Rapid diagnostic testing enables life-saving scale up of HIV diagnosis but is vulnerable to false positive results. Confirmation testing can be impractical or cost prohibitive in resource-limited settings. Retesting a diluted blood sample is evaluated and proposed, at a proof of concept level, as a simple cost-effective HIV confirmation methodology.

by Derryck Klarkowski and Dr Erwan Piriou

Background
The diagnosis of HIV infection in developed countries is based on initial screening for HIV antibodies, and if detected, confirmation with nucleic acid testing (NAT) [1]. This ensures high sensitivity and specificity. However, the current World Health Organization (WHO) HIV Testing Services Guidelines do not include specific confirmation testing for the diagnosis of HIV across large population groups in resource-limited settings [2]. Instead WHO recommends that diagnosis be made on the basis of rapid diagnostic tests (RDTs) only (or equivalent enzyme immune assay tests) requiring a minimum of two positive test results, using test devices from different sources, for a positive diagnosis (or three in low prevalence settings) [2]. Although the WHO strategy has enabled life-saving scale-up of HIV diagnosis the significant compromise is that without confirmation there is a risk that patients/clients can be falsely diagnosed as HIV positive [3]. This is also well demonstrated in the study discussed now in this article where the WHO RDT algorithm resulted in 6.8% false positive results (n = 2897). Incorrect HIV diagnosis can have devastating consequences for the individual as well as wasting often over-stretched resources required for treatment and care.

Médecins Sans Frontières (MSF) has strongly advocated for the use of serological HIV confirmation testing in resource-limited settings when it is impractical to perform NAT [4–6]. Commercial confirmation kits are available that detect individual specific HIV antibodies, such as gp40, gp120, p24 and p32, that significantly increase the accuracy of testing at a considerably lower cost than NAT, and this type of confirmation testing can be performed by non-specialized laboratories. The downside, however, is that commercial confirmation kits nevertheless add cost, albeit reduced compared to NAT, and logistical complications that restrict their widespread use.
To address this, MSF has recently published a simplified confirmation approach based on antibody dilution requiring only the use of an additional routinely used RDT test device [7]. This study has been published as a ‘proof of concept’ paper and requires further testing across different settings for refinement before it can be generally recommended.

Causes of false positive HIV antibody detection
As with all tests, false positive HIV RDTs can be caused by user error (clerical mistakes, incorrect test performance, misinterpretation and cross-contamination). Other causes for HIV tests include nonspecific IgG binding [2], cross reactivity [2, 5], contaminating proteins [2] and pseudo-antigens created during the manufacturing process [5]. However, a key additional vulnerability for HIV antibody detection testing is that all commonly available HIV RDTs share a common gp41 detection antigen. Therefore, a cross-reactive antibody interacting with gp41 will act as a pan-cross-reactive antibody across multiple test devices [5].

The WHO algorithm is based on the assumption that HIV RDTs that use different antigen preparations are independent and, therefore, by requiring two positive tests (at a prevalence >5%) before reporting HIV positivity, the algorithm assumes that the second test confirms the result of the initial screening test [2]. However, in one MSF published study 50% of false positive samples had cross-reactive anti-gp41 activity, identified by Western blot (WB), that was the likely cause of the double false positive reactions with the two independent RDTs used in the testing algorithm [4].

MSF has proposed that early-immune-response broad-specificity polyclonal B-lymphocyte antibodies are a potential source of HIV RDT cross-reactive interfering antibodies [4]. These antibodies are likely to have increased frequency and intensity in resource-limited settings because of the higher prevalence of concomitant infections [8–10]. Additionally, displaced populations and individuals, such as caused by oppression, conflict and famine, are likely to have a greater vulnerability to cross-reacting infections than stable communities.

