Over the last few years, hepatitis C virus (HCV) infection has emerged as one of the most significant causes of chronic liver disease worldwide, with estimated prevalence ranging from 2.2 to 3.0%. Since January 2011, the Infectious Diseases Department of San Raffaele Scientific Institute in Milan carried out a prevention programme called ‘EASY test project’, to diagnose the HCV infection. In these four years a total of 35,000 subjects have been approached to inform them about HCV infection and other sexually transmitted diseases. Of the total eligible volunteers, 6500 (18,6% of contacted subjects) performed HCV tests on saliva and completed the interview in the alternative ‘street lab’. We believe that increasing awareness of these alternative tests among individuals at risk and providers may be an appropriate strategy to increase the number of people who know their serological status and who could be linked to care and engaged in care!

by M.R. Parisi, Dr L. Soldini, Dr G. Vidoni, Dr K. Schlusnus, Dr F. Dorigatti, and Prof. A. Lazzarin

Background
Over the last few years, hepatitis C virus (HCV) infection has emerged as one of the most significant causes of chronic liver disease worldwide, with estimated prevalence ranging from 2.2 to 3.0% (1).

In our country, the proportion of subjects infected with HCV is approximately 2% of the general population with a gradient that increases from the north to the south and the islands and with age (60% of patients with hepatitis C are over 65 years old). It is estimated that about 1 million people in Italy are ill with hepatitis C (2).
As acute HCV infection is usually asymptomatic, early diagnosis is rare. Those people who are developing chronic infection, even though undiagnosed, may suffer serious liver damage. In fact, a significant proportion of HCV infected subjects will ultimately progress to liver cirrhosis and/or hepatocellular carcinoma, making chronic HCV infection a major health problem (3, 4).

Despite the excellent accuracy of the tests currently available for the detection of anti-HCV antibodies (anti-HCV), the delay in reporting the results, the need for specialized equipment for processing the samples and interpreting the results, as well as the need to transfer individuals to sample collection and processing centres, limit their use as screening tools. Serologic points-of-care tests (POCTs) have several advantages, namely that they require little specialized apparatus, can be brought to the individuals who are to be tested and allow diagnosis in as little as a few minutes in different clinical settings (5). These advantages might be translated into increased testing opportunity and, ultimately, identification of more patients who could benefit from antiviral treatment (6). Over the last few years, several tests for rapid detection of anti-HCV have been developed and are currently in use in various countries; however, only recently, the first POCT was approved by the U.S. Food and Drug Administration (7). The investigation of the diagnostic accuracy of POCTs and rapid tests for the detection of anti-HCV is a highly relevant topic. As well as the great importance of the issue in terms of public health, there is a lack of studies evaluating the performance of several of the currently used tests.

EASY test project
Since January 2011, the Infectious Diseases Department of San Raffaele Scientific Institute in Milan carried out a Prevention Program called “EASY test project”, using the new oral test (rapid and non-invasive) the OraQuick® HCV Rapid Antibody Test (OraSure technologies, Inc.) to diagnose the HCV infection. The test is a single-use, immunoassay for the qualitative detection of antibodies to hepatitis C virus (anti-HCV) in oral fluid, fingerstick whole blood, venipuncture whole blood and plasma specimens. The HCV rapid test received the FDA approval for use with oral fluid on 28 June 2010.

The clinical sensitivity and specificity of the OraQuick HCV test using oral fluid were 97.8% (95% confidence interval [CI]) and 100% (95% CI, 98.4-100%), respectively (8). 

The main objective of the project is to evaluate the acceptability of an alternative, free and anonymous HCV test offer, available in different settings (in Points of Care, STDs Prevention clinics and General Practitioner surgeries) (9, 10). Furthermore, contacting the ‘hard-to-reach’ people with this anonymous and free test offer could reduce or stop this public health problem, by making an easy link to healthcare.

Subjects who underwent the test were asked to complete an anonymous questionnaire, through which it has been possible to collect a series of data on risk behaviours of the population tested. The questionnaire was devised with the intention of collecting demographic and risk behaviour data, as well as previous HCV/HIV testing experience, information about sex, drug use, educational level, nationality, general behaviours, use of HIV/HCV prevention services, previous surgical practices, invasive diagnostic practices, dental cares, tattoos or sexually transmitted diseases. Post-test counselling has been provided to all HCV reactive and non-reactive subjects, by the Infectious Diseases Department physicians involved in the study. The test was been carried out by a biologist or a practitioner, following the manufacturer’s procedures.

If the HCV oral test provided a preliminary positive result, a venipuncture was performed immediately for standard test confirmation, supported by the post-test counselling.

