Use of psychoactive substances by goods carriage drivers associated with Kerala, India

Abstract Objective This study aimed to measure the prevalence of drugs of abuse (DOA) among the goods carriage drivers associated with the southern State of India, Kerala. Methods Point-of-collection testing (POCT) of oral fluid collected from the participants (n = 249) was done using the Evidence MultiSTAT DOA Oral Fluid II Assay kits and the Evidence MultiSTAT analyzer. Results Out of the total samples, 53 (21.29%) were positive for one or more DOA. A high prevalence of tetrahydrocannabinol (THC) (10.04%) and synthetic cannabinoids were detected in the samples. The use of ketamine, alpha-PVP, LSD, methamphetamine, opiate, 6-MAM, benzodiazepines I, methadone, PCP, tramadol, and amphetamine was also detected and their frequency of use ranged between 4.02 and 0.80%. An association between drug abuse and distance of travel was found in drivers in this study, χ 2 (5, N = 249) = 123.5, p < 0.001. Confirmatory analysis using ultra-high performance liquid chromatography-tandem mass spectrometry showed excellent agreement with the results of the screening test. Conclusions This was the first study conducted among drivers in India for the detection of DOA. Tetrahydrocannabinol (THC) was used more by the goods carriage drivers associated with Kerala State, India. The use of psychoactive substances significantly increased with the distance of travel. Point-of-collection testing (POCT) by the biochip array technology is an efficient method for the detection of these substances.


Introduction
Psychoactive substances and drugs have a significant impact on human life, but their abuse poses a substantial threat to global security and stability.The production and consumption of illicit drugs and psychoactive substances have increased worldwide due to factors, such as free markets, free trade, and improved communication.The United Nations Office on Drugs and Crime (UNODC) estimates that between 2010 and 2019, the number of drug users ranged from 226 million to 274 million, with a particular attraction to drugs among individuals aged 15-64 (Heikkilä et al. 2021).Notably, the highest level of drug use was found among individuals aged 18-25, encompassing adolescence and early adulthood, which are critical periods of physical and psychological development.In India, the use of psychoactive substances is prevalent among various population groups, with adult men being most affected by substance use disorders, according to the national survey report "Magnitude of Substance Use in India, 2019" (Ambekar et al. 2019).
In recent decades, the drug market has witnessed the introduction of a diverse range of substances, including both plant-based and synthetic drugs (Bewley-Taylor and Nougier 2018).This diversification has given rise to a group of drugs known as New Psychoactive Substances (NPS).NPS are substances that are not internationally controlled but mimic illicit drugs under international control, such as Cannabis, Cocaine, MDMA, and LSD.They are also referred to as designer drugs, legal highs, herbal highs, bath salts, or research chemicals (King and Kicman 2011).NPS present analytical challenges due to their diverse chemical structures, unknown purity and composition, and lack of regulation and testing.The UNODC reported the emergence of 950 new psychoactive substances in January 2020, highlighting the need for further research on their short-term and long-term effects (Heikkilä et al. 2021).
Detecting and identifying NPS poses significant challenges due to the vast range of chemical structures involved.Various techniques, including colorimetric, immunochemical, and chromatographic-mass spectrometry methods, have been developed for the rapid identification of NPS.Immunoassay screening technologies have advanced, leading to the development of user-friendly test platforms suitable for non-laboratory environments.These rapid screening tools find applications in seized products analysis, emergency rooms, workplace and roadside drug controls, drug addiction treatment clinics, law enforcement and health interventions, and postmortem and criminal casework (McLaughlin et al. 2013(McLaughlin et al. , 2019)).
The use of psychoactive substances in the workplace leads to severe occupational health problems.Vehicle drivers, in particular, face high levels of stress due to factors, such as irregular working schedules, night shifts, long periods away from family, and short deadlines.The stressful conditions faced by drivers often drive them to use psychoactive substances to reduce sleepiness during trips and increase their willingness to work and socialize (McLaughlin et al. 2019).However, the use of such substances impairs brain functionality and poses risks to the individual and society at large.Psychoactive substance use while driving is a major public health concern, as it affects drivers' health and safety, increasing the risk of injuries and traffic accidents.Fatalities and injuries caused due to road traffic accidents are major and growing public health problems in India (Singh 2017).The number of "Traffic Accidents" in India has increased from 368,828 in 2020 to 422,659 in 2021.The Indian State, Kerala ranks fifth among the States with the maximum increase in the number of traffic accident cases (Bureau 2021).
Commonly used substances among drivers include cannabis, alcohol, amphetamines, cocaine, benzodiazepines, opioids, and others (Girotto et al. 2014).Traditional methods of drug detection in the context of law enforcement primarily involve blood collection, which is invasive and often results in high refusal rates when participation is voluntary (Gjerde et al. 2011).Urine can also be used for drug intake verification, but it can detect drugs consumed several days or even weeks prior.Therefore, a positive drug finding in urine does not necessarily indicate that the driver was under the influence while driving (Kelley-Baker et al. 2014).Oral fluid has emerged as an alternative matrix for drug detection, as a positive drug finding in oral fluid indicates that the drugs are likely to present in the blood.Oral fluid collection is fast, easy, noninvasive, and difficult to adulterate, making it an effective option for drug testing (Vindenes et al. 2012;Moore et al. 2013).
To address the issue of drug use among drivers and promote safer driving practices, there is a growing interest in developing noninvasive and rapid procedures to detect drug use.The Randox biochip array technology enables the testing of oral fluid samples for the presence of drugs of abuse in point-of-collection testing (POCT).This technology can aid authorities in identifying drug use among drivers and implementing evidence-based measures and drug testing policies for occupational driving regulation (Dasgupta 2019).The Randox immunoassay screening system utilizes biochip technology, allowing for the detection of multiple drugs simultaneously (Ellefsen et al. 2014).The present study aimed to determine the prevalence of DOA and its relation with the distance of travel among goods carriage drivers associated with Kerala State, India.By filling the knowledge gap through this research, effective countermeasures can be implemented to address the drug abuse issue among drivers.