Theoretical basis for dilution methodology
Confirmation by dilution is based on the established sensitive/less sensitive (S/LS) methodology developed to identify recent HIV infection for the purposes of incidence surveys [11–15]. This methodology is based on the principle that HIV antibody titres increase over a period of several months after initial infection. Samples are initially tested using a high sensitivity HIV enzyme immunoassay (EIA) and if reactive are then further tested by the same EIA assay but using a diluted sample and reduced incubation time to reduce sensitivity. Samples testing positive on the sensitive (S) test but negative on the less sensitive (LS) test are designated a recent infection. The methodology has been successfully extended to the use of RDTs [13–15]. Confirmation by dilution adapts the S/LS principle to differentiate between high titre true HIV antibodies and low titre cross-reacting antibodies.

One postulated source of cross-reactive antibodies are broad spectrum antibodies produced in the early immune response to a wide range of infectious disease antigens, and these antibodies can cause nonspecific cross reactivity in HIV serological testing [5]. In proposing dilution as a methodology to confirm HIV infection, we postulate that cross-reacting antibodies will have a low titre relative to specific HIV antibodies.

Cross-reacting antibodies can generally be expected to have low avidity, as has been demonstrated by work in blood donors [16] and in MSF findings [4]. This will result in weakly positive results that can provide an alert for the tester; however, manufacturers generally state that any positive test line independent of strength should be interpreted as a positive result. Cross-reactive antibodies can also have high avidity as shown in a previous MSF publication where 7 of 24 (29.2%) false positive samples (total sample size 229) had strongly positive test lines in two RDTs but had a low titre relative to the confirmed true HIV antibodies [4].

The use of dilution as a supplementary confirmatory test by using antibody relative titres has been previously reported by Urwijitaroon et al. [17]. In another study, 41 samples were found positive using the HIV RDT Determine™ and 23 were negative on dilution [18]. Only 1 of these 23 samples was confirmed to be positive using serological confirmation (INNO-LIA™).

Field testing
A study was conducted at two sites in north western Ethiopia in programmes covering both residents and seasonal migrant workers. Seasonal workers are transient and, as postulated by MSF, may potentially have a higher risk of false positivity caused by cross-reacting antibodies [4, 5].

The study recruited 2897 individuals, and 265 (9.1%) samples tested as positive using two HIV RDTs from different manufacturers and would have been interpreted as HIV positive using the WHO algorithm [2]. Of the negative samples, 229 (approximately every 11th sample) were selected as a control. All algorithm-positive and negative control samples were further tested by dilution in situ, and additional confirmation testing performed by reference laboratories using WB and NAT for indeterminate WB samples.

All negative samples were confirmed as negative (100% sensitivity). However, 18/265 (6.8%) algorithm ‘positive’ samples were identified as HIV negative (false positive) by either WB or NAT.

Dilution testing was performed by titrating the patient’s plasma using confirmed seronegative plasma from healthy blood donors using a micropipette. Ten microlitres of patient plasma was first diluted 1 : 10 in 90 µL of negative plasma followed by a serial 4-fold dilution from 1:40 to 1 : 10,240. Testing was performed using Determine™ HIV-1/2 (Alere Laboratories, Japan) following manufacturer’s instructions. Tests were interpreted as positive if there was any colouration of the test line and the highest dilution that gave a positive result was recorded. Where the lowest dilution (1 : 10) was negative, the sample was reported as negative.

Findings and conclusion
In this study, based on a specific population group over a specific time period, repeating the RDT test using the sample diluted 1 : 160 identified all false positive results and misidentified one true positive (see Table 1). However, there is a safety net that any sample with a reactive HIV RDT test that is not resolved as a true positive at the time of testing is not reported as negative but as inconclusive [2]. The patient/client is advised that testing has been inconclusive and testing should be repeated at a later time; WHO recommends retesting after 14 days. This allows time for true HIV antibodies to increase in titre.

The discriminatory threshold dilution may vary between different settings. In an earlier MSF study, a dilution of 1 : 1000 differentiated 229 true HIV positive from 27 HIV false positive samples (unpublished data, for further details see Klarkowski et al. [4]).