The results were received in two working days. At this point, the HCV-positive patient was contacted directly by the infectious diseases specialist for the visit and the diagnostic procedures to define the liver disease status and eventually to start the treatment, according to the guidelines for when HCV viral load and genotype are identified.
In these four years a total of 35,000 subjects have been approached to inform them about HCV infection and other sexually transmitted diseases. Of the total eligible volunteers, 6500 (18.6%) performed HCV tests on saliva and completed the interview in the alternative ‘street lab’. From the questionnaires we know that this initiative has been much appreciated.

Discussion
In recent years, advances in detection technology made available a range of POCTs for different infectious diseases. It is now possible to screen and diagnose those conditions at primary healthcare settings, using minimally invasive tests. In the present study, a new POCT for HCV infection has been performed on oral fluid. The use of oral fluid is an attractive alternative based on the fact that collection of plasma or serum samples requires equipment and training, and is more time consuming.

The FDA-approved OraQuick HCV Rapid Antibody Test (OraSure Technologies) is one of the most studied rapid tests for the diagnosis of HCV infection.

The development of rapid alternative tests for the diagnosis of HCV infection is to facilitate access to testing to reduce the individual risk of disease progression and social costs.

Despite the excellent sensitivity and specificity of third-generation enzyme immunoassays (EIAs), the turnaround time for reporting test results is at least one day, thereby making it difficult to deliver the results to tested individuals at first visit. Rapid tests are formatted such that they do not require complicated instrumentation or testing by skilled technical staff. They potentially generate results within an hour and therefore may be used for point-of-care testing.  Rapid tests are obviously more expensive than conventional immunoassays and are not designed for testing large batches of specimens. However, in no-clinical settings and laboratories that conduct low-volume testing, adoption of rapid oral testing can be cost-effective. CDC guidelines formulated for confirming screening anti-HCV results remain to be refined to accommodate rapid anti-HCV testing. It is important to emphasize that OraQuick HCV test has not been approved for general screening. A positive result of a rapid anti-HCV positive test is indicative of the presence of anti-HCV and, again, does not indicate active infection (11).

We successfully conducted this rapid HCV testing and counselling programme with the goal of spreading the use of saliva test anonymously and free of charge. We aim to facilitate access to testing in alternative settings, in order to understand if the ‘hard-to access’ population would access salivary rapid testing versus the conventional settings.

Increasing awareness of these alternative tests among individuals at risk and providers may be an appropriate strategy to increase the number of people who know their serological status and who could be linked to care and engaged in care!

The recent introduction of rapid oral HCV antibody test could completely change the HCV diagnosis approach by facilitating the possibility of testing millions of people worldwide (in particular in the developing countries).

For these reasons, we hope the oral-fluid based rapid HCV tests could become the ‘gold standard’ to facilitate the HCV screening access and become the standard of care and the basis for the national HCV testing algorithm in many countries with spread HCV epidemic, also in the dental care surgeries.

References
1. Lavanchy D. The global burden of hepatitis C. Liver Int. 2009; 29: 74–81.
2. Istituto Superiore di Sanita (ISS). Available at: www.iss.it.
3. Hoofnagle JH. Hepatitis C: the clinical spectrum of disease. Hepatology 1997; 26: 15S–20S.
4. Hutin Y, Kitler M, Dore G, Perz J, Armstrong G, Dusheiko G, et al. Global burden of disease (GBD) for hepatitis C. J Clin Pharmacol. 2004; 44: 20–29.
5. Ferreira-Gonzales A, Shiffman ML. Use of diagnostic testing for managing hepatitis C virus infection. Semin Liver Dis. 2004; 24: 9–18.
6. Tucker JD, Bien CH, Peeling RW. Point-of-care testing for sexually transmitted infections: recent advances and implications for disease control. Curr Opin Infect Dis. 2013; 26: 73–79.
7. Food and Drug Administration. Available at: http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfClia/detail
8. OraQuick HCV Rapid Antibody Test. Available from:
http://www.fda.gov/MedicalDevices/productsandMedicalProcedures/DeviceApprovalsandClearances/Recently-approved-Devices
9. Parisi MR, Soldini L, Di Perri G, Tiberi S, Lazzarin A, et al. Offer of rapid testing and alternative biological samples as practical tools to implement HIV screening programs. New Microbiol. 2009; 32(4): 391–396.
10. Parisi MR, Soldini L, Vidoni GM, Clemente F, Mabellini C, Belloni T, Nozza S, Brignolo L, Negri S, Rusconi S, Schlusnus K, Dorigatti F, Lazzarin A. Cross-sectional study of community serostatus to highlight undiagnosed HIV infections with oral fluid HIV-1/2 rapid test in non-conventional settings. New Microbiol. 2013; 36(2): 121–132.
11. Center for Disease Control and Prevention. Recommendations for prevention and control of hepatitis C virus (HCV) infection and HCV-related chronic disease. Available from: http://www.cdc.gov/hepatitis/hcv/Management.htm.

