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

by Dr Gary McAuliffe

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Questions about gene testing were highlighted dramatically this summer after Hollywood superstar Angelina Jolie announced she had undergone a preventive double mastectomy. The reason: gene tests showed she carried the breast cancer-linked BRCA1 mutation. In an Op-Ed piece in the ‘New York Times’, the actress encouraged other women, who believed they were also at risk, to also get tested.
Ms. Jolie’s decision has been hailed by some, criticized by others. However, it may well mark a watershed, when gene testing began a paradigm shift to the mass market. Her announcement, for example, led to a doubling of cancer checks at top clinics in London.

US Patent ruling will bring costs down
Such trends are likely to be reinforced, strongly, by a US Supreme Court ruling in June 2013 (shortly after Ms. Jolie’s announcement) that human genes cannot be patented. The decision reversed three decades of US intellectual property case law, and within days, several US labs announced they would be offering BRCA tests. The latter could previously only be tested for by a single company, Myriad Genetics. Though patent laws are national matters, it is likely that the US court ruling will make an impact elsewhere. In Europe, the EU Biotech Directive allows patenting of gene tests, while Myriad itself recently won a Federal Court ruling in Australia upholding its BRCA patents.

Revenues from genetic screening were $5.9 billion in the US in 2011, according to a study by the respected Battelle Institute. To put the figure in perspective, this is about 10% of the total US clinical testing market. Globally, sales of genetic tests could be conservatively estimated at $10-$15 billion. Scores of vendors already offer a range of tests – from selective screening for some hundred-odd major disease genes to complete sequencing of a person’s genome.

The once-prohibitive costs of gene tests have seen downwards pressure over the past decade. As with other consumer technology cycles, lower prices are expected to drive an expansion in affordability, in users and revenues, in a virtuous cycle. One of the key market catalysts has been direct-to-consumer testing (DTC) companies. US DTC leader 23AndMe has seen its gene tests used by about 200,000 consumers. For just $99, the company provides information on 50 carrier traits, 20 drug classes and disease risk information. 23AndMe is currently seeking FDA certification. European firms are less visible. A leading vendor, deCode Genetics, shut down its DTC service after being acquired by Amgen in late 2012. The Iceland-based firm had been offering its deCodeme personal genomic scanning service for just under $1,000, as well as screening for cardiovascular diseases and common cancers –  in a package for $350. Other major DTC players in Europe are also from the US, among them Navigenics, DNADirect and Genelex.

Price falls are now almost certain to accelerate after the US Supreme Court decision on gene patents. Myriad, for example, was using its monopoly on BRAC to charge $3,000 and more for a test. After the Court ruling, the test is projected to see a steep fall in its price to just $100.

Drivers of consumer tests
The key reason for the growth of DTC is that genetic testing has so far largely been restricted to specialist labs and top academic medical centres. In spite of a sharp rise in the number of registered tests to over 7,500, most have yet to be translated into clinical applications.

A study by United Health, the US managed health group, found 63% of physicians saying that screening provided them “the ability to diagnose conditions that would otherwise be unknown.” However, a larger number, about three of four, also noted there were patients in their practices “who would benefit from a genetic test but have not yet had one.” United Health estimates that the US testing market alone would reach about $15 to $21 billion by 2021. In Europe too, an increase in formal healthcare settings for gene testing is likely to be welcomed, given growing concerns about DTC. A recent survey of clinical geneticists found 84% of respondents expressing concern about “replacing face-to-face supervision by a medical doctor with supervision via telephone” through DTC testing firms. A little less than half the respondents said they had at least one patient make contact with them after they had undergone a DTC genetic test, and 86% said they would provide post-test counselling to such patients. The survey posed the likelihood of a ‘cascade effect’ in the future, particularly should physicians spend more time on patients with DTC test results that are not medical priorities.  As a result, it seems market growth will be accompanied by the encouragement of general hospitals and physician practices to do gene testing.

The emergence of personal medicine
The impact of mass gene testing will clearly be enormous. One new frontier is personal medicine, where medicine choice and dosages would be prescribed according to a patient’s specific genetic profile. Further down the horizon may be an end to several inherited diseases. In January 2009, the UK saw the birth of the first baby “tested preconceptionally for a genetic form of breast cancer.” The baby was born at University College London (UCL) Hospital, using Preimplantation Genetic Diagnosis, which involves undertaking an in vitro fertilization treatment cycle to have several embryos available for genetic tests.