One strength of this MSF study is that NAT testing was available to resolve indeterminate WB samples which made it possible to rule out early seroconversion as a potential cause of false positive results. The limitation is that the findings are restricted to a single cohort with a single RDT and should be viewed as a ‘proof of concept’. More experience is needed in different settings and by different workers before the dilution methodology can be considered for potential scale up. It is proposed that the methodology has potential for use as a supplementary test in a confirmatory algorithm, whereby double positive RDT results are tested by dilution, with positive results above a determined threshold confirming HIV infection. Dilution results below the threshold would require further testing, such as repeat testing at a later time or NAT, to rule out false negative results either due to seroconversion or misclassification by the lower sensitivity dilution test.

References
1. Centers for Disease Control and Prevention and Association of Public Health Laboratories. Laboratory testing for the diagnosis of HIV infection: updated recommendations. 2014; http://stacks.cdc.gov/view/cdc/23447.
2. World Health Organization. Consolidated guidelines on HIV testing services. 2015; http://www.who.int/hiv/pub/guidelines/hiv-testing-services/en/.
3. Johnson C, Fonner V, Sands A, Tsui S, Ford N, Wong V, Obermeyer C, Baggaley R. Annex 14 A report on the misdiagnosis of HIV status. In: World Health Organization. Consolidated Guidelines on HIV Testing Services. 2015; http://www.who.int/hiv/pub/guidelines/hiv-testing-services/en/.
4. Klarkowski DB, Wazome JM, Lokuge KM, Shanks L, Mills CF, O’Brien DP. The evaluation of a rapid in situ HIV confirmation test in a programme with a high failure rate of the WHO HIV two-test diagnostic algorithm. PLoS One 2009; 4(2): e4351.
5. Klarkowski D, O’Brien DP, Shanks L, Singh KP. Causes of false positive HIV rapid diagnostic test results. Expert Rev Anti-infect Ther. 2013; 12(1): 49-62
6. Shanks L, Klarkowski D, O’Brien DP. False positive HIV diagnoses in resource limited settings: operational lessons learned for HIV programmes. PLoS ONE 2013; 8(3): e59906.
7. Shanks L, Siddiqui MR, Abebe A, Piriou E, Pearce N, Ariti C, Masiga J, Muluneh L, Wazome J, Ritmeijer K, Klarkowski D. Dilution testing using rapid diagnostic tests in a HIV diagnostic algorithm: a novel alternative for confirmation testing in resource limited settings. Virol J. 2015; 12: 75.DOI 10.1186/s12985-015-0306-4
8. Messele T, Abdulkadir M, Fontanet AL, Petros B, Hamann D, Koot M, Roos MT, Schellekens PT, Miedema F, Rinke de Wit TF. Reduced naive and increased activated CD4 and CD8 cells in healthy adult Ethiopians compared with their Dutch counterparts. Clin Exp Immunol. 1999; 115(3): 443–50.
9. Clerici M, Butto S, Lukwiya M, Saresella M, Declich S, Trabattoni D, Pastori C, Piconi S, Fracasso C, Fabiani M, Ferrante P, Rizzardini G, Lopalco L. Immune activation in Africa is environmentally-driven and is associated with upregulation of CCR5. Italian-Ugandan AIDS Project. AIDS 2000; 14(14): 2083–2092.
10. Clerici M, Declich S, Rizzardini G. African enigma: key player in human immunodeficiency virus pathogenesis in developing countries? Clin Diagn Lab Immunol. 2001; 8(5): 864–866.
11. World Health Organization Technical Working Group on HIV Incidence Assays. When and how to use assays for recent infection to estimate HIV incidence at a population level. 2011; http://www.who.int/diagnostics_laboratory/hiv_incidence_may13_final.pdf 2011.
12. Constantine NT, Sill AM, Jack N, Kreisel K, Edwards J, Cafarella T, Smith H, Bartholomew C, Cleghorn FR, Blattner WA. Improved classification of recent HIV-1 infection by employing a two-stage sensitive/less-sensitive test strategy. J Acquir Immune Defic Syndr. 2003; 32: 94–103.
13. Soroka SD, Granade TC, Candal D, Parekh BS. Modification of rapid human immunodeficiency virus (HIV) antibody assay protocols for detecting recent HIV seroconversion. Clin Diagn Lab Immunol. 2005; 12: 918–21.
14. Kshatriya R, Cachafeiro AA, Kerr RJS, Nelson JA, Fiscus SA. Comparison of two rapid human immunodeficiency virus (HIV) assays, Determine™ HIV-1/2 and OraQuick Advance Rapid HIV-1/2, for detection of recent HIV seroconversion. J Clin Microbiol. 2008; 46(10): 3482–3483.
15. Girardi SB, Barreto AM, Barreto CC, Proietti AB, Carvalho SM, Loureiro P, Sabino EC. Evaluation of rapid tests for human immunodeficiency virus as a tool to detect recent seroconversion. Braz J Infect Dis. 2012; 16(5): 452–456.
16. Bouillon M, Aubin E, Roberge C, Bazin R, Lemieux R. Reduced frequency of blood donors with false-positive HIV-1 and -2 antibody EIA reactivity after elution of low-affinity nonspecific natural antibodies. Transfusion 2002; 42(8): 1046–1052.
17. Urwijitaroon Y, Barusrux S, Romphruk A, Puapairoj C, Thongkrajai P. Anti-HIV Antibody Titer: An Alternative Supplementary Test for Diagnosis of HIV-1 Infection. Asian Pac J Allergy Immunol. 1997; 15:193–198.
18. Duedu KO, Hayford AA and Sagoe KW. Misclassification of recent HIV-1 seroconversion in sub-Saharan Africa using the sensitive/less sensitive technique. Virol J. 2011; 8: 176.

