clostridium difficile is a Gram-positive anaerobe and the causative agent of C. difficile infection (CDI). CDI is a major healthcare problem with a total of 14,687 cases reported in patients aged 2 years and over in England between April 2012 and March 2013 [1]. C. difficile is a spore-forming bacterium; dormant spores are resistant to antibiotics, heating and chemicals such as disinfectants and, therefore, can persist on surfaces and survive for long periods in the environment [2]. Ingestion of spores and their subsequent germination in the gut allows the proliferation of C. difficile in patients whose normal gut flora has been severely reduced following antibiotic treatment. Following germination, vegetative C. difficile can produce toxins and is susceptible to antibiotic treatment. Pathogenic C. difficile releases two types of toxins, toxin A and toxin B, and it is these toxins that cause the symptoms associated with CDI [3]. Clinical symptoms of C. difficile infection (CDI) include mild to severe diarrhoea, which can lead to pseudomembranous colitis and death. Diagnosis of CDI includes both clinical manifestations of symptoms supported by laboratory findings.
Diagnosis of C. difficile infection
Rapid diagnosis of CDI is essential to allow the most appropriate treatment to be prescribed, to enable proper use of hospital isolation facilities and to reduce the spread of the infection. Routine diagnostic methods in clinical microbiology laboratories employ a variety of techniques for diagnosis of CDI. These include immunoassays for detection of glutamate dehydrogenase (GDH) antigen and toxins, polymerase chain reaction (PCR) for detection of toxin B or isolation of C. difficile by culture (followed by confirmation of toxigenicity, e.g. using PCR). Immunoassays and PCR methods are typically highly automated and deliver rapid results and these have largely replaced the traditional cell cytotoxicity assay, which requires propagation of cell lines and takes days rather than hours to provide results.
Toxin immunoassays, although relatively inexpensive with rapid turnaround time, have limitations in terms of their sensitivity and specificity leading to false-negative results and false-positive results [4]. Immunoassays for GDH are typically highly sensitive for detection of C. difficile but lack specificity. Culture media for the isolation of C. difficile typically incorporate the antibiotics D-cycloserine and cefoxitin which suppress commensal bacteria; such media are based on the formulation recommended by George et al. [5]. Isolation of C. difficile via culture is sensitive but can take several days to obtain results and there may be a heavy growth of other fecal bacteria, particularly when reduced antibiotic concentrations are used [6]. Recent developments in C. difficile detection include using a chromogenic substrate which is structurally similar to naturally occurring substrate used by C. difficile toxins [7]. This allows the detection of toxigenic C. difficile, i.e. only pathogenic strains are targeted.
Identification of C. difficile-positive stool samples using gas chromatography has previously been explored [8]; volatile organic compounds (VOCs) such as p-cresol and short chain fatty acids were identified as potential markers for C. difficile. However, these methods suffered from a lack of specificity, particularly due to the high number of false positives obtained following the detection of p-cresol and isocaproic acid in stool samples without C. difficile. As a consequence, gas chromatography methods were deemed unsuitable for C. difficile detection [8]. More recent attempts to use bacterial VOC analysis as a tool for C. difficile identification have used headspace solid-phase microextraction (HS-SPME) as a VOC collection method coupled with gas chromatography–mass spectrometry (GC-MS) for VOC separation and detection [9].
Development of a novel method for detection of C. difficile in stools
A novel method for rapid identification of C. difficile in stool samples has been developed using the analysis of VOCs. Use of synthetic enzyme substrates is an effective means of differentiating bacteria, for example in chromogenic culture media [10]. These types of culture media incorporate chromogenic enzyme substrates where the action of a specific enzyme on the substrate liberates a molecule that is detectable visually, allowing the detection of pathogenic bacteria [10]. The philosophy behind the use of substrates in culture media can be applied to the analysis of bacterial VOCs, where a substrate is incorporated into a clinical sample inoculated in liquid media; the cleaved product is volatile and detectable using an analytical method such as HS-SPME-GC-MS. The detection of VOCs liberated following enzyme activity increases the specificity of bacterial VOC profiles, as these liberated VOCs act as markers for a particular species, hence aiding identification of bacteria. This approach was applied to the detection of C. difficile in stool samples.
p-Cresol is formed in C. difficile by the decarboxylation of p-hydroxyphenylacetic acid. The enzyme responsible for this decarboxylation is p-hydroxyphenylacetate decarboxylase. It has been established that the hydroxyl group in the para position on the phenyl ring is an essential requirement for decarboxylation to occur [11]. C. difficile is almost unique in its ability to form p-cresol using this pathway, with the exception of a Lactobacillus strain [12]. This was exploited in the development of the novel method that allows successful differentiation between C. difficile culture-positive and -negative stool samples based on VOC generation from an enzyme substrate [13]. 3-Fluoro-4-hydroxyphenylacetic acid was used as a substrate for p-hydroxyphenylacetate decarboxylase; the evolution of the VOC 2-fluoro-4-methylphenol indicated the presence of C. difficile (Fig. 1). VOCs were detected using HS-SPME-GC-MS.
The lack of specificity of previous GC methods was often due to the detection of VOCs in C. difficile culture-negative stool samples as these VOCs were generated by commensal bacteria. Techniques employed to reduce background flora, and therefore improve the selectivity and sensitivity of methods, include alcohol shock [14] and the inclusion of antibiotics [5]. The antibiotics D-cycloserine, cefoxitin and amphotericin were added to the sample matrix and an alcohol-shock step was included to suppress background flora present in stool samples. Alcohol shock kills vegetative cells but does not affect the viability of spores and incorporation of sodium taurocholate in a culture medium can subsequently aid germination of spores [6, 15]. Inoculation of stool samples into such a culture medium allows bacterial growth and concomitant generation of VOCs.
The method was tested with 100 stool samples, of which 77 were C. difficile culture-positive and 23 culture-negative. The generation of 2-fluoro-4-methylphenol indicated the presence of C. difficile after overnight incubation. Method specificity and sensitivity were 100 % and 83.1 %, respectively, using 2-fluoro-4-methylphenol as a marker for C. difficile identification (Table 1). The VOCs isocaproic acid and p-cresol were useful indicators for C. difficile-positive stool samples, although were insufficient for identification purposes. Both VOCs, particularly p-cresol, were generated by C. difficile-negative samples; this is in agreement with previous studies [8].
Advantages and disadvantages of VOC method
The method allows the detection of C. difficile with a very high specificity (100%), i.e. 2-fluoro-4-methylphenol was not generated by C. difficile culture-negative stool samples tested. Rapid detection of VOCs was possible with confirmation of the presence of C. difficile within 18 hours. This indicates that the method could be used to screen for C. difficile in stools allowing the prompt diagnosis of culture-positive samples by the detection of 2-fluoro-4-methylphenol. However, a study on method sensitivity in terms of the number of bacterial cells required to generate a positive signal confirmed that identification of C. difficile was possible provided the stool sample contained at least 150 colony forming units (CFU). It is entirely possible that some stool samples will contain much fewer CFU and therefore 2-fluoro-4-methylphenol would not be detected and a false-negative result would be obtained. This limitation is reflected in the method sensitivity (83.1%) after evaluation with 100 stool samples. The method targets all strains of C. difficile and further testing would be required (e.g. using PCR or immunoassay) to distinguish whether positive stool samples contain toxigenic strains. As a result, it is recommended that VOC analysis should be used alongside conventional methods for C. difficile detection, including toxin detection methods, which would allow any false negative results to be eliminated.