More recently, UCL announced that its scientists had developed a microchip test to analyse 35 different genetic mutations linked to cancer, and enable doctors to identify and target specific genes from a small sample of tissue. UCL Professor Charles Swanton said the test marked the beginning of tailored cancer care in the NHS.

Ethical questions remain
Nevertheless, there is some way to go. One barrier consists of still-lingering questions about the ethical implications of gene tests.
Here, the first issue is uncertainty. Even now, gene testing (including that for the high-profile BRCA 1 and 2) only predicts an increase in risk, not certainty of disease. This transfers the choice and responsibility for an irreversible prophylactic intervention to a patient, and to his or her best guess. It also rules out the possibility of effective, new and less-invasive surgical interventions emerging in the future.

Such technology evolution challenges – of better choices becoming available – apply broadly to all genetic testing. Some tests do not (as yet) identify all possible gene mutations which lead to a particular disease, or have only limited predictive value. Finally, it remains unclear whether a mutation is not just a symptom of a disease, rather than being a cause.

For example, in cystic fibrosis (CF), there is still no way to predict disease severity, even when a fetus has inherited two mutations. Parents thus face the dilemma of deciding whether to continue or end a pregnancy without full knowledge. In the meanwhile, even as data on CF mutations grows steadily by the year, promising new drug therapies are becoming available. For example, Ivacaftor (Vertex Pharmaceuticals), which addresses the G551D mutation affecting 4% of CF patients, is now being evaluated for the more prevalent F508del mutation.

The above dilemmas are aggravated by the question of false positives and false negatives. In spite of being at the cutting edge of mass screening techniques for Down’s syndrome and neural tube defects, Quad tests for pregnant women still retain a 5% false positive and 20% false negative rate. Elsewhere, while metabolic genetic disorders such as phenylketonuria can be identified by fetal gene tests and then addressed by dietary changes, many others lack treatment options.
The broader debate on gene tests and its ethics is unlikely to go away soon, but policy makers are broadly swinging to accept its inevitability. The Human Genetics Commission in Britain stated in April 2011 that there were “no ethical barriers preventing the use of genetic testing in couples before they conceive.” Within months, the German parliament enacted a law to allow testing fertilized embryos for possible life-threatening genetic defects, via Preimplantation Genetic Diagnosis (like that launched by University College London in early 2009).  Critics in Germany have been especially vociferous, calling the move “a step toward designer babies.”
One of the biggest concerns about genetic testing is the emergence of ‘a la carte’ health insurance, providing choice of cover and premium based on a person’s particular disease risks and (eventual) treatment requirements, rather than loading the highest-risk beneficiaries atop the lower-risk ones. 

In the US, resulting concerns about discrimination due to genetic testing led to the 2008 Genetic Information Nondiscrimination Act (GINA), which bars denial of health insurance or employment because of a genetic predisposition to a particular disease.

In Europe, different laws and regulations in the Member States seek to address ethical questions. A major hurdle here is the lack of an “approved definition of a genetic test,”  in spite of the EU-funded project EuroGentest. One of the latter’s goals was to “try to develop at least some key elements for a working definition” of a gene test.

Patients are routinely monitored for levels of trace elements to investigate situations of deficiency or toxicity. This article covers some of the reasons why trace elements are investigated in the clinical setting and discusses, with examples, how the measurements are carried out using advanced analytical instrumentation. It then goes on to suggest some important new developments in the field of inorganic mass spectrometry, which could have an important impact on future clinical assays.

by Dr Chris Harrington

Trace elements are defined as having a concentration of less than 100 µg/g or 100 mg/L and traditionally there are two main reasons for their measurement in a clinical setting: for the determination of deficiency or toxicity.