The authors
Derryck Klarkowski* MAppSc, Erwan Piriou PhD
Médecins Sans Frontières, Amsterdam, The Netherlands

*Corresponding author
E-mail: derryck.klarkowski@gmail.com

Professor Jordi Vila, Head of Department of Clinical Microbiology, Hospital Clinic, School of Medicine, University of Barcelona, Spain, describes how a new, fully automated molecular diagnostic system, has the potential to improve productivity and turnaround times at his busy organ transplant reference laboratory in Barcelona, Spain.

The Hospital Clinic of Barcelona serves a local population of 540,000, in addition to being a National and International Centre of reference, providing the full range of medical and surgical specialties.  The Hospital’s Department of Clinical Microbiology is also a reference laboratory for organ transplantation. 

Operating 24 hours a day, seven days a week, the laboratory has experienced a growing workload in recent years, mainly associated with an increase in molecular biology assays, including viral loads for Human Immunodeficiency Virus type 1 (HIV-1), Hepatitis C Virus (HCV), Hepatitis B Virus (HBV) and Cytomegalovirus (CMV).  In particular, the laboratory has observed an increase in HCV viral load requests, related to new treatment regimens, as well as an increase in CMV viral load requests for organ transplant patients. 
The total number of viral load assays performed annually in the Barcelona laboratory for HIV-1, HCV, HBV and CMV are shown in figure 1.
 
The need for workflow improvements
Like many laboratories throughout Europe, the Virology Section at the Hospital Clinic of Barcelona must cope with this growing workload without any increase in staffing levels.  As a result, there is a strong interest in workflow improvements as a means to increase productivity within the laboratory and to ensure the quality of generated results. 

Speed and efficiency are particularly important when clinical decisions are dependent on the result, and an increase in automation, particularly in the disciplines of serology, molecular diagnostics and bacteriology, have played an important role in achieving greater speed and efficiency in the Clinical Microbiology Laboratory.