The authors
Maria Rita Parisi^*1 MSc, Laura Soldini² MD, Gianmarino Vidoni³ MD, Karin Schlusnus⁴ PhD, Fernanda Dorigatti² MD, Adriano Lazzarin¹ MD
¹Division of Immunology, Transplantation and Infectious Diseases, Vita-Salute University, San Raffaele Scientific Institute, Milan, Italy
²Laboraf Diagnostic and Research OSR S.p.A., San Raffaele Scientific Institute, Milan, Italy
³Prevention Department, Reference Centre for HIV and STDs, Local Public Health Unit, Milan, Italy
⁴ANLAIDS Lombardia Onlus, Milan, Italy

*Corresponding author
E-mail: parisi.mariarita@hsr.it

New psychoactive substances (NPS) reach the recreational drugs market at a fast pace and are of concern because of potential health risks. In addition to not being legally regulated, NPS escape detection in standard drug tests. Drug testing laboratories, therefore, must adapt their analytical methods to also cover these new substances. For screening and confirmation of NPS, mass-spectrometric multicomponent methods are useful.

by Prof. Olof Beck and Prof. Anders Helander

New psychoactive substances
The emergence of new drugs of abuse that are designed to circumvent narcotics legislation by slight chemical structural modifications of already classified drugs represents an ever increasing problem [1, 2]. Nowadays, this phenomenon is commonly termed ‘new psychoactive substances’ or ‘NPS’, but also other names such as designer drugs, legal highs, research chemicals, smart drugs, bath salts, and spice have been and are used. The NPS problem is of global concern but may vary in extent between countries, partly due to national differences in legislation and drug culture. Statistics from the EU Early Warning System operated by the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) and Europol on the number of NPS reported for the first time in Europe on a yearly basis gives a good insight on the progress of this phenomenon (Fig. 1) [2]. Over the past 6 years particularly, it has escalated to the level of more than 100 new substances in 2014 (i.e. about two new substances each week on average). The NPS market was long dominated by stimulants and synthetic cannabinoids but currently comprises all classes of abused substances [2].

Problems related to NPS
NPS are of particular concern because they can be sold openly in web-based shops and elsewhere and thereby reach new drug users that are attracted by their ‘legal’ status. Of public concern are the unforeseen toxic effects of NPS, as using these uncontrolled and unsafe substances and products may lead to severe intoxication and even death [1, 3]. In Sweden, the progress of the NPS phenomenon and associated harmful effects has been followed in a collaborative project between the Department of Laboratory Medicine at the Karolinska University Hospital and the Karolinska Institutet, and the Swedish Poisons Information Center [3, 4]. This project, named STRIDA, enrolls patients with suspected NPS intoxication presenting in emergency departments all over the country. By combining the results from laboratory investigations of serum and urine samples with clinical information, new knowledge about NPS prevalence and toxicity is compiled. Since the start in 2010, the STRIDA project has documented over 2000 non-fatal but often severe acute intoxication cases involving a large number of different NPS. Polydrug use is commonly seen in these cases [3].

NPS in drug screening
One reason for using NPS instead of conventional drugs of abuse may be that NPS often remain undetected in standard drug testing procedures. Accordingly they are especially attractive alternatives for individuals who want to minimize the risk of being detected, such as in workplace drug testing and drug rehabilitation programmes.

The established procedure for drug testing is to use initial screening by immunoassays and then to confirm positive samples using methods based on the more sensitive and selective mass spectrometry (MS) technique. On one hand, the NPS present a challenge for the immunoassay screening, as available methods are typically directed only towards the conventional substances, e.g. amphetamines (amphetamine and methamphetamine), tetrahydrocannabinolcarboxylic acid (THC, cannabis), morphine (heroin), and benzoyl ecgonine (cocaine). On the other hand, as NPS are often designed to mimic and are chemical derivatives of conventional drugs, there is a possibility that certain NPS will also bind to (i.e. cross-react with) the antibodies used in immunoassay screening methods. And this is indeed the case. However, when these ‘false-positive’ screening results are subjected to confirmatory analysis by methods based on MS detection, they will turn out negative (i.e. ‘false-negative’ for drug use), if the MS method is only directed toward the standard set of abused drugs.

