Hair analysis for forensic diagnostics is gaining popularity in both research and applied settings. Commercially available dynamic multiple reaction monitoring (Dyn-MRM) software applied to hair samples can provide drug-use history for several months. The cost-effective drug test using Dyn-MRM software facilitates analysis of over 200 analytes in a 10-minute chromatographic run.
by Professor D. P. Naughton and Professor A. Petróczi
Background
Considerable global efforts are expended to address substance abuse which has major effects on public health and quality of life, as well as on economic and societal prosperity. This grand challenge impacts on a wide range of healthcare, regulatory and research endeavours. Key examples include destruction of lives through abuse of class A drugs, efforts to reduce doping in sport, attempts to address alcohol abuse and the lethal dangers of ‘legal highs’ (novel psychoactive substances).
Healthcare and regulatory officials engage in a wide range of activities to combat substance abuse. These include criminalization, banning substances in sport, education programs to prevent and efforts to understand and alter drug-related behaviour. However, in many cases there is an unmitigated failure to address the issues around substance misuse or abuse.
For example, for doping in sports, where vast efforts and resources are expended, indirect assessment of doping produces prevalence figures some 10-fold higher than positive doping test rates [1]. These frequent prevalence reports, at odds with figures from analytical tests, corroborate the belief that current anti-doping testing regimes are far from adequate [1]. Using doping in sport as an exemplar, major improvements in approaches to test for prohibited substances are needed. The advent of more advanced instrumentation aids testing in a number of ways. Increased sensitivity and affordability are very important but so are software developments that provide capability to monitor several hundred substances in one liquid chromatography-tandem mass spectrometry (LC-MS/MS) cycle of less than 10 minutes. These advances bring opportunities that require both instrumentation updates and frequent training updates for staff.
Hair analysis
Despite major advances in instrumentation and software, there are still obstacles to performing successful drug tests that benefit the drug taker. In sport, doping practices are frequently highly advanced, with some athletes taking heed of in-depth knowledge about most key parameters including generic testing methods, masking drugs and advances in detection for specific substances. The burden of proof has shifted to acquiring samples both in and out of competition as well as ensuring that appropriate tests are performed on each sample [2]. Owing to varied pharmacokinetics, analyte distribution and analytical procedures used, testing for a wide range of drugs in all samples is prohibitive [3]. Current testing approaches using biofluids impart considerable practical and financial consequences for the testing regime. Furthermore, cases of microdosing, masking and using novel substances make life challenging for the anti-doping officials.
Drug testing based on biofluids presents a number of issues that add considerably to cost but also are restrictive in terms of the number of tests required to cover a suitable duration owing to the pharmacokinetic profiles of many drugs. Where drugs or their metabolites are washed out efficiently after cessation of use, detection is less viable. The relatively short half-lives of many substances means the window for detection can be limited, which affects the success of occasional testing. The cost of supervised sampling along with the requirement for biofluid storage and handling to avoid sample corruption or infection is prohibitive for major levels of testing. Focusing on doping in sport, further complexities arise through variations in the lists of prohibited substances for testing in and out of competition [4]. The advantage of a longer window of detection via hair-analysis is suited to out of competition testing where a cumbersome system currently exists for sampling which is intrusive and controversial [5]. Thus, new approaches that allow a single test to be conducted simultaneously for (i) a wide range of substances and (ii) covering a prolonged period such as a 3-month window, would be valuable in sport for out of competition testing but also, beyond sport, for social drugs and new psychoactive substances.
In contrast to drug tests based on biofluids, hair analysis provides a range of advantages including: ease of sampling, ability to conduct multiple tests on one cut-hair sample to cover a prolonged duration (a typical 3-cm hair sample is equivalent to approximately 3 months’ growth), lack of issues with infection risk, facile storage at room temperature, lack of requirement to process tissue containing genetic data, and good stability of many drugs and metabolites in the hair matrix.