The aims of the laboratory’s investigations into increased automation and workflow improvements were to reduce turnaround times, to reduce waste (of time and reagents), to maximise the use of staff, space and equipment, to increase productivity and to reduce opportunities for error.

Limitations of current methods
When looking at potential areas for improvement, a number of drawbacks were observed in the current methods used for obtaining HIV-1, HCV, HBV and CMV viral loads.  These methods require separate platforms for nucleic acid extraction, amplification and detection. Numerous steps are required to achieve the final result, which are quite labour intensive.  In order to be cost effective, assays are performed in batches (table 1), which limits the number of assay runs performed in a week.  This has a major impact on result turnaround times and has significant cost implications for urgent samples. 

In addition to these limitations, all of the existing equipment and sample preparation is located in a small room where space is of a premium.  As a result, working conditions are very crowded and some tasks, for example reagent preparation, need to be performed in an adjacent room, which is not ideal.

A new, fully automated system
An independent time/workflow analysis study was performed at the Hospital Clinic of Barcelona Virology Laboratory by Nexus Global Solutions (Plano, Texas, USA).  This study compared workflows and time to results between current viral load methods and the new, fully automated DxN VERIS Molecular Diagnostics System (Beckman Coulter Inc.).

Launched at ECCMID 2015, the DxN VERIS Molecular Diagnostics System consolidates DNA extraction, amplification and detection on a single automated instrument.  By reducing manual intervention and automating the process from sample loading to reporting of results, this system has the potential to transform virology laboratory workflows.

DxN VERIS assays are supplied in a unique, single cartridge system and all consumables and reagents are stored on-board the system, which reduces preparation time and effort. Unlike traditional plate-based systems, there is no need to batch assay runs and there are no empty wells, which reduces wastage and consumable costs. With true single sample random access, the DxN VERIS platform allows viral load assays to be performed as soon as they arrive in the laboratory and the short assay runtimes ensure rapid turnaround times.

Comparative performance studies at several DxN VERIS evaluation sites[1-13]  have shown that the VERIS HBV, HCV, HIV-1 and CMV assays demonstrate comparable precision, sensitivity and linearity to a range of alternative, commercially available viral load methods.

Workflow study results
It was decided to run DxN VERIS samples as single sample random access, as intended by the manufacturers.  This meant that samples could be loaded straight on to the DxN VERIS when they arrived in the laboratory, which is much faster than daily batch testing.  The results of the comparative workflow study at the Hospital Clinic of Barcelona are shown in table 2 and figures 2 and 3. 

In particular, the DxN VERIS workflow involved far fewer steps, especially pre-analytical steps, reduced hands-on time and fewer consumables.  The time to the first result is greatly reduced compared to current methods and, notably, subsequent results are available every 2.5 minutes.  For the current methods, results are not available until the end of the run.
During a normal working week, the DxN VERIS system allowed much faster turnaround of results, with all results being reported in under 24 hours (figure 3).
 
Workflow improvements
The DxN VERIS Molecular Diagnostics System offers some important workflow advantages compared to current methods for the determination of viral loads for HIV-1, HCV, HBV and CMV.  For example, the DxN VERIS system allows continuous loading of samples, which eliminates the need for batching and, with true, single sample random access, it allows urgent samples to be added at any time.  This is a particularly important aspect for us as a reference centre where urgent test requests can arrive at the laboratory at any time of day.  The DxN VERIS system allows laboratories to perform assays for several viruses at the same time, on the same platform, which allows flexibility, and with adaptable racks, it also has the versatility to accept a variety of sample tube types.

As a fully automated system, the DxN VERIS system decreases the potential for human error and reduces turnaround times considerably compared to the current methods, which allows much faster reporting of results to service users.  Unlike current methods, technicians are not required to pipette samples and reagents, which is an important ergonomic advantage.  By reducing manual time requirements it will allow laboratories to achieve the most from existing staffing levels, helping to maximize productivity within the laboratory.