Cross-reactivity of NPS in immunoassays
When ecstasy (3,4-methylenedioxymethamphetamine, MDMA) became established as a street drug, interest emerged to detect it in immunoassay screening. MDMA and its metabolite 3,4-methylenedioxyamphetamine (MDA) were found to be detectable in existing assays for amphetamine and methamphetamine, due to a high degree of cross-reactivity for these compounds [5]. Likewise, also other new amphetamine-like substances were detectable [6].

However, although many NPS showed low cross-reactivity in commercial immunoassays [7, 8], the stimulant methylenedioxypyrovalerone (MDPV) was reported to cross-react in the CEDIA phencyclidine test [9]. A study from the authors’ laboratory comprising 45 NPS confirmed that several possessed chemical similarities leading to high cross-reactivity in the immunochemical screening tests commonly employed in routine urine drug testing [10]. The detectability of NPS observed to possess cross-reactivity was further confirmed by analysis of urine specimens from authentic intoxication cases included in the STRIDA project (Table 1). Given a more widespread use of new drugs among individuals subjected to drug testing, an increased number of unconfirmed positive screening results may occur.
The cross-reactivity for NPS in current screening assays may be seen as a problem or as a possibility to detect more substances. One possibility for improved drug testing is to include the most common new substances in the confirmation methods. As ecstasy became established as an illicit drug, new immunochemical screening tests for amphetamine/methamphetamine were developed that also included MDMA and MDA. Authentic case samples were used to demonstrate the capability of several commercial amphetamine class screening tests to detect MDMA/MDA. At that time, cross-reactivity towards the new ‘amphetamine’ analytes was wanted [5]. With the advent of the large number of NPS, both legal and illegal, the strategy to also cover new substances in the screening assays for classical narcotic drug substances may not be feasible. For example, the multitude of new synthetic cannabinoids (‘spice’) have not been incorporated in screening tests for THC, but resulted in the development of new independent tests [11].

One approach put forward to understand the potential of immunoassays to detect NPS is to use molecular similarity models [12]. Interestingly, the work of Petrie and co-workers [13] included such a molecular modelling method to predict the cross-reactivity of 261 amphetamine-like compounds. However, when comparing the theoretical data with our experimental data for one compound, the predicted reactivity for butylone was 10 times lower than that observed. In a more recent publication, it was proposed that molecular similarity models could be used to design new immunoassays with sensitivity for a larger number of target compounds [14].

NPS analysis by mass spectrometry
Another analytical strategy to cover NPS in drug testing is to employ MS-based ‘screening’ methods. As part of the STRIDA project, a multicomponent analytical MS method for NPS analysis in urine and serum specimens has been developed [15]. The method uses MS in combination with liquid chromatography (LC-MS/MS in selected-reaction monitoring mode) and is continuously updated as new NPS appear. There are also other methods for multicomponent screening of drugs in urine and plasma/serum, which proves that this technology can be employed in routine drug testing [16].

The LC-MS/MS technique has great potential for drug testing and for clinical laboratories in general. There are examples of laboratories that have already successfully replaced immunoassay screening by MS methods, also for the conventional drugs of abuse [17]. One way to make this possible and cost-effective is to use simple sample preparation procedures, e.g. a simple dilution of urine with internal standards [16]. When studying the cross-reactivity of 30 NPS in commercial ELISA tests for serum and blood, only a few were found to display cross-reactivity, and it was therefore proposed that MS methods should be used in future drug screening [18]. One attraction of MS-based screening is that accurate results are already obtained from the initial analytical step, which may be especially important in cases of acute intoxication (Fig. 2).

Potential of high-resolution MS
One promising technique for drug screening is high-resolution MS (HRMS) [19]. In the HRMS technique, the acquisition of data can be made with an untargeted design. Thousands of substances can be monitored at the same time without the need for optimizing MS parameters for each compound. In addition, new compounds can be searched for retrospectively.

Conclusion
The NPS present a challenge for drug testing laboratories and calls for novel drug screening strategies. It is likely that the current broader spectrum of abused psychoactive drugs will persist in at least in the foreseeable future. This new drug situation has put the performance of drug testing into focus and indicates that drug testing laboratories will play a more important role, as on-site drug screening using dipsticks is likely to lose significance.