Sample collection
The study protocol was approved by the Human Ethical Committee, University of Calicut, Kerala, India, and informed written consent was obtained from all individual participants included in the study.The collection of oral fluid samples was performed by the authors at the check posts of the Kerala State Excise department located on the national highways passing through Walayar, Palakkad district; Tholpetty, Wayanad district; Manjeshwar, Kasaragod district in the Kerala State.The collection of samples was done between 7 pm and 12 am from July to September 2021.Equal numbers of samples were collected from each check post by collecting a minimum of 25 samples per day for 4 consecutive days.The location and date of sample collection were previously chosen after discussion with the officials of the Kerala State Excise Department.Excise officers randomly stopped goods carriages entering through the above-mentioned borders of Kerala State and invited them to participate in the study.At a time three goods carriages belonging to the category of heavy motor vehicle (HMV) and light motor vehicle (LMV) each were stopped for sample collection.The drivers were informed about the study and those who agreed to participate signed an informed consent form.All drivers who agreed to participate were included in the study.Age, type of goods carriage, place of departure, destination, and travel length were recorded.
Oral fluid samples were collected using the Neosal Oral Fluid Collection Device (Neogen, MI, USA).The NeoSal Oral Fluid Collection System consists of an absorbent pad (Collection Pad) affixed to a plastic handle and a proprietary buffer solution in a plastic tube (Buffer Tube).The NeoSal Collection Pad was placed between the cheek and gum of the participants which enhanced the flow of oral fluid onto the absorbent Collection Pad.The Sample Volume Adequacy Indicator (SVAI) was built into the device to ensure a suitable volume of oral fluid was collected and had a visible line in the clear plastic window before the collection of the sample.This line turned distinctly blue when a sufficient sample was collected.After collecting the sample, the saturated Collection Pad was inserted into the Buffer Tube and the handle was tightly screwed into the Buffer Tube.After the Collection Pad had been secured in the Buffer Tube, the tube was gently inverted 4 or 5 times thoroughly to distribute the buffer solution into the collection pad.The samples were labeled with a number linking the sample to the information collected.The collected samples kept inside the icebox were transported to the testing facility nearby and analyzed by the authors.

Screening test
The samples were tested for the presence of drugs using the Evidence MultiSTAT analyzer, a fully automated Biochip Array System that performed simultaneous detection of multiple analytes from a single sample.The Evidence MultiSTAT DOA Oral Fluid II Assay kits were used for testing the qualitative determination of the parent molecule and metabolites of drugs in the oral fluid collected.They are competitive enzyme immunoassays run on the automated biochip array analyzer, Evidence MultiSTAT.The core technology is the Randox Biochip, a solid-state device containing an array of discrete test regions containing immobilized antibodies specific to the different drug of abuse (DOA) compound classes.A competitive chemiluminescent immunoassay was employed for the DOA assays with the drug in the specimen and the drug labeled with horseradish peroxidase (HRP) was in direct competition for the antibody binding sites.Increased levels of a drug in a specimen lead to reduced binding of the drug labeled with HRP and thus a reduction in chemiluminescence being emitted.The light signal generated from each of the test regions on the biochip was detected using digital imaging technology and compared to that from the cutoff material.The classification of the test analyte present in the sample was determined from the cutoff material.