Conclusion
C. difficile is a common cause of nosocomial infections and therefore rapid, accurate diagnosis of CDI is of extreme importance for infection control and patient care. There are currently a number of methods used in hospital laboratories for the diagnosis of CDI; however, each method has its drawbacks. A novel approach has been developed for the identification of C. difficile in stool samples that involves the incubation of stool samples in the presence of 3-fluoro-4-hydroxyphenylacetic acid which acts as a substrate for the enzyme p-hydroxyphenylacetate decarboxylase. The success of this new approach is evaluated by its application to 100 stool samples and its ability to differentiate between C. difficile culture-positive and -negative stool samples. It is envisaged that the identification of C. difficile culture-positive stool samples by the analysis of VOCs could allow rapid diagnosis of CDI. In addition, the novel approach of using enzyme substrates that release VOCs that are not normally generated by bacteria, for example fluorinated VOCs, may find application in the identification of other bacterial pathogens in clinical microbiology.
References
1. Public Health England. Summary Points on Clostridium difficile Infection (CDI). 2013; http://www.hpa.org.uk/webc/HPAwebFile/HPAweb_C/1278944283388.
2. Kuijper EJ, Coignard B, Tull P. Emergence of Clostridium difficile-associated disease in North America and Europe. Clin Microbiol Infect. 2006; 12(Suppl 6): S2−S18.
3. Voth DE, Ballard JD. Clostridium difficile toxins: mechanism of action and role in disease. Clin Microbiol Rev. 2005; 18: 247–263.
4. Eastwood K, Else P, Charlett A, Wilcox M. Comparison of nine commercially available Clostridium difficile toxin detection assays, a real-time PCR assay for C. difficile tcdB, and a glutamate dehydrogenase detection assay to cytotoxin testing and cytotoxigenic culture methods. J Clin Microbiol. 2009; 47: 3211–3217.
5. George WL, Sutter VL, Citron D, Finegold SM. Selective and differential medium for isolation of Clostridium difficile. J Clin Microbiol. 1979; 9: 214–219.
6. Nerandzic MM, Donskey CJ. Effective and reduced-cost modified selective medium for isolation of Clostridium difficile. J Clin Microbiol. 2009; 47: 397–400.
7. Darkoh C, Kaplan HB, DuPont HL. Harnessing the glucosyltransferase activities of Clostridium difficile for functional studies of toxins A and B. J Clin Microbiol. 2011; 49: 2933–2941.
8. Levett PN. Detection of Clostridium difficile in faeces by direct gas liquid chromatography. J Clin Pathol. 1987; 37: 117–119.
9. Garner CE, Smith S, Costello BL, White P, Spencer R, Probert CSJ, Ratcliffe NM. Volatile organic compounds from feces and their potential for diagnosis of gastrointestinal disease. Faseb J. 2007; 21: 1675–1688.
10. Orenga S, James AL, Manafi M, Perry JD, Pincus DH. Enzymatic substrates in microbiology. J Microbiol Meth. 2009; 79: 139–155.
11. Selmer T, Andrei PI. p-Hydroxyphenylacetate decarboxylase from Clostridium difficile. A novel glycyl radical enzyme catalysing the formation of p-cresol. Eur J Biochem. 2001; 268: 1363–1372.
12. Yokoyama MT, Carlson JR. Production of skatole and para-cresol by a rumen Lactobacillus sp. Appl Environ Microbiol. 1981; 41; 71–76.
13. Tait E, Hill KA, Perry JD, Stanforth SP, Dean JR. Development of a novel method for detection of Clostridium difficile using HS-SPME-GC-MS. J Appl Microbiol. DOI: 10.1111/jam.12418.
14. Clabots CR, Gerding SJ, Olson MM, Peterson LR, Gerding DN. Detection of asymptomatic Clostridium difficile carriage by an alcohol shock procedure. J Clin Microbiol. 1989; 27: 3286–3287.
15. Wilson KH, Kennedy MJ, Fekety FR. Use of sodium taurocholate to enhance spore recovery on a medium selective for Clostridium difficile. J Clin Microbiol. 1982; 15; 443–446.
The authors
Emma Tait1 PhD, Stephen P. Stanforth1 PhD, John D. Perry2 PhD and John R. Dean1* DSc, PhD
1Faculty of Health & Life Sciences, Department of Applied Sciences, Northumbria University, Newcastle-upon-Tyne, UK
2Department of Microbiology, Freeman Hospital, Newcastle-upon-Tyne, UK
*Corresponding author
E-mail: John.dean@northumbria.ac.uk
Uncovering molecular biomarkers in gastric adenocarcinoma
Gastric adenocarcinoma is usually diagnosed at an advanced stage, which portends a poor prognosis. Molecular biomarkers are important tools to understand the underlying biology of its aggressive behaviour and to discover new targets for therapeutic agents. Microarray analyses and next generation sequencing are leading to a deeper understanding of tumour biology and the development of new biomarkers, offering hope for better treatment approaches in the future.
by Dr I. Snitcovsky, Dr F. Solange Pasini and Dr G. de Castro Jr
Background
Gastric cancer is the fourth most common cancer in the world and is especially prevalent in East Asia and South America [1]. Adenocarcinoma accounts for the great majority of these tumours, which are classified as intestinal or diffuse type. The pathogenesis is incompletely understood, but it is associated with Helicobacter pylori infection and dietary salt and nitrosamines, particularly in intestinal type tumours. In these cases, chronic inflammation is thought to lead to preneoplastic lesions that may progress to invasive cancer in a stepwise fashion. In a minority of cases, germline mutations of P53, CDH1 and mismatch repair genes are associated with familial cases. The most important prognostic factor is the tumour TNM stage, since the only curative approach is surgical resection, followed (or not) by adjuvant therapies. Thus, locally advanced and metastatic disease portends a poor prognosis, with a median survival of less than one year. Unfortunately, most patients in Western countries are diagnosed with advanced disease, and, in these cases, chemotherapy can palliate symptoms and prolong overall survival but it is not curative [2]. In patients with metastatic disease, the only biomarker routinely tested for in gastric adenocarcinoma is HER2 (human epidermal growth factor 2 receptor; by immunohistochemistry), which is associated with poor outcomes and is also predictive of the anti-tumour efficacy of the humanized anti-HER2 antibody, trastuzumab [3].
The development of innovative treatment approaches begins with the identification of molecular biomarkers relevant to tumour biology. The next step is clinical validation, usually by showing that the studied biomarker has a prognostic value. Finally, a targeted agent is developed and shown to prolong survival in phase III clinical trials. Genome-wide studies are revealing potential biomarkers for targeted therapies and immunotherapy. This review will focus on recently identified candidate biomarkers in gastric adenocarcinoma with potential clinical applications.