There are about 10 inorganic micronutrients essential for human health which include the transition elements Cu, Co (as vitamin B12), Cr, Fe, Mn, Mo and Zn, the metalloid Se and the halogen elements F and I. The human body also contains As, B, Ni, Si, Sn and V, but there is no firm evidence any of these are essential for health. These elements have a number of biochemical roles, e.g. co-factors for different enzymes; constituents of important molecules, such as the thyroid hormones; and electron transport, due to their redox chemistry. The toxic effect of any trace element is dose-dependent, but there are a number which exert toxicity at low concentration and examples of these include: Hg, Ti, Pb, Cd and As. The degree of harmful toxicity will not only depend on the concentration, but also on the actual chemical form and exposure time. In the case of an element such as As, it is highly toxic when present as arsine (AsH3) because it is a gas, but when exposure is via a more complex organometallic compound such as arsenobetaine (C5H11AsO2) which is common in fish and seafood, an equivalent dose of As would be harmless. Commonly exposure to a toxic trace element is determined by analysis of a urine sample normalized to the creatinine concentration and comparison to an established guidance value such as a biological exposure index (BEI). However, clearly in the case of As, a measurement of total As in the urine will not differentiate between different chemical forms. To achieve this aim each of the separate As-containing species will need to be determined using methods based on elemental speciation, whereby a chromatographic separation is coupled to a suitable atomic spectroscopic detector, for example HPLC-ICP-MS (inductively coupled plasma mass spectrometry in combination with HPLC).

A more recent development in the clinical measurement of trace elements relates to the orthopedic area and the increasing use of metal alloys containing Cr, Co, Mo and Ti as the components of metal-on-metal (MoM) hip replacements. As a result of complications with the use of such implants and the potential for failure requiring revision surgery, all patients in the UK with MoM replacements are now monitored on an annual basis. The guidelines issued by the UK Medicines and Healthcare products Regulatory Agency (MHRA) in 2010 [MDA/2010/033] and subsequently updated in 2012 [MDA/2012/008 and 036] provide advice to healthcare professionals involved in the management of patients implanted with MoM hip replacements. The initial alert recommended that all patients should be followed up regularly by measurement of Co and Cr in whole blood samples and that this should be carried out most frequently on patients with symptoms consistent with high rates of failure. The medical device alert stipulates that if either element was elevated above a concentration of 7 µg/L (134 and 119 nmol/L for Cr and Co respectively), then further tests should be performed including imaging, to identify patients with potentially failing MoM hip joints. Whereas there are already action limits for these elements relating to occupational exposure, the concentration of 7 µg/L was chosen after consultation with orthopedic clinicians and using information from the National Joint Registry for England and Wales, as a level at which the joint was not showing optimum performance. It was not set as an indication of toxicity but rather as an indicator of joint performance and is thus interpreted with this in mind.

Internal quality control and external quality assurance are important prerequisites for measuring trace elements and making appropriate diagnosis or treatment decisions. A good example of this is the routine annual follow-up of patients with MoM hip-replacements, where clinicians need to make sure that their decisions are based on well controlled analytical measurements. How, for instance, can a clinician decide if an increase in concentration of Co or Cr results from a change in the particular joint and does not arise from a change in the laboratories measurement performance? We recently looked at data [1] from the UK National External Quality Assessment Scheme for trace elements (TEQAS). This supplies whole blood specimens which are spiked with known amounts of a number of trace elements including Co and Cr. The mean recovery over the samples measured in the 2011–12 scheme year was 96.4% (SD 2.23, CV 2.3%) for Co and 96.1% (SD 3.19, CV 3.3%) for Cr. The excellent agreement between the amounts in the specimens and the mean value indicates the results are accurate, and agreement between the pools distributed on different occasions shows they are reproducible over time. This should provide the necessary confidence to the clinical decision maker that the laboratories providing the Co and Cr results are competent and the results are suitably accurate.

Analytical instrumentation
The instrumental mainstay of clinical laboratories which specialise in the measurement of trace elements is inductively coupled plasma mass spectrometry (ICP-MS), which is a form of inorganic MS measuring elemental ions rather than molecular ions. Developed as a commercial analytical technique in the early 1980s it was initially used in environmental and geological laboratories, but after instrumental improvements it is now gaining popularity in the clinical area. This is mainly because it is multi-elemental in nature.
A review of new research and instrumental approaches in the elemental analysis of clinical and biological materials, foods and beverages is published annually as an Atomic Spectrometry Update [2].