In addition to this, consolidation of extraction, amplification and detection for these four targets onto a single platform is an important consideration for laboratories, like this, where space is very limited. 

The implementation of automated methodologies, such as this, has the potential to improve the quality and delivery of virology services and, for patients, it allows infectious disease results to be obtained at the earliest opportunity with high sensitivity and specificity.

^{For further information about the DxN VERIS Molecular Diagnostics System and the DxN VERIS assays currently available, please contact: Tiffany Page, Senior Pan European Marketing Manager Molecular Diagnostics, Email: info@beckmanmolecular.com or visit www.beckmancoulter.com/moleculardiagnostics.}

References
1. Williams, JA, Rodriguez, J, Wang, Z et al (2014) Poster presentation, ESCV, Prague.
2. Drago, M, Franchetti, E, Fanti, D and Gesu, GP (2015) Poster presentation, EuroMedLab, Paris.
3. Zurita, S, Gutiérrez, F, Folgueira, MD et al (2015) Poster presentation, EuroMedLab, Paris.
4. Christenson, R, Maggert, K, Ruiz, RM et al (2015) Poster presentation, ECCMID, Copenhagen.
5. Trimoulet, P, Tauzin, B, Belloc, E et al (2015) Poster presentation, EuroMedLab, Paris.
6. Gilfillan, R, Wang, Z, Xu, Y et al (2014) Poster presentation, ECCMID, Barcelona.
7. Xu, Y, Gilfillan, R, Wang, Z et al (2014) Poster presentation, ESCV, Prague.
8. Mengelle, C, Sauné, K, Haslé, C et al (2014) Poster presentation, RICAI.
9. Mengelle, C, Sauné, K, Haslé, C et al (2015) Poster presentation, ECCMID, Copenhagen.
10. Silvestro, A, Duan, H, Lim, S et al (2014) Poster presentation, ECCMID, Barcelona.
11. Li, Q, Williams, J, Maggert, K et al (2014) Poster presentation, ECCMID, Barcelona.
12. Xu, Y, Dineen, S, Annese, V et al (2014) Poster presentation, ESCV, Prague.
13. Williams, JA, Rodriguez, J, Wang, Z et al (2014) Poster presentation, ECCMID, Barcelona.

This contribution describes the possibility of applying liquid chromatography coupled to tandem mass spectrometry for analysing sewage in order to track down the use of new psychoactive substances.

by J. Kinyua, Prof. A. Covaci, Prof. A. L. N. van Nuijs

Introduction
Sewage-based epidemiology (SBE) is an alternative method of monitoring population drug use by the analysis of excretion products of drugs in sewage (Fig. 1). SBE has been applied since 2005 as a complementary approach to classical investigation methods, such as interviews with users, medical records, population surveys, and crime statistics for estimating illicit drug use in communities [1–3]. Data obtained from SBE provide information on drug use in a direct, quick and objective way.

New psychoactive substances (NPS) are substances that are not controlled by the 1961 United Nations Single Convention on Narcotic Drugs or the 1971 Convention on Psychotropic Substances and that may pose a threat to public health [4, 5]. These compounds mimic effects of illicit drugs like cocaine, cannabis and amphetamines and are produced to evade law enforcement by introducing slight modifications to chemical structures of controlled substances [6]. Currently, more than 450 NPS are being monitored by the European Monitoring Centre for Drug and Drug Addiction (EMCDDA) with 101 new substances reported for the first time in 2014 to EU Early Warning System (EWS). Synthetic cannabinoids and synthetic cathinones are the largest groups in the NPS scene [7]. NPS are easily acquired through online vendors and in smart shops where they are sold with misleading information about their effects and safety [8]. They are considered a growing problem in many communities and are responsible for numerous fatal intoxications [9]. SBE has the potential to be usefully applied for the detection and quantification of NPS to document their occurrence to appropriate authorities. Being an emerging issue, only a few studies have applied SBE for the analysis of NPS [10–13]. In this contribution, the optimization, validation and application of an analytical method using liquid chromatography coupled to positive electrospray tandem mass spectrometry (LC–ESI-MS/MS) for the determination of seven NPS in sewage: methoxetamine (MXE), butylone, ethylone, methylone, methiopropamine, 4-methoxymethamphetamine (PMMA), and 4-methoxyamphetamine (PMA) is described together with a critical evaluation of the methodology.