Biomarkers in the pre-genomic era
Cancer cells are characterized by self-sufficiency in growth signals, insensitivity to anti-growth signals, evasion of apoptosis, limitless reproductive potential, sustained angiogenesis and tissue invasion and metastasis [4]. Accordingly, in gastric adenocarcinoma, a great number of studies focused on the prognostic role of single molecules. They included, but were not restricted to, growth factors and their receptors [HER2, IGFR (insulin-like growth factor 1 receptor)], cell cycle regulators (p53), angiogenesis controllers [VEGF (vascular endothelial growth factor)] and matrix metalloproteinases, with so far no impact in patient management, with the exception of HER2.
The epidermal growth factor receptor (EGFR) family includes HER1 (EGFR), HER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). These molecules form dimers on the cell surface after ligand binding, which leads to intracellular signalling that modulates cell proliferation, metastasis and angiogenesis. HER2 has no known ligands, but forms heterodimers with other members of the HER family and potentiates signalling. In breast cancer, HER2 overexpression is related to poor prognosis and the humanized anti-HER2 monoclonal antibody trastuzumab prolongs survival in those patients with HER2 positive tumours [5]. In gastric adenocarcinoma, HER2 overexpression is detected in 9–35% of cases and implies a worse prognosis in some studies. A phase III trial that included patients with HER2-overexpressing gastric adenocarcinoma found that the addition of trastuzumab to chemotherapy resulted in an overall survival benefit of about two months, as compared to chemotherapy alone [3]. In contrast, phase III studies evaluating anti-angiogenic agents in unselected gastric adenocarcinoma patients presented conflicting results [6, 7].
Gene panels and next generation sequencing
Gastric adenocarcinoma is a heterogeneous disease, thus the simultaneous determination of several biomarkers may be more informative then single ones, nowadays possible by high-throughput technologies as microarray platforms and next generation sequencing. Chen et al. [8] proposed a prognostic three-gene model, derived from gene expression profiling in eighteen paired samples. Marchet et al. [9] proposed another three-gene model predictive of lymph node involvement in a cohort of 32 patients. Another prognostic four-gene signature was also described [10]. Little overlap was observed among these above-mentioned signatures, which is not informative in terms of advancing in the cancer biomarker development.
A study comparing 248 gastric adenocarcinoma tumour samples was able to classify tumours in three subtypes, based on gene expression patterns: proliferative, metabolic and mesenchymal. In addition, these subtypes were shown to have differences in molecular and genetic features, and response to therapy [11]. Next generation sequencing is providing a deeper level of understanding the tumour biology. Genetic alterations were observed in Wnt, Hedgehog, cell cycle, DNA damage and epithelial-to-mesenchymal-transition pathways by analysing the genome and the transcriptome in 50 adenocarcinoma samples. About 20% of these alterations could be considered as potential targets for drugs that are already available [12]. Novel fusion genes were identified, especially DUS4L-BCAP29, when transcriptome sequencing was performed in 12 gastric adenocarcinoma cell lines. Knockdown of this transcript inhibited cell proliferation, thus validating its functional role [13].
Immune biomarkers
Cancer, including gastric adenocarcinoma, is viewed as a tissue disease. This implies that the microenvironment plays a key role in tumour biology [4]. Thus, immune cell infiltrate has been shown to be of prognostic value in gastric adenocarcinoma. As depicted in Figure 1, tumour-associated macrophages present two different polarizations: classical (M1) characterized by immunostimulation activity and tumour suppression; and alternative (M2) characterized by tumour promotion and immune suppression. A higher ratio of M1/M2 macrophages was associated with a favourable prognosis [14]. The underlying mechanism is complex, but may involve growth factor modulation [15]. We conducted a gene expression study, including a total of 51 freshly frozen tumour samples from patients with gastric adenocarcinoma treated with surgery. An immune-related gene trio (OLR1, CXCL11 and ADAMDEC1) was identified as an independent biomarker of prognosis. We proposed that immune dampening in the tumour microenvironment was present in patients with poor prognosis. Three main observations supported our hypothesis. First, the expression levels of genes belonging to the functional group of immune/inflammatory response were markedly reduced as a whole. Second, a network analysis suggested an unwired inflammatory response, and third, a decreased expression of type-1 helper lymphocyte (Th1) and other immune activating genes was found [16]. The biomarkers we identified may be good candidates for selecting patients for immunomodulation therapies, including immune checkpoint inhibitors [17].
Conclusions and perspectives
Gastric adenocarcinoma needs better treatment approaches. New technologies are offering the necessary tools to identify molecular biomarkers, leading to a deeper understanding of tumour biology and the development of innovative treatment strategies, and we are entering an era of cautious optimism. Considering the tumour heterogeneity and the limited survival gains with targeted agents in solid tumours, it is possible that patient selection by immune biomarkers and the use of immune checkpoint inhibitors are promising alternatives. The impressive response rates and overall survival benefits observed in patients with squamous cell lung cancer and melanoma, two notoriously chemoresistant tumours, when treated with anti-PD1 or anti-PD1L are good examples [18].
References
1. Ferlay J, et al. GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11; 2013. (http://globocan.iarc.fr)
2. Waddell T, et al. Gastric cancer: ESMO-ESSO-ESTRO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2013;24(Suppl 6):57.
3. Bang YJ, et al. Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer. Lancet 2010;376: 687.
4. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144:646.
5. Figueroa-Magalhães MC, et al. Treatment of HER2- positive breast cancer. Breast 2013;doi:10.1016/j.breast.2013.11.011.
6. Ohtsu A, et al. Bevacizumab in combination with chemotherapy as first-line therapy in advanced gastric cancer: a randomized, double-blind, placebo-controlled phase III study. J Clin Oncol. 2011;29:3968.
7. Fuchs CS, et al. Ramucirumab monotherapy for previously treated advanced gastric or gastro-oesophageal junction adenocarcinoma (REGARD): an international, randomised, multicentre, placebo-controlled, phase 3 trial. Lancet 2014;383:31.
8. Chen CN, et al. Gene expression profile predicts patient survival of gastric cancer after surgical resection. J Clin Oncol. 2005;23:7286.
9. Marchet A, et al. Gene expression profile of primary gastric cancer: towards the prediction of lymph node status. Ann Surg Oncol. 2007;14:1058.
10. Xu ZY, et al. Gene expression profile towards the prediction of patient survival of gastric cancer. Biomed Pharmacother. 2010;64:133.
11. Lei Z, et al. Identification of molecular subtypes of gastric cancer with different responses to PI3-kinase inhibitors and 5-fluorouracil. Gastroenterology 2013; 145:554.
12. Holbrook JD, et al. Deep sequencing of gastric carcinoma reveals somatic mutations relevant to personalized medicine. J Transl Med. 2011;9:119.
13. Kim HP, et al. Novel fusion transcripts in human gastric cancer revealed by transcriptome analysis. Oncogene 2013;doi:10.1038/onc.2013.490.
14. Pantano F, et al. The role of macrophages polarization in predicting prognosis of radically resected gastric cancer patients. J Cell Mol Med. 2013;17:1415.
15. Cardoso AP, et al. Macrophages stimulate gastric and colorectal cancer invasion through EGFR Y(1086), c-Src, Erk1/2 and Akt phosphorylation and smallGTPase activity. Oncogene 2013;doi:10.1038/onc.2013.154.
16. Pasini FS, et al. A gene expression profile related to immune dampening in the tumor microenvironment is associated with poor prognosis in gastric adenocarcinoma. J Gastroenterol. 2013;doi:10.1007/s00535-013-0904-0.