The instrumentation itself will not be discussed as many texts [3] deal with the fundamentals of ICP-MS. However, the significant strengths of the technique include: multi-elemental detection in a single run; wide elemental coverage up to m/z 254 (UO+); high sensitivity with low limits of detection, down to sub ng/L levels (limited by purity of the reagents); fast analysis times as a result of the scanning speed of the quadrupole analyser; wide linear working range, up to 9 orders of magnitude in the same run; isotopic information, making high accuracy calibration via isotope dilution mass spectrometry available; and it can be used for specialist applications such as speciation analysis, where it is used as a chromatographic detector for HPLC, GC, CE or GE separations. The main weaknesses of the technique are: the presence of isobaric interferences on some elements, which mean the isotope of one element is at the same m/z ratio for the analyte of interest, for instance Ca has an isotope at m/z 48 which is the same as the most abundant isotope for Ti, making the measurement of Ti in clinical samples problematic; the formation of polyatomic ions from sample matrix and atmospheric ions can impinge on the m/z for the analyte of interest, an example would be the measurement of Cr at its most abundant isotope at m/z 52 which has a major interference from the formation of ArC; and the formation of doubly charged ions, for instance Gd2+ can interfere with Se at m/z 78. Luckily instrumental developments based on the use of a reaction/collision cells containing a suitable gas have been introduced to overcome the problems due to polyatomics and doubly charged ions. These work by using a reactive gas, e.g. H2 in the cell and removing the interference by reactively neutralising it, which we have recently demonstrated for the removal of the Gd2+ interference from the measurement of Se [4], or using a collision gas, e.g. He to remove the larger polyatomic ions by collision induced kinetic energy discrimination. These newer instruments are extremely robust and can rapidly deliver highly accurate measurements for multi-elements at low concentrations in difficult matrices such as whole blood, serum or urine.

Future trends and developments
Over the last 5–10 years the capabilities of ICP-MS for the detection of molecules that do not contain a trace element have been investigated. By using a reagent with specificity for the analyte and which carries a metal or nanoparticle tag, the molecule of interest becomes visible for detection by ICP-MS. The reagents used are often antibodies, so the protocols often mimic those developed for immunochemical assays and, in theory, can be applied to the determination of the same analytes, including peptides, proteins and other specific biomarkers. So why would this be advantageous compared to conventional immunochemical assays? Most importantly this approach has a greater potential for multiplexing than spectroscopic methods; there are a large number of elemental tags to choose from and no overlap between them. As illustrated in Figure 1 this is not the case with fluorescence signals.

Other advantages include: analyte quantification with high precision; low detection limits; large dynamic range; low matrix effects from other components of the biological sample (i.e. contaminating proteins in the sample have no effect on elemental analysis); low background from plastic plates (i.e. plastic containers do not cause interference on elemental detection as it can with fluorescence), and superior spectral resolution. As can be seen in Figure 1 this has generated a new approach to flow-cytometry based on ICP-MS and it’s very likely that other new approaches to other biochemical analytes will shortly become commercially available.

Acknowledgements
Scott Tanner, University of Toronto for permission to use the figures from the CyTOF flow cytometer based on ICP-ToF-MS (DVS Sciences Inc).

References
1. Harrington CF, Taylor A. BMJ 2012; 344: e4017.
2. Taylor A, Day MP, Hill S, Marshall J, Patriarca M, White M. J Anal At Spectrom. 2013; 28: 425–459.
3. Inductively Coupled Plasma Mass Spectrometry Handbook. Ed. Nelms S, Blackwell 2005.
4. Harrington CF, Walter A, Nelms S, Taylor A. Ann Clin Biochem. 2013; submitted.
 
The author
Chris Harrington PhD, MRSC
SAS Trace Element Laboratory, Faculty of Health and Science, University of Surrey, Guildford, Surrey, GU2 7XH, UK
E-mail: Chris.harrington1@nhs.net

Leaders from the medical diagnostics, laboratory medicine, and healthcare fields convened in Houston, Texas, July 28 – August 1 for the AACC annual meeting, the world’s largest diagnostics conference and expo. Over 17,000 attendees took part in the event and the exhibit totalled more than 625 companies. A selection of research papers presented in Houston are summarized below.

