Evaluation of the immune system status and hematological dyscrasias, among amphetamine and cannabis abusers at Eradah Hospital in Qassim, Saudi Arabia

This cross-sectional study aims to evaluate the immune system status and hematological disturbances among individuals who abuse amphetamines and cannabis. Substance abuse, particularly of amphetamines and cannabis, has been associated with various adverse effects on the body, including potential impacts on the immune system and hematological parameters. However, limited research has been conducted to comprehensively assess these effects in a cross-sectional design. Additionally, fungal infections are on the rise internationally, and immune-compromised people are particularly susceptible. The study will recruit a sample of amphetamine and cannabis abusers (n = 50) at the Eradah Hospital in the Qassim Region of Buraydah and assess their sociodemographic and biochemical variables, including blood indices and differential WBC indices, liver, and kidney profiles. Additionally, 50 sputum samples in total were cultured for testing for fungus infections. To obtain the descriptive statistics, the data was imported into Microsoft Excel and subjected to statistical analysis using SPSS 22.0. Amphetamine and cannabis abuser's sociodemographic variables analysis observed that the majority (52%) were aged 18–30, with 56% in secondary school. Unemployment was a significant issue, and most had no other health issues. The majority (50%) had 5–10 years of abuse, while 32% had less than 5 years, and only 18% had been drug abusers for more than 10 years. There were significant changes (p < 0.001) in all different leukocyte blood cells, including neutrophils, lymphocytes, monocytes, eosinophils, and basophils. Furthermore, a microscopic examination of blood films from individuals who misuse the combination of the medications "amphetamine and cannabis" reveals hazardous alterations in Neutrophils. Out of 50, 35 sputum samples showed positive growth on Sabouraud dextrose agar (SDA) with chloramphenicol antibiotic, indicating a unicellular fungal growth. The present study explores the immune system and hematological disturbances linked to amphetamine and cannabis abuse, providing insights into health risks and targeted interventions. The findings complement previous research on drug users' hematological abnormalities, particularly in white blood cells. Routine hematological tests help identify alterations in homeostatic conditions, improving patient knowledge and preventing major issues. Further research is needed on multi-drug abuse prevention, early detection, and intervention. The cross-sectional design allows for a snapshot of the immune system and hematological status among abusers, laying the groundwork for future longitudinal studies. Key Words: Drug Effect, Immunity, Epidemiology, Oxidative Stress, Inflammation.


Sample collection and biochemical analysis
Three ml of whole blood samples were taken in Ethylenediaminetetraacetic acid (EDTA) tubes, which were drawn by the nurses in the hospital to measure the Complete Blood Count (CBC) with blood differential by "Sysmex xn series" for these parameters (WBC, RBC, HB, HCT, MCV, MCH, MCHC, PLT, NE, LYM, MON, EO, BASO), and then also blood film observed under a microscope (Light microscope (OLYMPUS), to look at leukocyte cell structure.The prepared blood films were stained with 10% Giemsa stain for 10 min after completion of fixation by methanol.Additionally, the "Dimension & Cobas" (Roche, Switzerland) machine used blood samples in a heparin tube for testing ALT, AST, ALP, Creatinine, and BUN to check the kidney and liver functions among participants.Before being processed in automated analyzers, the blood samples underwent centrifugation (1500 rpm for 10 min) to separate their constituents, yielding clear plasma for precise and reliable results.The conducted tests were executed in strict compliance with established quality control standards, encompassing the calibration and maintenance procedures of utilized equipment to uphold precision and reliability in the obtained results.Furthermore, adherence to laboratory safety protocols was maintained, along with the sustenance of a hygienic and systematically arranged operational setting.After the observation and collection of results, appropriate disposal measures were undertaken, with all samples handled with precision.Contaminated cultures and pipettes were specifically disposed of in impermeable red bag containers bearing biohazard symbols to ensure proper containment.
To quantify immunological function in amphetamine and cannabis users and screen the sample for fungal infections, the study calls for the collection of sputum samples.Participants cough up sputum samples from deep within the chest into a sterile plastic collection bottle, which are then cultivated on Sabouraud dextrose agar with Chloramphenicol, at 25 °C for one week of incubation in a hospital microbiology lab.After incubation, fungi were identified through macroscopic examination by observing the colony morphology, color, texture, and growth pattern which provide initial clues about the fungi present.Further, the microscopic examination was conducted using a stain (Crystal violet), to analyze the fungal structures.Before commencing a culture, the suitability of a sputum sample for fungal analysis was substantiated, ensuring it exhibits purulent characteristics indicative of lower respiratory tract origin and contains a sufficient quantity for culturing purposes, while also being free from saliva contamination.