17. Eggermont AM, et al. Immunotherapy and the concept of a clinical cure. Eur J Cancer 2013;49: 2965.
18. Brahmer JR, et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med. 2012;366:2455.
The authors
Igor Snitcovsky1,2 MD, PhD; Fátima Solange Pasini1,2 PhD; and Gilberto de Castro Jr*1,3 MD, PhD
1 Departamento de Radiologia e Oncologia, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brazil
2 Centro de Investigação Translacional em Oncologia, Instituto de Câncer do Estado de São Paulo (ICESP), São Paulo, Brazil
3 Oncologia Clínica, Instituto do Câncer do Estado de São Paulo (ICESP), São Paulo, Brazil
*Corresponding author
E-mail: gilberto.castro@usp.br
Measuring renal function: traditional methods and iohexol clearance
An accurate measure of glomerular filtration rate (GFR) is required in a variety of clinical situations. Plasma creatinine and creatinine-based equations have served as convenient estimates of GFR but they are subject to limitations. Formal assessment of GFR using clearance methods traditionally necessitates the use of radioactive traces such as 51Cr-EDTA. More recently, iohexol has become increasingly well recognized as an alternative exogenous marker which can now be used routinely, providing a safe, efficient and cost-effective measure.
by Zoe Maunsell and Prof. Tim James
Background
Glomerular filtration rate (GFR) describes the amount of fluid filtered by the glomerulus per unit time, and is therefore a useful indicator of renal function. Expressed in mL/min, it can be accurately measured from the rate of disappearance of an injected substance from the plasma or by collecting urine over a defined time period. The clearance formula, UV/P (where U represents urine concentration, V is urine volume per unit time and P is plasma concentration) can be used to calculate GFR when an ideal marker is used. Such a marker should be freely filtered at the glomerulus, and not secreted, absorbed or metabolized by the renal tubules. Since renal function is proportional to kidney size (which is proportional to body surface area), when estimating GFR, values are usually standardized to an average body surface area of 1.73 m2. Having an accurate yet convenient means to monitor renal function is extremely important in clinical practice. A compromise exists between highly accurate but time consuming, technically difficult reference methods and more accessible, readily available markers of renal function.
Gold standard methods
The ‘gold standard’ for the measurement of GFR is inulin clearance. Inulin is a polysaccharide derived from plants, which can be introduced into the body either by intravenous infusion or bolus dose and its rate of clearance measured. Since inulin is freely filtered by the glomerulus, is not absorbed, secreted or metabolized by the tubules, its elimination is proportional to GFR. Although it is the ‘gold standard’ for assessment of GFR, assay of inulin is technically demanding, and so this technique is not suitable for routine clinical practice.
Similarly, the radioactive tracer 51Cr-EDTA can be administered intravenously and its elimination rate monitored. This marker is widely used to measure GFR, but presents a risk to patients and healthcare workers since it involves exposure to ionizing radiation.
Iohexol clearance is an alternative to inulin clearance [1]. Iohexol is an iodine-containing, non-isotopic contrast agent, and studies have shown close agreement between GFR values obtained by iohexol and inulin clearance.
Creatinine and endogenous markers
Creatinine is a widely used clearance marker but suffers from well-described problems including the difficulty and inconvenience of 24-hour urine collection.
GFR has therefore been estimated without urine collection using endogenous markers, the most widely used of which is plasma/serum creatinine. Improvements have been made to the methodology used for creatinine measurement, including standardization of kinetic Jaffe creatinine assays, defining their traceability to isotope-dilution reference assays. However, the Jaffe method remains subject to numerous interferences and therefore laboratories may select the more specific enzymatic creatinine methods. Using creatinine as a marker of GFR has several limitations, including the relationship of creatinine concentration to muscle mass, meaning that estimation of GFR using creatinine alone is particularly a problem in children. It is for this reason that corrections for body surface area have been made. The major limitation of creatinine alone as a marker of GFR is that it is a relatively insensitive marker, and a large decline in renal function can occur before a change in plasma creatinine concentration is observed.
Cystatin C is another endogenous marker that can be used for the estimation of GFR. It is a cysteine protease inhibitor, is produced at a constant rate (independent of muscle mass, sex, age when older than 12 months and inflammatory conditions) and is freely filtered by the kidneys. It is almost completely reabsorbed by the proximal renal tubular cells so that little is normally excreted in the urine. The reciprocal of plasma cystatin C concentration has been shown to be correlated with GFR. Cystatin C has significant advantages over creatinine as a marker of renal function, since increases in serum concentration become apparent with mild renal impairment, such as GFR of 60–90 mL/min, and may be more useful than creatinine in detecting acute kidney injury. Like creatinine, the measurement of cystatin C suffers to some extent from problems with standardization, in particular with reported differences [2] in measured concentrations using turbidimetric versus nephelometric assays by different manufacturers, presumably due to the use of different antibody and detection systems. The availability of an international reference preparation (ERM–DA471/IFCC) is likely to lead to greater agreement between cystatin C methods.
Calculated estimates of GFR
In order to overcome the limitations of measuring creatinine and cystatin C concentrations alone, calculations have been devised. These involve corrections for body surface area, sex, age and ethnicity. Among the most well known are the 4- or 6-parameter MDRD (Modification of Diet in Renal Disease study), Cockroft–Gault (which estimates creatinine clearance) and CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) equations for adults and the Schwartz and the Counahan–Barratt prediction equations for children.
The Cockroft–Gault equation [3] provides an estimate of creatinine clearance. Devised in the 1970s, the equation was calculated by examining the mean of two 24-hour creatinine clearances from 249 adult men.
The 4-variable MDRD equation [4] (incorporating creatinine, age, sex and race) is the most widely used in the UK, being reported by laboratories in the form of eGFR, and forms the basis of the staging system for CKD. The formula was developed from the Modification of Diet in Renal Disease study, using data from 1628 patients. The patients had already been diagnosed with CKD, and the study aims were to slow the progression of CKD with a low protein diet and blood pressure control. However, significant limitations to the use of the equation exist, mainly connected with the use of creatinine as a marker of GFR. These include inaccuracies of the equation at extremes of body type such as malnourished or amputee patients and the unsuitability of the equation for use in pregnancy. The formula includes a correction factor for use in African American populations but its validity in other ethnic groups has not been established. The MDRD formula tends to underestimate function at normal GFR, therefore slight reductions in eGFR should not be over-interpreted and reporting eGFR >90mLs/min/1.73m2 is not recommended.
The CKD-EPI equation was developed in 2008 [5] and updated in 2012 [6]. The 2008 equation was developed using data from over 8000 patients from 10 studies. The 2012 versions used over 5000 patients from 13 studies. Importantly, both studies included patients with normal GFR, as well as those with CRF. It was found that the CKD-EPI equations performed better than the MDRD equation, particularly at higher levels of estimated GFR. The limitations of the studies include a limited number of elderly patients and those from ethnic groups other than black race.
In terms of estimation of GFR in children, the Schwartz [7] and Counahan–Barratt [8] equations were developed in the 1970s and have been widely used to estimate GFR from serum creatinine and height (length in infants). The Schwartz equation was modified in 2009 [ 9, 10]. Based on new studies of iohexol clearance, the original formula was found to overestimate GFR. It was postulated that this was in part due to the use of new, standardized creatinine assays. In 2009 the equation was updated, based on studies of 349 patients aged 1–16 years with mild to moderate chronic renal disease. The formula uses creatinine determined using an enzymatic method, urea, height and cystatin C.