References
1. Lewin AH, Seltzman HH, Carroll FI, Mascarella SW, Reddy PA. Emergence and properties of spice and bath salts: A medicinal chemistry perspective. Life Sci. 2014; 97: 9–19.
2. EMCDDA. New psychoactive substances in Europe. An update from the EU Early Warning System (March 2015). 2015. Available at: http://www.emcdda.europa.eu/attachements.cfm/att_235958_EN_TD0415135ENN.pdf.
3. Helander A, Bäckberg M, Hultén P, Al-Saffar Y, Beck O. Detection of new psychoactive substance use among emergency room patients: results from the Swedish STRIDA project. Forensic Sci Int. 2014; 243: 23–29.
4. Helander A, Bäckberg M, Beck O. MT-45, a new psychoactive substance associated with hearing loss and unconsciousness. Clin Toxicol. 2014; 52(8): 901–904.
5. Hsu J, Liu C, Hsu CP, Tsay WI, Li JH, Lin DL, Liu RH. Performance characteristics of selected immunoassays for preliminary test of 3,4-methylenedioxymethamphetamine, methamphetamine, and related drugs in urine specimens. J Anal Toxicol. 2003; 27: 471–478.
6. Apollonio LG, Whittall IR, Pianca DJ, Kyd JM, Haher WA. Matrix effect and cross-reactivity of select amphetamine-type substances, designer analogues, and putrefactive amines using Bio-Quant direct Elisa presumptive assays for amphetamine and methamphetamine. J Anal Toxicol. 2007; 31: 208–213.
7. Kerrigan S, Mellon MB, Banuelos S, Arndt C. Evaluation of commercial enzyme-linked immuno assays to identify psychedelic phenethylamines. J Anal Toxicol. 2011; 35: 444–451.
8. Bell C, George C, Kicman AT, Traynor A. Development of a rapid LC-MS/MS method for direct urinalysis of designer drugs. Drug Test Anal. 2011; 3: 496–504.
9. Macher AM, Penders TM. False-positive phencyclidine immunoassay results caused by 3,4-methylenedioxypyrovalerone (MDPV). Drug Test Anal. 2012; 5: 130–132.
10. Beck O, Rausberg L, Al-Saffar Y, Villen T, Karlsson L, Hansson T, Helander A. Detectability of new psychoactive substances, ‘legal highs’, in CEDIA, EMIT, and KIMS immunochemical screening assays for drugs of abuse. Drug Test Anal. 2014; 6: 492–499.
11. Arntson A, Ofsa B, Lancaster D, Simon JR, McMullin M, Logan B. Validation of a novel immunoassay for the detection of synthetic cannabinoids and metabolites in urine specimens. J Anal Toxicol. 2013; 37: 284–290.
12. Krasowski MD, Pizon AF, Siam MG, Giannoutsos S, Iyer M, Ekins S. Using molecular similarity to highlight the challenges of routine immunoassay-based drug of abuse/toxicology screening in emergency medicine. BMC Emerg Med. 2009; 9: 5.
13. Petrie M, Lynch KL, Ekins S, Chang JS, Goetz RJ, Wu AHB, Krasowski MD. Cross-reactivity studies and predictive modeling of “Bath Salts” and other amphetamine-type stimulants with amphetamine screening immunoassays. Clin Toxicol. 2013; 51: 83–91.
14. Krasowski MD, Ekins S. Using cheminformatics to predict cross reactivity of “designer drugs” to their currently available immunoassays. J. Cheminform. 2014; 6: 22.
15. Al-Saffar Y, Stephanson NN, Beck O. Multicomponent LC-MS/MS screening method for detection of new psychoactive drugs, legal highs, in urine – experience from the Swedish population. J Chromatogr B 2013; 930: 112–120.
16. Beck O, Ericsson M. Methods for urine drug testing using one-step dilution and direct injection in combination with LC-MS/MS and LC-HRMS. Bioanalysis 2014; 6 : 2229–2244.
17. Eichhorst JC, Etter ML, Rousseaux N, Lehotay DC. Drugs of abuse testing by tandem mass spectrometry: A rapid, simple method to replace immunoassays. Clin Biochem. 2009; 42: 1531–1542.
18. Swortwood MJ, Hearn WL, DeCaprio AP. Cross-reactivity of designer drugs, including cathinone derivatives, in commercial enzyme-linked immunosorbent assays. 2014; 6: 716–727.
19. Maurer HH. What is the future of (ultra) high performance liquid chromatography coupled to low and high resolution mass spectrometry for toxicological drug screening? J Chromatogr A 2013; 1292: 19–24.

The authors
Olof Beck*^1,3 PhD and Anders Helander^2,3 PhD
¹Department of Clinical Pharmacology, Karolinska University Laboratory Huddinge, Sweden
²Department of Clinical Chemistry, Karolinska University Laboratory Huddinge, Sweden
³Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden

*Corresponding author
E-mail: olof.beck@karolinska.se