Instrumentation advances
We recently reported a hair-based method, using liquid chromatography–tandem mass spectrometry (LC-MS/MS), for the analysis of substances of forensic nature [6]. The multi-drug/metabolite assay employs a dynamic multiple reaction monitoring (Dyn-MRM) method using proprietary software [7, 8]. It allows both screening and validated confirmatory analysis depending on the focus of the investigation. This approach has several benefits: (a) the Dyn-MRM software is suited to screen over 200 compounds on a single chromatographic run of under 10 minutes, (b) full validated methods for compounds can be incorporated into the software, (c) hair samples provide the opportunity to cover longer windows for detection in one test (e.g. approximately 3-month history covered in a 3-cm sample), and (d) the software is designed to allow ready adoption of new compounds of interest. The advantage of Dyn-MRM is that multiple reaction monitoring is employed with a focus on scanning for specific peaks at their selected elution times. This efficient method allows the analysis of large numbers of analytes simultaneously in a short run (Fig. 1). In our report, the proprietary software has been extended and applied to cover a range of drugs and metabolites of interest to forensic investigations including cognitive enhancers, amphetamines, barbiturates, benzodiazepines, cannabinoids, cocaine, opioids, steroids and sedatives. The chromatographic run is calibrated by a test mixture containing approximately 20 substances and further tailoring would be required to match a specific remit such as the WADA (World Anti-Doping Agency) prohibited list more closely [4].
Conclusion and future perspectives
The application of Dyn-MRM software to screen for a large range of drugs brings considerable advantages to laboratories involved in drug testing. The ease of use and ability to add new compounds to the screening database are noteworthy. Coupling this commercially available software to hair analysis adds the extra dimension of being able to screen for drug use over several months in one hair sample. This advance will add considerably to the efficiency of drug testing but will remain as an adjunct to other testing methods for out of competition testing in sport as it will not cover all analytes of interest to anti-doping officials [4]. Some substances are unlikely to be found in hair (e.g. performance enhancing peptides) and for other substances there will be issues with establishing a threshold – either for endogenous substances (e.g. testosterone) or for substances consumed through diet (e.g. drugs used in farming). Further limitations are that (i) a single use of a drug may be undetectable owing to the low levels deposited in hair, and (ii) more research is warranted to ascertain the effects of hair type and colour on analyte uptake and stability. In spite of these limitations, hair analysis coupled to modern advances in instrumentation sensitivity and software capabilities is promising in many scenarios especially to obtain a prolonged history of abuse and where ‘zero tolerance’ is applied (e.g. for synthetic steroids). In addition, hair analysis may have a role in support of the Athlete Biological Passport through analysis of indirect biomarkers of doping [9].
References
1. de Hon O, Kuipers H, van Bottenburg M. Prevalence of doping use in elite sports: A review of numbers and methods. Sports Med. 2015; 45(1): 47–69.
2. World Anti-Doping Agency (WADA). International Standards. 2015; https://www.wada-ama.org/en/what-we-do/international-standards.
3. Maennig W. Inefficiency of the anti-doping system: Cost reduction proposals. Subst Use Misuse 2014; 49(9): 1201–1205.
4. WADA. List of prohibited substances and methods. 2015; http://list.wada-ama.org/.
5. Hanstad DV, Loland S. Elite athletes’ duty to provide information on their whereabouts: Justifiable anti-doping work or an indefensible surveillance regime? Eur J Sport Sci. 2009; 9(1): 3–10.
6. Shah I, Petroczi A, Uvacsek M, Ranky M, Naughton DP. Hair-based rapid analyses for multiple drugs in forensics and doping: application of dynamic multiple reaction monitoring with LC-MS/MS. Chem Cent J. 2014; 8(1): 73.
7. Agilent Technical Overview. Ion optics innovations for increased sensitivity in hybrid MS systems. Agilent Technologies USA 5989-7408EN. 2007; http://www.chem.agilent.com/Library/technicaloverviews/Public/5989-7408EN_HI.pdf.