Various modifications of these equations, using creatinine, cystatin C [11] or both [12] have been published and optimized in various patient cohorts. When using creatinine-based assays, it is important to know which creatinine assay is being used, since equations have been devised which include different coefficients depending on the methodology used.
Iohexol clearance: a gold standard method in routine use
Oxford University Hospitals NHS Trust has been routinely offering an iohexol clearance service for the measurement of GFR since June 2011. The service is widely used by the hospital’s pediatric service, and is used in a variety of clinical situations, including determining renal function in surgical patients and for chemotherapy dosing. Before the introduction of this service, GFR was assessed using 51Cr EDTA clearance. Although this provided an accurate measurement of GFR, a long waiting list and difficult patient preparation made this technique suboptimal for use in children. In addition, where GFR results are required before administration of chemotherapy agents, timely result availability is critical to prevent delayed treatment. With iohexol clearance we have been able to improve GFR turnaround time compared to 51Cr-EDTA clearance and audit of our first 2 years of service demonstrated that 89% of results were reported within 2 working days and 99% within 3 working days. Iohexol clearance also avoids the use of radioactive isotopes, reducing exposure for patients, carers and hospital staff. Therefore the service has several distinct advantages over conventional isotopic clearance methods (Table 1).
Iohexol clearance is measured according to a standardized protocol, as described previously [13]. In summary, the patient is cannulated, a baseline blood specimen is collected and a standard dose of Omnipaque containing 300 mg iodine/mL is administered through the cannula, (2 mL in patients weighing <40 kg, 5 mL in patients weighing >40 kg). Blood (1 mL) is collected into lithium heparin tubes at 120, 180 and 240 min after iohexol administration.
The iohexol concentration in each specimen is measured by a UPLC (ultra-high performance liquid chromatography) method involving precipitation of plasma samples with equal volumes of perchloric acid and injection of the supernatant onto a Waters 50 x 2.1 mm 1.8 μm reversed phase column with isocratic acetonitrile-based solvent elution. The assay uses a one-point (604 mg/L) calibration through zero. The assay is straighfoward, reliable and rapid; chromatography time is 6 min per specimen. The assay demonstrates excellent inter-assay CVs: 2.2%, 1.9% and 1.9% at 39, 163 and 322 mg/L respectively. The laboratory participates in external quality assessment through the Scandinavian EQUALIS scheme.
The two-point model is used to calculate iohexol clearance, according to the Brochner–Mortensen method [14]. The model assumes a one-compartment model where iohexol is cleared by first-order kinetics. The estimated clearance (Cl ) of the GFR marker is expressed as:
Cl= Q• b/c1 (ml/min)
Where Q = amount of injected marker, b = disappearance rate of marker (min−1), c1= intercept on the y-axis.
The clearance correction for non-immediate mixing of the tracer substance is expressed as:
Cl = 0.990778 x estimated Cl−0.001218• estimated Cl2
Alternatively, single point estimates of GFR at appropriate time points can be calculated using the Jacobsson model. This method can be used in cases where the two-point method cannot be used, for example when contamination has occurred due to poor flushing of lines between sample collection. Here:
GFR = ln(D/VCt) / (t/V + 0.0016)
Where V = volume of distribution in mL (V = 187 x weight in kg + 732), t = time of sampling in min, D = dose of iohexol administered, Ct = concentration of iohexol at time t.
Iohexol clearance is corrected for actual body surface area, using the Dubois and Dubois method, calculated as 0.007184 x (weight in kg)0.425 x (height in cm)0.72, to yield a measure of GFR in mL/min/1.73 m2.
Since initiating the iohexol GFR service in 2011, we have analysed over 400 sets of patient samples. The use of UPLC over HPLC allows for smaller sample volumes to be collected, making the technique particularly suited to pediatrics. Ongoing close liaison with clinicians and nursing staff has revealed that patients and their families have responded favourably to the new procedure and in particular prefer being able to remain on the Daycare Ward throughout the investigation, which is a child-friendly environment.
Iohexol clearance has the added benefit of being significantly less costly than 51Cr-EDTA clearance. The approximate cost is £80 compared to £250 for 51Cr-EDTA.
Conclusions
The measurement of glomerular filtration rate, GFR, is important in a number of clinical scenarios. Much work has been undertaken to develop methods of estimating GFR in order to avoid the time-consuming and relatively invasive formal measurement of renal function using clearance methods. These include the development of eGFR equations based on factors such as plasma creatinine, cystatin C, age, race and body surface area. Equations such as the MDRD and CKD-EPI formulae for adults, and Schwartz and Counahan–Barratt equations for children have been widely used in clinical practice.
However, despite their widespread use, limitations of these equations have been described, including the problems of creatinine-based equations tending to underestimate GFR at normal GFR levels and the issue of creatinine-based formulae being unsuitable for use in patients with non-normal muscle mass.
For the formal measurement of GFR, clearance studies must be performed which have traditionally used radioactive tracers such as 51Chromium-EDTA. However, in recent years the use of iohexol has increased. Thanks to its stability in vitro and relative ease of measurement, the assay of iohexol is rapid, reliable and can be performed on small specimens. This fits in particularly well with the clinical needs of a pediatric service for GFR measurement and is cheaper, safer and more convenient than traditional methods.
References
1. Ng DK, Schwartz GJ, Jacobson LP, Palella FJ, Margolick JB, Warady BA, Furth SL, Muñoz A. Universal GFR determination based on two time points during plasma iohexol disappearance. Kidney Int. 2011; 80: 423–430.
2. Herget-Rosenthal S, Bökenkamp A, Hofmann W. How to estimate GFR-serum creatinine, serum cystatin C or equations? Clin Biochem. 2007; 40: 153–161.
3. Cockcroft DW, Gault MH. Prediction of creatinine clearance from serum creatinine. Nephron 1976; 16: 31–41.
4. Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D. A more accurate method to estimate glomerular filtration rate from serum creatinine: A new prediction equation. Modification of Diet in Renal Disease Study Group. Ann Intern Med. 1999; 130: 461–470.
5. Levey AS, Stevens LA, Schmid CH, Zhang YL, Castro AF III, Feldman HI, Kusek JW, Eggers P, Van Lente F, Greene T, Coresh J, for the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI). A new equation to estimate glomerular filtration rate. Ann Intern Med. 2009; 150: 604–612.
6. Inker LA, Schmid CH, Tighiouart H, Eckfeldt JH, Feldman HI, Greene T, Kusek JW, Manzi J, Van Lente F, Zhang YL, Coresh J, Levey AS, for the CKD-EPI Investigators. Estimating glomerular filtration rate from serum creatinine and cystatin C. N Engl J Med 2012; 367: 20–29.
7. Schwartz GJ, Haycock GB, Edelmann CM Jr, Spitzer A. A simple estimate of glomerular filtration rate in children derived from body length and plasma creatinine. Pediatrics 1976; 58: 259–263.