New biomarkers for prostate cancer
Dimitra Georganopoulou, PhD, Ohmx Corporation, presented results of a pilot study to find a new biomarker for prostate cancer aggressiveness. The researchers measured the enzyme activity of prostate-specific antigen (PSA), termed the “aPSA”, in patient specimens that had been removed by radical prostatectomy. They wanted to determine if this activity level could be a clue to how aggressive the cancer was. The team found that there was a significant negative correlation between prostate cancer progression and the aPSA in prostatic fluid. Patients with the least amount of aPSA (PSA activity) had the most aggressive prostate cancer. Tests for an “aggressiveness biomarker” would provide critical information for making decisions about when clinical treatment should occur or when it could be postponed. Many men might be able to avoid radical treatments if their cancer was known to be non-aggressive. Likewise, men whose cancer was too aggressive to employ the “active surveillance” or “watchful waiting” approach would have more information to help them make meaningful personal decisions with the help of their doctors about what level of treatment was right for them. The findings from this study could lead to the development of a new tool to use along with existing screening tests.

PSA Enzymatic activity: A new biomarker for assessing prostate cancer aggressiveness.
Dimitra Georganopoulou, PhD, OHMX Corporation, Evanston, Ill., U.S.A.


Diagnosing cystic fibrosis at the point of care
Xuan Mu from Peking Union Medical College presented test results from cystic fibrosis patients using an exciting new point-of-care method. Microfluidics and colour changes within a Band-Aid type of adhesive strip on the skin allow the new device to rapidly, accurately, and quantitatively diagnose cystic fibrosis in a small amount of sweat. Detecting sweat chloride has been the gold standard in diagnosing cystic fibrosis for more than 50 years. The new test detects increased chloride in sweat using a colour change in paper on an adhesive strip when a very small amount of sweat is absorbed. The intensity of changed colour is recorded with a cell phone camera, and is then measured against a colour model. Cystic fibrosis in an inherited disease of the body’s mucus glands. Technically a rare disease, the incidence of cystic fibrosis varies around the world and by ethnic group. Different mutations in the CFTR gene cause the severity and symptoms of CF to vary considerably. Respiratory and digestive systems are affected, as well as sweat glands and reproductive systems.  The new point-of-care test device can distinguish healthy people from cystic fibrosis samples and conveniently integrates the many separate steps of current sweat chloride tests whose results take several hours to obtain. Treatment advances have increased the life expectancy of cystic fibrosis patients over the past several decades from the mid-teens in the 1970s to more than 36 years today in the U.S. An early diagnosis and a comprehensive treatment plan can improve both survival and quality of life of patients. This new method demonstrates a fast and cost-effective opportunity in diagnosing cystic fibrosis. 

On-site colorimetric detection of sweat chloride ion for diagnosing cystic fibrosis.
Xuan Mu, Peking Union Medical College, Beijing, China

Determining the safety of olanzapine for schizophrenia and bipolar disorder
AACC member Werner Steimer from Munich, Germany presented the results of research showing that study patients who carried a specific genetic variation in an antipsychotic-metabolizing enzyme developed significantly higher serum concentrations of the drug olanzapine. The increased drug concentrations were still noteworthy even when researchers accounted for differences in the patient’s age, sex, weight, and other medications that they used. This is the first study to demonstrate that this polymorphism influences serum levels of olanzapine, and the study is extremely timely in the context of the recent FDA safety alert on the injectable form of olanzapine, an “atypical” or second generation antipsychotic medication. Under investigation are two unexplained deaths of patients who received an intramuscular injection of Zyprexa Relprevv (olanzapine pamoate) and showed very high blood levels of the drug, although they had received appropriate doses. They died 3-4 days after injection. Olanzapine is approved by the U.S. FDA for treating schizophrenia and bipolar disorder in adults and children older than 13, and is one of the most widely prescribed of the atypical antipsychotics. Olanzapine is available in tablet, injectable, and long-acting “depot” formulations. Long acting medications can be more tolerable to some patients and help them adhere to treatment. Olanzapine is metabolized in the liver by specific cytochrome P450 enzymes. Some individuals have genetic variations – polymorphisms – of cytochrome enzymes. These can impact the way that drugs are broken down and distributed throughout the body and sometimes even the strength or effectiveness of treatment.

The CYP1A2*1D Polymorphism has a significant impact on Olanzapine serum concentrations.
Werner Steimer, MD, Klinikum Rechts der Isar – Technische Universität München, Munich, Germany.