8. Stone P, Glauner T, Kuhlmann F, Schlabach Tim, Miller K. New dynamic MRM mode improves data quality and triple quad quantification in complex analyses. Agilent Technologies USA 5990-3595EN. 2009; http://www.chem.agilent.com/Library/technicaloverviews/Public/5990-3595en_lo%20CMS.pdf.
9. Vernec AR. The athlete biological passport: an integral element of innovative strategies in antidoping. Br J Sports Med. 2014; 48(10):817–819.
The authors
Declan P. Naughton* PhD, Andrea Petróczi PhD
School of Life Sciences, Kingston University, London, UK
*Corresponding author
E-mail: D.Naughton@kingston.ac.uk
In spite of major medical advances in diagnosis and treatment, cardiovascular disease (CVD) is still the leading cause of mortality in the Western world accounting for 51 percent of female and 42 percent of male deaths. Around half of these deaths are due to coronary heart disease, and it has been recognized for more than two decades that the outcome for women with acute coronary disease (ACD) is worse than it is for men. Quite apart from the fact that surveys show older women are less aware of their risk of myocardial infarction (MI) than men, women presenting with MI are less likely to be appropriately diagnosed.
Various explanations have been given for this disparity. Clinical symptoms of ACD in women may not be the ‘typical’ sudden severe chest pain; physicians have even attributed female symptoms of more diffuse pain, dyspnea and fatigue to falling levels of estrogen and progesterone. And although sex differences in electrocardiography (ECG) were first reported around 90 years ago, with recent studies emphasizing that normal values of the adult ECG should be both age- and sex-specific, the use of sex-specific diagnostic criteria is still not routine in many hospitals. In addition clinical research into ACD was biased towards men in the past, resulting in predictive values for analytes that are not necessarily appropriate for women; results of diagnostic tests should of course take sex-related differences into consideration. Given that the diagnosis of MI relies on a combination of clinical examination, suggestive ECG abnormalities and a rise and fall of key cardiac biomarkers, it is not surprising that myocardial infarction in women is still under-diagnosed.
However, the results of a recently published study in the BMJ should be a step towards more effective diagnosis. The troponin I level of 1126 consecutive patients presenting at a regional cardiac centre with suspected MI, 46% of whom were women, was measured using a high sensitivity assay and sex-specific diagnostic thresholds (men 34 ng/L, women 16 ng/L) in place of the current recommended threshold of 50 ng/L for both sexes. There was a significant increase in the number of women diagnosed with MI (from 11% to 22%) but the increase in men (19% to 21%) was not significant. Although studies continue to show that even when diagnosed women are less likely to undergo percutaneous coronary interventions or bypass surgery, receive prompt thrombolytic therapy or even be prescribed statins on discharge from hospital, more accurate diagnosis should go a long way towards redressing sex inequalities.
Bacterial resistance to antibiotics is a major health and economic problem recognized today by national and international policy makers. The Enterobacteriaceae belong to the commensal human gut flora and are frequently the cause of community- and healthcare-associated infections (HAI). Infections with Klebsiella pneumoniae are usually hospital-acquired and occur primarily in patients with impaired host defences while Escherichia coli are mostly involved in urinary tract infections. Acinetobacter and Pseudomonas species are opportunistic pathogens frequently isolated from healthcare settings where they cause life-threatening infections particularly in immunocompromised patients.
For several years, in Europe and worldwide, Enterobacteriaceae, mainly K. pneumoniae and E. coli but also non-fermenting bacteria (Acinetobacter baumannii and ) have become resistant to the main antibiotics, i.e. ß-lactam antibiotics, fluoroquinolones and aminoglycosides [1]. In particular, the US “Center for Disease Control and Prevention” (CDC) pointed the carbapenemase-producing Enterobacteriaceae (CPE) (or Carbapenem Resistant Enterobacteriaceae, CRE) among the three microorganisms exhibiting the most urgent health risk. Carbapenemases are indeed enzymes that inactivate ß-lactam antibiotics like carbapenems that currently constitute the last resort for treating multi-drugs resistant Gram-negative bacteria. Moreover, production of carbapenemases in these bacteria is most often associated with the expression of additional resistance mechanisms to other classes of antibiotics such as aminoglycosides, fluoroquinolones and cotrimoxazole, leading to bacteria resistant to all available antibiotics, so-called pan-resistant bacteria [2].