8. Counahan R, Chantler C, Ghazali S, Kirkwood B, Rose F, Baratt TM. Estimation of glomerular filtration rate from plasma creatinine concentration in children. Arch Dis Child 1976; 51: 875–878.
9. Schwartz GJ, Muñoz A, Schneider MF, Mak RH, Kaskel F, Warady BA, Furth SL. New equations to estimate GFR in children with CKD. J Am Soc Nephrol. 2009; 20: 629–637.
10. Schwartz GJ, Work DF. Measurement and estimation of GFR in children and adolescents. Clin J Am Soc Nephrol 2009; 4: 1832–1843.
11. Grubb A, Nyman Un, Björk J, Lindström V, Rippe BG, Christensson A. Simple cystatin C–based prediction equations for glomerular filtration rate compared with the modification of diet in renal disease prediction equation for adults and the Schwartz and the Counahan–Barratt prediction equations for children. Clinical Chemistry 2005; 51: 1420–1431.
12. The Swedish Council on Health Technology Assessment (SBU). Methods to estimate and measure renal function (glomerular filtration rate). Stockholm: The Swedish Council on Health Technology Assessment (SBU). 2012; Yellow 214.
13. James TJ, Lewis AV, Tan GD, Altmann P, Taylor RP, Levy JC. Validity of simplified protocols to estimate glomerular filtration rate using iohexol clearance Ann Clin Biochem. 2007: 44; 369–376.
14. Bröchner-Mortensen J. A simple method for the determination of glomerular filtration rate. Scand J Clin Lab Invest. 1972; 30(3): 271–274.
The authors
Zoe Maunsell* MBiochem MSc DipRCPath and Tim James PhD
Department of Clinical Biochemistry, Oxford University Hospitals NHS Trust, Oxford, UK
*Corresponding author
E-mail: zoe.maunsell@ouh.nhs.uk
H. pylori infection: a laboratory perspective
Infection with Helicobacter pylori is indicated in disorders of the upper gastrointestinal tract, from dyspepsia and ulcer disease to gastric carcinoma. It can be detected during endoscopy or non-invasively using breath, stool or blood samples, each of which has advantages and limitations depending on patient and population circumstances.
by Sarah Knowles and Julia M. Forsyth
Helicobacter pylori
Helicobacter pylori is probably the most common cause of bacterial infection in humans, present in up to 50% of the world’s population [1]. The presence of such a microorganism in the stomach was first reported almost 100 years ago [2] but was not taken seriously until Marshall and Warren demonstrated a strong association between the presence of an unidentified curved bacillus and inflammation on a gastric biopsy [3]. The organism was initially placed in the Campylobacter genus but as further morphological, structural and genetic information was determined a new genus called Helicobacter was created. After identification, Marshall demonstrated the role of H. pylori in antral gastritis by self-administration of the bacteria and also showed that it could be cleared by the use of antibiotics and bismuth salts.
H. pylori and disease
Infection with H. pylori is known to be a contributing factor in producing gastritis [4]. Studies have shown that infection with H. pylori leads to increased release of gastrin by the antral mucosa (through a mechanism that is as yet undefined), which resolves on eradication [5, 6]. There is a high prevalence of H. pylori positive chronic gastritis in patients with duodenal and gastric ulcers (70% and 90% respectively) [6, 7]. These ulcer patients have been shown to have an exaggerated response to the increased gastrin, even compared to asymptomatic H. pylori-positive patients [6], leading to excess acid production and a deterioration from the initial inflammation of gastritis to mucus layer erosion by peptic acid. H. pylori is also linked with gastric carcinomas and mucosa-associated lymphoid tissue (MALT) lymphomas with one study showing that 62% of patients with low-grade gastric MALT lymphoma entering complete remission 12 months after H. pylori eradication therapy [8]. The vast majority of individuals with H. pylori infection, however, do not have ulcerative disease, but are symptomless carriers [4].
The survival capabilities of H. pylori in the stomach make it difficult to eradicate. Effective treatment requires a multidrug ‘triple therapy’ regime consisting of two antibiotics from clarithromycin, metronidazole, amoxicillin and tetracycline combined with an acid suppressant or bismuth compounds [9], and in areas of high clarithromycin resistance, quadruple therapy including two antibiotics, proton pump inhibitors (PPIs) and bismuth [10].
Testing for H. pylori
The definitive identification of H. pylori is by histological examination of biopsies stained with Giesma stain or Warthin–Starry silver impregnation [4]. Other biopsy immunohistochemical techniques using specific antibodies against H. pylori are also available along with rapid biopsy urease tests (otherwise known as the CLO-test), where a biopsy specimen is placed in a urea broth containing a pH indicator. Testing by any of these methods requires endoscopy which, apart from being very unpleasant for the patient, carries risk and is very costly, and is therefore unsuitable for routine screening for H. pylori in patients with a low risk of cancer. In these cases a ‘test and treat’ strategy is recommended using non-invasive tests (Fig. 1) [10].
There are three common non-invasive tests available for the routine detection of H. pylori:
1. The urea breath test (UBT)
2. Stool test for H. pylori antigen
3. Serum test for H. pylori antibody.
Urea breath test
The UBT is generally considered to be the ‘gold standard’ non-invasive test and is recommended as the first-choice screening test both in the diagnosis of infection and post-eradication testing [9–11].
Urea labelled with 13C is taken as a drink or a capsule and urease produced by H. pylori acts upon the urea to produce 13C-labelled carbon dioxide. This is absorbed into the blood stream and exhaled in the breath. The ratio of 13CO2 : 12CO2 is measured using an isotope ratio mass spectrometer (IRMS), and the ratios before and after the administration of urea are compared to give the result.
Apart from being the ‘gold standard’ test the other main advantage of the UBT is the stability of the breath samples once collected. This allows storage without refrigeration and transportation to the central referral laboratory for analysis. In the UK this permits sample collection at local health centres rather than hospital settings, which is convenient for patients. Worldwide this can extend to large, remote areas with limited facilities or courier transport links to laboratories, for example rural Africa and Australia, permitting testing in all communities. Local studies have also shown the test to be very popular with patients as sample collection is simple and pain free [12].
A disadvantage of the UBT is that PPIs and antibiotics need to be withheld for 2 and 4 weeks respectively before testing to avoid potential false-negative results as a consequence of decreased bacterial load [10]. PPIs particularly are commonly prescribed to alleviate the symptoms of dyspepsia and patients can be reluctant to withhold such medication pending tests. As with all testing cost is also an important issue. IRMS is a specialized laboratory technique requiring expensive equipment and highly trained laboratory personnel. These fixed costs can make the UBT an expensive option but running costs and reagents for such equipment are relatively low, and therefore centralized high workload laboratories can provide a very cost-effective service [12].
Stool antigen test
The stool antigen test uses enzyme-linked immunosorbent assay (ELISA) methodology to detect H. pylori antigen. ELISA technology is available in many laboratories, thus making the test easy to implement at local laboratories. It is also reported to be more cost effective than the UBT [13], although local method validation of commercially available ELISA kits (for a workload of 3500 tests per year) showed the costs to be almost identical when samples were analysed in duplicate [12]. Duplicate analysis was recommended due to poor replicate precision, probably as a result of the heterogeneity of stool in the samples. As workload increases, e.g. at centralized laboratories, the UBT actually becomes the cheaper alternative because of the low running/reagent costs [12]. A final advantage of the stool antigen test is that there is no need to attend a clinic or phlebotomy appointment for sample collection. The patient can collect the sample at home and deliver it to their GP/local courier collection or laboratory at their convenience.