Assay protocol
Ready-to-use reconstitution buffer of 1 ml, each was added to the lyophilized cutoff and control powders and kept for 30 min out of bright light before use.USB provided along with the assay kit was inserted into the USB port of the analyzer.Then the cartridge barcode was scanned to import the batch details from the provided USB.The batch update was selected on the screen of the Evidence MultiSTAT analyzer.Then, 220 microliters of oral fluid cutoff and 220 microliters of the sample (buffer mixed with oral fluid) were pipetted out to the respective well of the cartridge.Along with this, positive control was also run as indicated in the assay protocol.It took 20 min to get the result on the display of the analyzer.The Evidence MultiSTAT DOA Oral Fluid II array Technology facilitates simultaneous screening of NPSs and/or their metabolites, such as Fentanyl, Ketamine, LSD, Methamphetamine, Barbiturates, Benzodiazepines I, Benzodiazepines II, Methadone, Opiate, PCP, BZG/Cocaine, Oxycodone, Tramadol, Cannabinoids (THC), Amphetamine, Buprenorphine, 6-MAM, Synthetic Cannabinoids (JWH-018), alpha-PVP, and Synthetic Cannabinoids (UR-144).

Confirmatory test
Confirmatory analyses of the saliva were performed at Forensic Fluids Laboratories, Michigan, United States, using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS).Chromatographic separation was performed on an Acquity UPLC system (Waters) equipped with an Acquity BEH (C-18, 100 × 2.1 mm, 1.7 µm) analytical column (Waters).Mobile Phase A was 0.1% formic acid in water:acetonitrile (95:5, v/v), and mobile Phase B was 0.1% formic acid in acetonitrile.The column and autosampler were kept at 50 and 15 °C, respectively.The flow rate was kept at 0.7 mL/min, and the total run time was 4.5 min.The gradient conditions were as follows: from time 0 to 0.5 min 60% A; from time 0.5 to 4.0 min, mobile phase A was decreased to 10%; from time 4.01 to 4.4 min, mobile phase A was further decreased to 0%.At 4.41 min, mobile phase A was returned to 60% and the column was equilibrated at the mobile phase ratio until 4.5 min.The injection volume was 5.0 µL.The needle was washed with 600 µL of DI water:acetonitrile (90:10, v/v) and 200 µL of methanol:isopropanol:acetonitrile solution (25:25:50, v/v) in between injections.
Electrospray ionization (ESI) mass spectrometry was performed on a TQD instrument (Waters).Analysis was performed in positive ionization (ESI+) and multiple reaction monitoring (MRM) modes.The source temperature was 150 °C, and the desolvation gas temperature was 450 °C.The flow rates of the desolvation gas (nitrogen) and the cone gas (nitrogen) were set at 850 and 50 mL/min, respectively.Collision gas (argon) flow was set at 0.18 mL/min.The collision cell pressure was 3.59E −3 mbar of argon.The MRM conditions were developed and optimized using the IntelliStart software (Waters).

Statistical analyses
Data were statistically analyzed using SPSS v. 21.Descriptive statistics were employed to understand the frequencies of age, type of goods carriage, place of departure, travel length, and DOA among the participants.A chi-square test was performed to understand the association between drug abuse and the distance traveled by the drivers.Significance is set at p < 0.05 to determine the value of the χ 2 test to understand the association.