Carbapenemases resistance
Carbapenems are a class of broad-spectrum ß-lactam antibiotics with very broad activity and have therefore become the empirical treatment of choice in countries where infections due to Extended-Spectrum ß-Lactamases-producing bacteria are common. Resistance to carbapenems in Enterobacteriaceae is linked to either decreased permeability because of porine alteration or loss combined with production of a ß-lactamase with poor carbapenemase activity or, more worryingly, to the enzymatic breakdown of the antibiotic by a carbapenem-hydrolyzing ß-lactamase [3].These so called carbapenemases can hydrolyse and hence deactivate several kinds of antibiotics ranging from penicillins to cephalosporins, monobactams and carbapenems.
The most frequent carbapenemases in Enterobacteriaceae reported in Europe belong to three molecular classes according to the Ambler classification:
– class A carbapenemases hydrolyse almost all ß-lactams including carbapenems. Klebsiella pneumoniae carbapenemase (KPC) are the most frequent enzymes of this group that show a very high spreading capability [4] . KPC, contrary to other class A ß-lactamase, is inhibited by boronic acid and its derivatives.
– class B carbapenemases, belonging to metallo-ß-lactamases, including Verona integron-encoded metallo-ß-lactamase (VIM), IMP and the New Delhi metallo-ß-lactamase-1 (NDM-1) can hydrolyse all ß-lactams but monobactams. These enzymes are inhibited in vitro by EDTA and dipicolinic acid that are used in confirmatory tests for the presence of metallo ß-lactamases (MBL). NDM is the most frequent MBL present in Enterobacteriaceae. Originally detected in the Asian subcontinent, NDM is now spreading worldwide and causing outbreaks in Western countries.
– class D carbapenemases including the OXA carbapenem-hydrolysing oxacillinases hydrolyse penicillins but show lower activity against carbapenems, and no activity against extended-spectrum cephalosporins [5)]. OXA-48 is the main enzyme of this family and is now spreading worldwide even in the community although only a few cases are reported in the USA. OXA-48 hydrolyses all penicillins including temocillin. The resistance to temocillin primarly evidenced by a Belgian group [6] is now recommended as a marker of the possible presence of OXA-48. Resistance to carbapenems in OXA-48-producing CPE is variable with minimal inhibitory concentration againt carbapenems varying between less than 0.5 µg/mL to more than 256 µg/mL. This mechanism is very difficult to detect and no confirmatory test currently exists as OXA-48 is not specifically inhibited by clavulanic acid, boronic acid or EDTA. To date, only expensive molecular tests are able to confirm the presence of OXA-48. The rapid and global expansion of CPE is a threat to healthcare and patient safety worldwide, as it seriously curtails the ability to cure infections. Infections due to CPE are associated with higher in-hospital morbidity and mortality [7].
Carbapenemases epidemiology
According to the report summarizing the results from 39 European countries [8], six levels of occurrence of CPE have been defined i.e. endemic situation (level 1), inter-regional spread (level 2), regional spread (level 3), sporadic hospital outbreaks (level 4), single hospital outbreaks (level 5) and sporadic occurrence (level 6).
Nevertheless, specific occurrence may also vary depending on the type of CPE.
Discussion
Carbapenemase-producing Enterobacteriaceae (CPE) are an emerging threat to healthcare and are frequently resistant to many other antibiotics besides carbapenems leaving few treatment options [9, 10]. Rapid diagnostic tests that can be performed directly on clinical specimens or blood cultures are urgently needed in order to save an average of 24 hours compared to the results obtained by culture. Some commercial tests of this type targeting carbapenemases from CPE currently exist and are based either on molecular amplification of specific genes associated with resistance or on molecular hybridization.
Some tests are able to target all carbapenemases of clinical interest, or other resistance mechanisms alongside with accurate species identification.