Current UK guidelines for the investigations do not recommend the stool antigen test in the post-eradication setting [9]. There is, however, no discrimination between polyclonal and monoclonal assays in these guidelines. Initial commercial ELISA assays were based upon polyclonal antibodies which showed reasonable performance in untreated patients with reported sensitivities of 91–93% and likewise specificities of 91–93% [14–16]. However, in the post-eradication setting results are less convincing with reported sensitivities ranging from 67–89% [14–19]. The introduction of monoclonal antibody assays has lead to an improvement in the diagnostic accuracy with reported sensitivities of 94–98% in both untreated adults and children [16, 20, 21], and pooled specificity of 97% [22]. Several more-recent studies using monoclonal antibody kits with favourable results have been published since the guidelines were drawn up with pooled sensitivities and specificities of 93% and 96% respectively [22]. This is reflected in the recent European study group findings which have recently been updated to include the monoclonal ELISA test as accurate in the post eradication setting, suitable for use if the UBT is not available [10], and local studies have validated the stool test as a suitable alternative to the UBT both in diagnosis and post-eradication settings [12].
The stool antigen test offers no further advantage to the UBT with regard to patient preparation as antibiotics and PPI medication should also be withheld before testing and locally the test is unpopular with patients who do not wish to provide stool specimens. Stability is also a disadvantage as specimens need to be refrigerated, and if not tested immediately, frozen within 48 hours [23]. Samples therefore need to reach the laboratory quickly or be transported frozen, providing logistical challenges in more remote locations.
Rapid point-of-care testing or ‘office’ stool antigen assays are available but have limited accuracy and are not recommended by current guidelines [10, 11].
Serum antibody test
The serum antibody test is an ELISA assay for IgG H. pylori antibodies, which again has the advantage of being a standard laboratory technique. It is also the cheapest testing option as reagent costs are low compared to the stool antigen test. Unfortunately this test has been shown to have poor diagnostic accuracy [11] especially in the post-eradication setting. This is thought to be due to the fact that the antibody levels persist in the blood for a long time and therefore lead to false-positive results after treatment. It may, however, have a place in the diagnosis of infection setting on occasions where PPI or antibiotic medications cannot be withheld, or for testing young children for whom the UBT is inappropriate and the likelihood of previous infection is low. Point-of-care or office-based serology tests are available, although their performance has been shown to be inadequate and they are not recommended for use in testing for H. pylori [10, 11].
Conclusion
In conclusion, H. pylori detection and eradication can lead to significant health benefits to the world’s population, not only alleviating life-restricting symptoms but also preventing the development of more serious disease. Several reliable methods of laboratory testing are now available, the choice of which depends on the patient population, facilities available, workload and networking possibilities and pre-existing medical conditions.
References
1. Cover TL, Blaser MJ. Adv Int Med. 1996; 41; 85–117.
2. Maden E, et al. Am J Clin Pathol. 1988; 90: 450–453.
3. Marshall BJ, Warren JR. Lancet 1984; 1(8390): 1311–1315.
4. Mera SL. Br J Biomed Sci. 1995; 52; 271–281.
5. Smith JTL, et al. Gut 1990; 31: 522–525.
6. El-Omar E, et al. Gut 1993; 34: 1060–1065.
7. Parsonner J, et al. N Engl J Med. 1991; 325: 1127–1131.
8. Fischbach W, et al. Gut 2004; 53: 34–37.
9. NICE. Dyspepsia. Management of dyspepsia in adults in primary care. Clinical Guideline 17 2004; http://www.nice.org.uk/nicemedia/pdf/CG017NICEguideline.pdf.
10. Malfertheiner P, et al. Gut 2012; 61: 646–664.
11. Malfertheiner P, et al. Gut 2007; 56: 772–781.
12. Research Studies, Pathology Department, Royal Derby Hospital. Data awaiting publication.
13. Elwyn G, et al. Br J Gen Pract. 2007; 57: 401–403.
14. Roth DE, et al. Clin Diagn Lab Immunol. 2001; 8: 718–723.
15. Gisbert JP, et al. Am J Gastroenterol. 2001; 96: 2829–2838.
16. Gisbert JP, et al. Helicobacter 2004; 9: 347–368.
17. Perri F, et al. Am J Gastroenterol. 2002; 97: 2756–2762.
18. Bilardi C, et al. Aliment Pharmacol Ther. 2002; 16: 1733–1738.
19. Erzin Y, et al. J Med Microbiol. 2005; 54: 863–866.
20. Koletzko S, et al. Gut 2003; 52: 804–806.
21. Weingart V, et al. J Clin Microbiol. 2004; 42: 1319–1321.
22. Gisbert JP, et al. Am J Gastroenterol 2006; 101: 1921–1930.
23. Oxoid Amplified IDEIA HPStAR Kit insert Ref K6630.
The author
Sarah Knowles MSc, FRCPath and Julia Forsyth MSc, FRCPath
Pathology Department, Royal Derby Hospital, Derby, UK
*Corresponding author
E-mail: Sarah.knowles@nhs.net
The rise and rise of vitamin D
With research demonstrating the impact of vitamin D on a range of conditions, the importance of adequate levels of vitamin D is often in the spotlight. This has resulted in increased rates of testing from both GPs and self-referring individuals.
by Robyn Shea and Dr Jonathan Berg
Background
Vitamin D is an essential nutrient required for bone health and calcium homeostasis. It is also described as a pro-hormone as it is the biologically inactive precursor to the active secosteroid hormone 1,25-dihydroxyvitamin D [1,25(OH)2D, also known as calcitriol] [1].
Vitamin D is found in two forms: vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Both forms are available as supplements or in a small number of foodstuffs, naturally occurring or fortified. However, the main source of vitamin D, around 90%, is through endogenous synthesis in the skin with the conversion of 7-dehydrocholesterol, via UVB radiation from the sun, into vitamin D3. Vitamin D2 or D3 are hydroxylated, first in the liver to form 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3] respectively, and secondly in the kidney to form the active hormone 1,25(OH)2D. The first hydroxylation step is unregulated and 25(OH)D levels therefore depend on the availability of the vitamin D substrate. The second hydroxylation step is tightly controlled via parathyroid hormone and through a variety of negative feedback mechanisms including calcium and phosphate levels as well as 1,25(OH)2D itself [2]. Comprehensive discussion about sources and metabolism of vitamin D are covered elsewhere in the literature [3, 4].
How is vitamin D measured?
25(OH)D is the best marker of vitamin D status. It is a difficult analyte to measure – it is very hydrophobic, is present in two forms [25(OH)D3 and 25(OH)D2] and is bound to vitamin D binding protein or albumin. Despite the difficulty in measuring it, a number of analytical platforms are available for 25(OH)D measurement:
- enzyme immunoassay (EIA)
- high pressure liquid chromatography (HPLC)
- radioimmunoassay (RIA)
- enzyme-linked immunosorbent assay (ELISA)
- liquid chromatography-tandem mass spectrometry (LC-MS/MS).