Statistical analysis
Microsoft Excel 2023 was used to input, tabulate, and analyze the gathered data.The quantity and percentage of categorical variables were determined to apply the descriptive analysis.To handle and analyze more data, the SPSS 22.0 application was used.The measured data distribution was represented by mean ± SD.The investigation of the link among the different factors was conducted by basic correlation analysis.The ANOVA test was employed to examine the average difference between the two groups under investigation.When the P value is less than 0.05, the correlation and difference between variables are deemed statistically significant.

Ethical considerations
Study approval was obtained from the Regional Research Ethics Committee, Registered at the National Committee of Bio & Med.Ethics [NCBE] Registration No. H-04-Q-001.Participants were allowed to provide informed consent before sample collection and were briefed on the study's objectives.Each participant completed informed consent forms, explicitly acknowledging their ability to withdraw from participation at any given time.Strict confidentiality measures were implemented to protect the dataset for research purposes only, ensuring participant anonymity by omitting sensitive identifiers such as names and addresses.All methods were performed according to the relevant guidelines and regulations, particularly with the Declaration of Helsinki, which states the ethical standards for medical research involving human beings.

Potential biases and limitations
In this cross-sectional study with a volunteer sampling technique, both sampling bias and confounding variables presented challenges.Participants self-selected, potentially distorting the population's view by overrepresenting or excluding certain groups.Additionally, confounding variables made it difficult to accurately attribute observed associations, limiting the study's ability to draw meaningful conclusions about cause and effect due to the lack of random assignment or control.

Results
The sociodemographic variables of the 50 amphetamine and cannabis abusers' participants were included in this study (Table 1).For age, it is noticed that the majority, about half (52%), were 18-30 years of age, fewer (36%) were 31-40 years of age, and only 12% were above the age of 41 years.Most of the abusers (56%) are in secondary school-level education; a few (14%) were in preparatory school, and a few (4%) were in elementary school, whereas 26 percent were college-educated.In addition, only 24% of abusers answered that they have other health issues, and 76% answered no.The data collected from the abusers about the number of years of addiction shows the following: 32% are less than 5 years, 50% are from 5 to 10 years, and 18% are more than 10 years.Whereas 92% of the abusers are unemployed and 8% are employed.
Table 2 presents a comprehensive hematological analysis of amphetamine and cannabis abusers (n = 50).For this study, the analysis of various blood cell counts and indices means, standard deviations, t-tests, and p values for hypothesis testing between the "normal" and "non-normal" groups.For instance, "Norm" which means normal range or values, and "Non-Norm" which means abnormal values to compare the biochemical parameters.The findings provide valuable insights into the distribution and significance of blood parameters between normal and non-normal categories, contributing to our understanding of the physiological variations in these parameters.
The study's most startling discovery is that amphetamine and cannabis abusers' peripheral blood smears frequently exhibit neutrophil cytotoxicity.We can interpret white blood cell properties (size, shape, and color) by looking into peripheral blood films.Panel A displays the typical features, while Panels B and C display harmful alterations (Fig. 1).During differential counting, the neutrophil showed cytotoxic impact, including nuclear crumbling, vacuolization, and defragmentation, which may suggest the impact of drug abuse involving some cytotoxic metabolites such as free radicals, ROS-reactive oxygen species, and cytokines, and specifically the vacuolization in neutrophils is strongly representative of infection.
Table 1.Distribution of Cannabis and Amphetamine abusers based on their demographic characteristics.www.nature.com/scientificreports/

Count
The study analyzed the impact of cannabis and amphetamine addiction on the liver and renal profiles (Table 3).Results showed a significant positive link between years of abuse and the liver profile (ALT-GPT), with a correlation coefficient of r = 0.453.The study also found a significant effect of years of abuse on ALT-GPT, accounting for 21% of the variation.The data also showed a positive relationship between years of abuse and AST-GOT, with a correlation coefficient of r = 0.641, suggesting that abuse over years accounts for 41% of the variation in AST-GOT.However, there was no statistically significant effect of years of misuse on alkaline phosphatase, with a p > 0.196.
Furthermore, there is a statistically significant and positive correlation between creatinine and BUN and several years of abuse.At a significance threshold of α ≤ 0.05, the correlation coefficient of 0.356 indicates that years of abuse had a substantial impact on creatinine.The p > 0.011, and the coefficient of determination (r 2 ) is 0.121.This implies that 12% of the fluctuation in creatinine over the years can be attributed to drug misuse.www.nature.com/scientificreports/Additionally, the study demonstrates a substantial influence on both variables and a positive correlation between the number of years of abuse and BUN.
To check differences in patients who take the two types of drugs and demographic variables, an ANOVA test has been used, where the null hypothesis refers to the fact that there are no significant differences in patients who take the two types of drugs and demographic variables represented in Table 4.There were established significant differences in MONO related to the age variable and blood variables, where the p-value of the test was 0.035 less than 0.05.Whereas, other significant differences were established in ALT-GPT and AST-GOT related to age variables and liver variables, where the p-values of these tests were 0.000 and 0.001, respectively.Therefore, the results conclude that the age variable affects the emergence and rate of the impact of drug abuse.However, this correlation does not imply causation, meaning it only refers to the variables solely based on an observed association or correlation between them.Additionally, there are no significant differences in other demographic variables correlated with blood variables, liver variables, or kidney variables.
A total of 50 sputum samples were cultured on "Sabouraud dextrose agar (SDA) with chloramphenicol" antibiotic to prevent the growth of a variety of Gram-positive and Gram-negative bacteria (Fig. 2a-c).After one week of incubation at 25 °C, the majority of the samples (35 cultures) recorded positive growth, while 15 cultures recorded negative growth (Table 5).Moreover, some of the positive cultures are then stained with "Crystal Violet" and examined under the microscope.Figure 2d shows a single or cluster of oval-shaped cells with budding characteristics, indicating a unicellular fungus.However, further studies should be performed to identify the specific type.