However, these tests require specific equipment and are extremely expensive (80 Euros being an average price).
Multiplex real-time PCR tests allow the detection of the genes encoding for the main carbapenemases directly from samples or feces but do not detect all variants of the genes of interest in a single operation.
Other molecular biology tests can be performed on isolated colonies from culture. These tests are costly (average cost of 40 €), quite labour-intensive and often do not deliver results before the next morning, when the susceptibility testing is already available. Molecular biology tests only partially meet the needs in carbapenemase identification. Either these tests do not cover the complete range of targets or cannot distinguish between different carbapenemases and, in any case, do not give information on the level of gene expression and thus the level of bacterial resistance. The high price of these techniques and/or the need for expensive equipment, dedicated areas and specially trained personnel restrict their use to a limited number of specialized laboratories.
Rapid phenotypic tests directly performed on bacterial colonies and based on the hydrolysis of a carbapenem with colorimetric shift are now available at a reasonable price. MALDI-TOF mass spectrometry is also proposed, however, this technology requires the use of expensive equipment together with specific software analysis. All the above phenotypic tests only partially meet the needs of clinical laboratories. On the one hand, most of them require the use of antibiotics with stability problems, and secondly, the time for obtaining a result with these tests is not totally satisfactory in terms of integration into the laboratory workflow that would ensure results in a short time and allow quick decision for optimal impact. On the other hand these phenotypic tests do not identify the exact type of carbapenemase and ideally require subsequent procedures using a molecular method to achieve identification. Among CPE, OXA-48 represents the most challenging resistance mechanism to be identified that would need a rapid and easy to use test to be performed in routine labs.
References
1. Carbapenemase-producing bacteria in Europe. Interim results from the European Survey on carbapenemase-producing Enterobacteriaceae (EuSCAPE) project 2013.
2. Souli M, Galani I, and Giamarellou H. Emergence of extensively drug-resistant and pandrug-resistant Gram-negative bacilli in Europe. Euro Surveillance 2008; 13(47).
3. Nordmann P, Naas T, and Poirel L. Global spread of carbapenemase producing Enterobacteriaceae. Emerging Infectious Diseases 2011; 17(10): 1791–1798.
4. Naas T, Cuzon G, Villegas M-V, Lartigue M-F, Quinn JP, and Nordmann P. Genetic structures at the origin of acquisition of the ß-lactamase blaKPC gene Antimicrobial Agents and Chemotherapy 2008; 52(4): 1257–1263.
5. Nordmann P, Naas T, and Poirel P. Global spread of carbapenemase producing Enterobacteriaceae. Emerging Infectious Diseases 2011; 17(10): 1791–1798.
6. Glupczynski Y, Huang TD, Bouchahrouf W, Rezende de Castro R, Bauraing C, Gérard M, Verbruggen AM, Deplano A, Denis O, Bogaerts P. Rapid emergence and spread of OXA-48-producing carbapenem-resistant Enterobacteriaceae isolates in Belgian hospitals. Int J Antimicrob Agents 2012; 39(2):168-72.
7. Borer A, Saidel-Odes L, Riesenberg K, Eskira S, Peled N, Nativ R, et al. Attributable mortality rate for carbapenem-resistant Klebsiella pneumoniae bacteremia. Infect Control Hosp Epidemiol 2009, 30:972–6.
8. Carbapenemase-producing Enterobacteriaceae in Europe: a survey among national experts from 39 countries, February 2013, Euro Surveill. 2013;18(28):pii=20525.
9. Cantón R, Akóva M, Carmeli Y, Giske CG, Glupczynski Y, Gniadkowski M, et al. Rapid evolution and spread of carbapenemases among in Europe, Clin Microbiol Infect. 2012;18(5):413–31.
10. Hawkey PM. The growing burden of antimicrobial resistance, J Antimicrob Chemother. 2008;62(Suppl 1):i1–i9.
The authors
Isabelle OTE, Laetitia AVRAIN, Pascal MERTENS, Thierry LECLIPTEUX
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