LC-MS/MS methodology
An LC-MS/MS method was developed and validated using a Phenomenex Luna HILIC (hydrophilic interaction liquid chromatography) 200A (150 x 3 mm, 5 µm) column, with a mobile phase composed of A) 5 mM ammonium acetate in ultrapure water and B) acetonitrile. The mass spectrometer compound dependent parameters, fragmentor voltage and collision energy, were optimized to acquire two multiple reaction monitoring (MRM) transitions (qualifier and quantifier) for each compound, and one MRM for the internal standards (IS). The method was validated, assessing accuracy and precision, using blank sewage (samples collected prior to 2009 in which NPS have not been detected). A linear range with lower limits of quantification (LLOQ) of 0.5 ng/L (MXE and methylone) and 2 ng/L (all other compounds) and upper limits of quantification (ULOQ) of 200 ng/L was achieved for investigated compounds. The limit of detection (LOD) was between 0.02 and 0.2 ng/L for all compounds.

Sample collection and preparation
24-h composite influent sewage samples were collected from different wastewater treatment plants (WWTPs) in Belgium and one WWTP in Zurich. Before sample extraction, 50 mL sewage was filtered through a 0.7 µm glass filter to remove solid particles. After filtration, the samples were brought to pH 2 using a 6 M HCl solution and spiked with deuterated IS at a concentration of 100 ng/L. Thereafter the solid-phase extraction (SPE) procedure was performed using a mixed-mode strong cation exchange sorbent-Oasis MCX (Fig. 2).

Application of the procedure
The method could reliably differentiate the analytes and IS from endogenous components. MXE, methylone and ethylone could be detected. The method revealed the presence of MXE in sewage from five urban centres within two counties in Belgium. Methylone was detected and quantified in only two samples from Switzerland at levels slightly higher than LLOQ (Fig. 3). The compounds that were not detected could be absent in the sewage or present in the form of metabolites which were not targeted in the present study.

Advantages/limitations of the SBE methodology
Phenylethylamine-based compounds (synthetic cathinones and amphetamine-like substances) form a large group of NPS and they are very polar. Hydrophilic interaction was found to be a good and robust LC stationary phase to obtain retention for these high-polarity compounds. Furthermore, we showed for the first time in SBE that the use of a more realistic matrix for method development, such as real sewage, can help in overcoming challenges associated with matrix effects in MS detection. The results from these samples demonstrate the importance of developing highly sensitive analytical methods that can detect and quantify NPS at very low concentrations (<10 ng/L). Limitations of the present methods
It is difficult to determine if the low drug concentrations in sewage are related to low popularity of the NPS or due to the presence of an unknown form of the parent drug in sewage, urinary metabolites or transformation product from other in-sewer processes. SBE requires a specific, reliable and stable biomarker for the NPS of interest. Further studies on the metabolism and in-sewer transformation processes (which may affect stability of drug residues) of NPS needs thus to be carried out to provide SBE with information regarding additional biomarkers of NPS parent drugs.

Future of SBE in NPS analysis
Concentrations of NPS in sewage may be low depending on the area served by the WWTP and on the prevalence of its use [14]. Therefore, pooled urine analysis would be useful in detecting the occurrence of NPS before dilution into sewage [15]. It would be a valuable approach to combine pooled urine analysis and SBE to track down the actual use of NPS in communities.