The agreement between the different methodologies is often poor and there can be quite large variation even within method groups, again highlighting how difficult it is to measure 25(OH)D. This has improved over time with the introduction of a NIST Standard Reference Material and the introduction of commercially available calibrators for chromatographic techniques [5].
Historically, manual techniques such as RIA and ELISA were used to measure 25(OH)D. Many laboratories have moved over to more automated random access immunoassays – either standalone automated analysers specifically for 25(OH)D analysis or as part of a large scale automated laboratory where 25(OH)D is just one test in a large repertoire – in order to cope with the ever increasing work load [6]. However, immunoassays, automated or not, have the disadvantages of performance changes over time, e.g. due to changes in kit reagents or antibody, and a large number of the assays are not able to detect both 25(OH)D3 and 25(OH)D2 and therefore may underestimate total 25(OH)D. Some of the immunoassays are also subject to interference from heterophilic antibodies and 24,25-dihydroxyvitamin D [5].
Direct detection methods such as HPLC and LC/MS-MS do not suffer from the disadvantages mentioned above; however, they can suffer from interference from the inactive 3-epi-25(OH)D isomer, which is often seen in high concentrations in infants. The equipment is expensive and specialist knowledge is required to set up and maintain a clinical service. However, the advantages of low consumables costs, potential for automation and high throughput, and the ability to detect both 25(OH)D2 and 25(OH)D3 explains its growing popularity with clinical laboratories [5].
Reasons for testing
Even though vitamin D can be made endogenously and is available through dietary sources, vitamin D deficiency is extremely common, verging on pandemic [7]. In inner-city Birmingham, 24% of the study population was found to be deficient [defined as having 25(OH)D levels <25 nmol/L], with 43% of Asian women deficient [8]. If vitamin D insufficiency is defined as having levels <75 nmol/L, then it has been estimated that 1 billion people worldwide are vitamin D deficient or insufficient [3].
Deficiency of vitamin D is most commonly linked with rickets in children and osteomalacia in adults, but in the last few years it has been associated with a range of non-musculoskeletal conditions such as diabetes, immune function, cardiovascular disease and cancer. As well as being deficient in vitamin D, it is possible to become intoxicated with vitamin D, often as the result of patients taking supraphysiological doses of vitamin D for a variety of reasons. Hypervitaminosis D can lead to hypercalcaemia as a result of increased intestinal calcium absorption and bone resorption, which can ultimately lead to kidney injury [9].
It is very hard to predict what a person’s vitamin D concentration is, even if you take into account factors such as age, supplement use, season, sun exposure, race and body mass intake [8]. However there are a range of risk factors and these can often help to identify people who are vitamin D deficient (Table 1) [7, 11–13].
Routine population screening is not advocated by most of the literature but there is still considerable debate as to when and who to test. The guidelines from the Endocrine Society suggest screening for vitamin D deficiency in individuals at risk for deficiency [7]. The National Osteoporosis Society guidelines suggest that vitamin D deficiency should be corrected before certain drug regimens begin, e.g. before starting treatment with a potent antiresorptive agent (zoledronate or denosumab) implying that 25(OH)D should be checked in these patients. They also recommend testing patients who have symptoms suggestive of osteomalacia or who have chronic widespread pain. They do not, however, recommend routinely testing 25(OH)D in asymptomatic individuals who may be at higher risk of vitamin D deficiency, but suggest that these individuals should have a higher intake of vitamin D [13]. The drawback of this approach is that the recommended daily allowance will not be enough to correct severe deficiency, and giving higher loading doses to those already replete may put them at risk of vitamin D intoxication.
Rate of testing
Many things in the area of vitamin D lack consensus, such as what is the optimal level of serum 25(OH)D; however, as far as the rate of testing goes the evidence is unequivocal: 25(OH)D testing is growing at a staggering rate. The author’s laboratory at City Hospital, Birmingham, UK, has seen a 75% increase in testing since 2012 and will measure around 80,000 serum samples this year for 25-hydroxy vitamin D (Fig. 1). This is even after demand management was introduced in 2010, whereby GPs could only have one 25(OH)D test per patient per year, unless there were strong clinical indications for measurement. Other laboratories have noticed an annual 80–90% growth in 25(OH)D test rates [14]. One report suggested that Quest laboratories in the USA were receiving 500,000 requests for 25(OH)D analysis per month [15].
Self-referral testing
Growth has come from testing in the GP population as well as hospital-based testing. In addition to this, our Dried Blood Spot (DBS) Vitamin D service, which is a direct-to-the-public service, has also grown steadily since its introduction in 2011. We have analysed close to 10,000 samples since the service inception and requests have been received from all over the world as well as the UK (Fig. 2).
The reasons why health professionals want to test 25(OH)D have been discussed but we have found that the reasons the general public want to test for 25(OH)D are very different. Many are knowledgeable about vitamin D and want to check their and their family’s levels are adequate. Many are taking supplements and they want to be sure that they are taking enough. Some have certain conditions, e.g. psoriasis, multiple sclerosis, cancer, and have read about connections between vitamin D and these conditions and want to make sure they have achieved recommended levels of 25(OH)D. Some are not able to get 25(OH)D tested through their GP. A small but significant proportion suspect that they have over-supplemented and they use the DBS service to check how high their levels have got [16].
The different reasons behind testing, as well as population differences such as ethnic distribution, have led to a significantly different distribution of 25(OH)D statuses in the two populations. During 2012, 54.7% of self-referred samples showerd adequate levels, compared to only 20.1% of GP samples were [25(OH)D >50 nmol/L, P<0.001, 95% confidence interval]. Only 0.1% of GP samples were high to toxic [25(OH)D >220 nmol/L] compared with 1% of the DBS samples (P<0.001, 95% confidence interval).
For people who want to be pro-active about their health, the self-referral service is giving them this opportunity. It is also allowing a proportion of the population who may have been inadvertently over-supplementing to realise this and act before any long-term harm has occurred. Clinical laboratories are struggling to cope with the increase in demand for vitamin D testing, not just with physically performing the work but also because many are not paid appropriately for the analysis and this has led to financial difficulties for some laboratories. However, the demand from the public is not going to abate, especially as the media and public awareness of vitamin D grows. Self-referral vitamin D testing gives people a viable alternative when they are not able to access the testing through their health care system.
Into the future…
Vitamin D testing is going to continue to rise and clinical laboratories are going to have to find strategies to cope. This may mean putting demand management strategies in place, negotiating prices with commissioners in order to be paid appropriately and looking at the methods used for measuring 25(OH)D to see if savings and efficiencies can be made.
As vitamin D remains in the media spotlight, and while vast numbers of papers relating to vitamin D continue to be published, hopefully the awareness of the importance of having adequate levels of vitamin D will continue to rise amongst healthcare professionals, and advice on what to do to achieve adequate levels will improve. One day we will have a national strategy on who to test, when and how often, but for now it seems that some of the population will continue to try to figure it out for themselves.
References
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The authors
Robyn Shea* BSc, MSc, MSc, DipRCPath and Dr Jonathan Berg BSc, PhD, MCB, FRCPath, MBA
Department of Clinical Biochemistry, Sandwell and West Birmingham Trust Hospitals NHS Trust, Birmingham, UK
*Corresponding author
E-mail: robyn.shea@nhs.net