Results
A total of 290 drivers were invited to participate, and of those, only 255 drivers participated in this study and all were men with an average age of 36.53 years.Since no oral fluids were obtained from 6 drivers, the total sample size was reduced to 249.Out of the 249 samples, 53 tested positive (21.29%) (Table A1, see online supplement).The majority of the participants belong to the age group of 36-46 (n = 90; 36.1%),followed by 26-35 (n = 83; 33.3%), 18-25 (n = 43; 17.3%), and 47-57 (n = 31; 12.4%).Only 0.8% (n = 2) of the samples were representing the age group of 58 years and above.The type of goods carriage and the place of departure of the vehicle are shown in Table A2 (see online supplement).
Among the positive samples, the multiple uses of THC + UR144 drugs were more prominent and this combination was found positive in 11 cases (4.42%).Four samples were positive for the multiple uses of THC + JWH-018 (1.61%), and two samples (0.80%) were positive for the multiple uses of THC + Methamphetamine and THC + Alpha-PVP.However, one sample each was found positive for the combination uses of THC + 6-MAM + JWH-018 + UR144; Ketamine + Methadone + Opiate; Ketamine + LSD; LSD + Opiate + Alpha-PVP; Ketamine + Tramadol; and Ketamine + PCP (Table A1, see online supplement).In samples identified with a single use of substances, THC, ketamine, and JWH-018 were prominent and found in six samples (2.41%), followed by UR-144 in four samples (1.61%), and Alpha-PVP in two samples (0.80%) (Table A1, see online supplement).
In the "Evidence MultiSTAT DOA Oral Fluid II Assay" there were twenty DOA that could be readily identified from the oral fluid sample.THC and ketamine were prominent in use among the participants either alone or as multiple drugs.Considering the frequency of the substance use, THC was the most preferred DOA, which was found in a total of 25 samples (10.04%).It was followed by UR-144 in 16 samples (6.43%), JWH-018 in 12 samples (4.82%), Ketamine in 10 samples (4.02%), and Alpha-PVP in six samples (2.41%) (Table A3, see online supplement).The use of other drugs, such as LSD, methamphetamine, opiate, 6-MAM, benzodiaz-epines1, methadone, PCP, tramadol, and amphetamine was also detected in oral fluid samples and their frequency of use ranging between 3.77 and 5.66%.However, fentanyl, barbiturate, benzodiazepines2, benzoylecgonine, oxycodone, buprenorphine were not detected in any oral samples collected for the present study.
Of the 53 tested positive samples in the screening test, a representative sample from single DOA and DOA combinations underwent confirmatory analysis.In the confirmatory analysis, only THC was detected in the three corresponding positive samples in which a combination of natural and synthetic cannabinoids was detected.Results of the screening test of the remaining samples showed 100% agreement with the confirmatory analysis.
The prevalence of drug use was higher among the drivers of the age group between 47 and 57 years (48.3%)than in the age groups of 36-46 years (34.4%),26-35 years (30.1%), and 18-25 years (23.3%)(Table A3, see online supplement).On the other hand, the consumption of different drugs was more prominent among drivers of 36-46 years than in the other age groups.No substance use was found in samples from drivers aged 58 years and above (Table 1).
In the age group of 47-57 years, the rate of usage of both THC and UR144 was the same and higher (12.9%) than the use of other drugs (Table A3, see online supplement).Likewise, the usage of THC was prevalent among the age groups of 36-46 years (11.1%),18-25 years (9.3%), and 26-35 years (8.4%).UR144 was the second most prominent drug in usage after THC in the age groups of 26-35 years (6.0%) and 36-46 years (7.8%).However, JWH-018 was the second most prominent drug in usage after THC in the 18-25 age group (4.7%) (Table 1).
An association between drug abuse and distance of travel was found in drivers in this study, χ 2 (5, N = 249) = 123.5,p < 0.001.The result indicated that drug use among drivers was increasing consistently with the distance traveled, where an increase from 72 to 100% in drug use was observed in drivers who traveled more than 1000 km (Figure 1).