Conclusion
In conclusion, SBE can help in revealing the occurrence of NPS within catchment areas of urban centres and showed the need to develop very sensitive analytical methods to detect NPS in sewage.

References
1. Bijlsma L, Sancho JV, Pitarch E, et al.  Simultaneous ultra-high-pressure liquid chromatography-tandem mass spectrometry determination of amphetamine and amphetamine-like stimulants, cocaine and its metabolites, and a cannabis metabolite in surface water and urban wastewater. J Chromatogr A 2009; 1216: 3078–3089.
2. Boleda MR, Galceran MT, Ventura F. Trace determination of cannabinoids and opiates in wastewater and surface waters by ultra-performance liquid chromatography-tandem mass spectrometry. J Chromatogr A 2007; 1175: 38–48.
3. Huerta-Fontela M, Galceran MT, Ventura F. Ultraperformance liquid chromatography-tandem mass spectrometry analysis of stimulatory drugs of abuse in wastewater and surface waters. Anal Chem 2007; 79: 3821–3829.
4. United Nations Office on Drugs and Crime (UNODC). Global synthetic drugs assessment. (United Nations publication, Sales No. E.14.XI.6), 2014. http://www.unodc.org/documents/scientific/2014_Global_Synthetic_Drugs_Assessment_web.pdf
5. King LA, Kicman AT. A brief history of ‘new psychoactive substances’. Drug Test Anal. 2011; 3: 401–403.
6. Dargan PI, Wood DM. Novel psychoactive substances classification, pharmacology and toxicology. Elsevier/Academic Press, 2013. ASIN: B00FK8HYY2.
7. European Monitoring Centre for Drugs and Drug Addiction (EMCDDA). New psychoactive substances in Europe. An update from the EU Early Warning System, 2015. http://www.emcdda.europa.eu/publications/2015/new-psychoactive-substances
8. EMCDDA. EMCDDA–Europol 2013 Annual Report on the implementation of Council Decision 2005/387/JHA, 2014. http://www.emcdda.europa.eu/publications/implementation-reports/2013
9. Vevelstad M, Øiestad E.L, Middelkoop G, et al. The PMMA epidemic in Norway: Comparison of fatal and non-fatal intoxications. Forensic Science International 2012; 219: 151–157.
10. Kinyua J, Covaci A, Maho W, et al. Sewage-based epidemiology in monitoring the use of new psychoactive substances: validation and application of an analytical method using LC-MS/MS. Drug testing and analysis 2015 ( In press).
11. Reid M.J, Derry L, Thomas K.V. Analysis of new classes of recreational drugs in sewage: Synthetic cannabinoids and amphetamine-like substances. Drug Test Anal. 2014; 6: 72–79.
12. Van Nuijs ALN, Gheorghe A, Jorens PG, et al. Optimization, validation, and the application of liquid chromatography-tandem mass spectrometry for the analysis of new drugs of abuse in wastewater. Drug Test Anal. 2014; 6: 861–867.
13. Kankaanpää A, Ariniemi K, Heinonen M, et al. Use of illicit stimulant drugs in Finland: a wastewater study in ten major cities. Sci Total Environ. 2014; 487: 696–702.
14. Archer JRH, Dargan PI, Lee HMD, et al. Trend analysis of anonymised pooled urine from portable street urinals in central London identifies variation in the use of novel psychoactive substances. Clinical Toxicol (Phila). 2014; 52: 160–165.
15. Archer JRH, Dargan PI, Hudson S, et al. Analysis of anonymous pooled urine from portable urinals in central London confirms the significant use of novel psychoactive substances. QJM 2013; 106: 147–152.

The authors
Juliet Kinyua MSc, Adrian Covaci PhD, Alexander L.N. van Nuijs* PhD
Toxicological Center, University of Antwerp, Belgium

*Corresponding author
E-mail: alexander.vannuijs@uantwerpen.be