Discussion
The main finding in this study was the high prevalence of the psychoactive ingredient of cannabis, the delta-9-tetrahydrocannabinol (THC) in the oral fluids of the participants followed by the presence of synthetic cannabinoids (UR-144 and JWH-018).A similar finding was obtained in another Indian study where the subjects were truck drivers in the state of Haryana (Punia et al. 2020).Cannabis is the most frequently used illicit psychoactive substance worldwide (Merz 2018).The National Survey on Extent, Pattern, and Trends of Drug Use in India also found cannabis is the most  common illicit substance of use in the country (Ray 2004).
The reason for this high prevalence of cannabis use is due to its relatively more availability and it is cheaper when compared to other illicit substances.Cannabis has a long history of cultural and medicinal use in many parts of the world and India, it has been used for various purposes, including relaxation, spiritual rituals, and pain relief, in different cultures throughout history (Zuardi 2006).This cultural significance and acceptance in certain societies may contribute to its popularity (Elsaid et al. 2019).Some individuals view marijuana as a relatively low-risk substance compared to hard drugs.This perception is due in part to its medicinal properties, reported milder side effects, and lower potential for addiction than substances, such as opioids or stimulants and such perceptions may lead to higher usage rates (Caulkins et al. 2016).Several studies revealed the acute intoxication effects of cannabis on the drivers' psychomotor and cognitive skills which makes it difficult in keeping vehicles on track due to impaired brain mechanisms leading to motor vehicle accidents (Nicholson et al. 2004;Ménétrey et al. 2005;Degenhardt and Hall 2012;Dahlgren et al. 2020).The higher prevalence of drug use among the drivers of ages between 47 and 57 was also noticed.This may be due to the reason that those individuals may have greater access to such substances due to their social networks, personal connections, or long exposure to specific environment and peer pressure.The usage of methamphetamine, alpha-PVP, and 6-MAM along with cannabis was also identified in this study which is alarming.Ketamine is the second most prevalent DOA among the participants in this study.Ketamine has hallucinogenic effects including analgesia and dissociation/disconnection from reality and is popular among youngsters as a party drug or as a sexual assault drug (Jansen 1993).Also, among the participants, the usage of ketamine was seen in combination with other psychoactive substances, such as methadone, opiate, LSD, tramadol, and PCP.Ketamine has both physical and mental effects which became a potential source of dangerous driving (Giorgetti et al. 2015).Similarly, Lysergic Acid Diethylamide (LSD) and α-Pyrrolidinopentiophenone (Alpha-PVP) were also used by the participants along with other DOA.Small doses of LSD can produce changes in perception, sense of time and space, and mood (Schechter 1998).
We observed an increase of cannabinoid-positive cases with age and a higher number of cannabinoid-positive cases in 26-57 years, which is similar to a previous study that described the incidence of drugs in the fatally injured driver's population in France (Labat et al. 2008).
In this study, out of twenty DOA, we could detect fourteen different drugs mentioned in the Evidence MultiSTAT DOA Oral Fluid II array, which shows the diversity of DOA among the participants.The use of psychoactive substances by drivers is reported in many countries, however, there are no systematic scientific studies conducted in India to understand the prevalence of drugs of abuse (DOA) among drivers (Das et al. 2012).As suggested by the WHO, this knowledge is important to improve public policies on traffic safety so that India could decrease the mortality rate from traffic accidents.Point-of-collection testing (POCT) is the need of the hour as the DOA and the usage of NPS are increasing alarmingly among different populations (Arkell et al. 2019).
The association between the consumption of psychoactive substances and long traveling distances among drivers was reported previously (Sinagawa et al. 2015;Bombana et al. 2017).A similar trend was observed in the present study.To stay awake for many hours, particularly during nighttime, drivers use these substances (Bombana et al. 2017).
The use of oral fluid has been found to offer significant promise since it is a noninvasive method and allows on-site detection of drugs.It is pertinent to mention that in the majority of the participants during oral fluid collection using the NeoSal Oral Fluid Collection System, the Sample Volume Adequacy Indicator (SVAI) formed a distinct blue line in the clear window after 10-15 min.However, in six participants, the line in the clear window of the Sample Volume Adequacy Indicator (SVAI) of the NeoSal Oral Fluid Collection System did not turn blue even after 20-30 min since the mouth was dry.The lowered salivary flow rate may be due to the perceived stress as the participants were stopped by the law-enforcement officers or due to xerostomia (Gholami et al. 2017).This may be due to the consumption of smokeless tobacco, such as paan and gutkha was very common among the participants and oral dryness may happen due to these habits (Scully Cbe 2003;Niaz et al. 2017).

Policy implications and future scope
This is the first study on DOA conducted among drivers in India.The Randox DOA Oral Fluid II Array panel offers law enforcement agencies, forensic scientists, and toxicologists, the opportunity to screen oral fluid samples for the presence of common therapeutic, abused drugs, and NPS.Driving under the influence of drugs (DUID) is a topic of concern to both the medical and legal fields and one for which science has not yet provided sufficient guidance to the policy-making community.India is reporting the highest number of road traffic crashes, related injuries, and deaths among all countries in the world (Das et al. 2012;World Bank 2021).The intake of psychoactive substances by drivers is a relatively frequent occurrence, although the prevalence varies according to the place and methodology employed.Furthermore, intake seems to be higher when working conditions are poor and can have a direct impact on the health of individual drivers and society as a whole due to the increase in traffic accidents.
Although from a scientific standpoint, the knowledge on the use of psychoactive substances by these professionals still needs to be broadened, the available evidence is more than enough to justify facing and addressing the problem.The tendency to use drugs of abuse (DOA) and new psychoactive substances (NPS) among youth is increasing day by day.Hence point-of-collection testing (POCT) at schools/colleges will help to generate scientific data about the prevalence of these substances in society and help law-enforcement agencies to develop preventive measures.

Figure 1 .
Figure 1.Drug use as distance traveled by drivers in the state of Kerala, india during 2021.The number inside the boxes represents the cases.

Table 1 .
Percentage of drug use in the different age categories of drivers (n = 249) who participated in the present the study from the state of Kerala, india in 2021.