Discussion
Amphetamine and cannabis are frequently abused substances, and experimental evidence shows that cannabis use is associated with negative risks for non-medical amphetamine users 22 .The purpose of this cross-sectional study is to assess the effects of cannabis and amphetamines on various sociodemographic and biochemical characteristics in individuals who abuse, specifically in the Qassim region of Saudi Arabia, and may not be generalizable to other populations due to cultural, environmental, and genetic differences that can influence substance abuse patterns and health outcomes.
The sociodemographic variables of the 50 amphetamine plus cannabis abusers' participants in this study (Table 1).For age, it is noticed that the majority, about half (52%), were 18-30 years of age, fewer (36%) were 31-40 years of age, and only 12 percent were above the age of 41 years.Most of the abusers (56%) are in secondary school-level education; a few (14%) were in preparatory school, and a few (4%) were in elementary school, whereas 26 percent were college-educated.It may suggest that adolescents often experiment with drugs due to rapid physical and intellectual development, which can be influenced by life events and social group affiliations, leading to an experimental attitude and increased drug use risk [23][24][25] .
A significant difference was observed in the employee ratio, where a considerable number of abusers are unemployed.Our findings are supported by a previous study 26 that revealed that people who were jobless and had the lowest degree of education were more likely to be heavy drinkers, smokers, and physically inactive.Other variables of the present study were whether the participants had other health issues, and the majority of the answers were no.Lastly, the number of years of abuse that constituted 50 percent were from 5 to 10 years, slightly less (32%) was less than 5 years, and just 18% were drug abusers for more than 10 years.
In addition, the microscopic view of blood film for patients who abuse the mixing drugs "Amphetamine and Cannabis", shows segmented neutrophils with toxic change (Fig. 1B,C) have chromatin that is uneven and less condensed in the lobes, as well as a large number of large purple or dark blue cytoplasmic granules (primary granules) brought on by the retention of ribosomal RNA and cytoplasmic vacuolization.As opposed to their abnormal counterparts, normally matured segmented neutrophils in Fig. 1A (control) possess comparatively small nuclei, chromatin that is densely packed, and white cytoplasm with a majority of fine pink secondary granules.The exact cause of neutrophil toxicity is not fully understood, but several theories include direct toxic effects on bone marrow cells, activation of the immune system, and abnormal maturation of neutrophils.These effects can lead to nuclear granulation, which is not exclusive to cannabis and amphetamine abuse but can also occur in other conditions like infections and autoimmune diseases.Cytokines, primarily correlated with C-reactive protein, can cause toxic changes in neutrophils.Furthermore, it may be suggested that it is due to oxidative stress, which is characterized by the release of excess free radicals, reactive oxygen species, and cytokines and can lead to neutrophil cell destruction, causing cytotoxicity 28,29 .Amphetamine abuse has been associated with liver damage, primarily due to the increased release of stress hormones and oxidative stress caused by the drug.Prolonged amphetamine abuse can lead to hepatotoxicity, characterized by elevated liver enzymes such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST).Cannabis abuse, on the other hand, has shown conflicting results in terms of its effects on the liver profile.Some studies suggest a minor increase in liver enzymes, such as ALT and AST, in cannabis users.However, these changes are typically mild and not associated with severe liver damage 30 .Our results suggest no significant difference was observed in AST-GOT, ALT-GPT, and alkaline phosphatase levels, as depicted in Table 3, further validating the previously reported results 30 .
Amphetamine abuse has been linked to kidney damage, largely due to its vasoconstrictive effects and increased release of stress hormones.Long-term abuse of amphetamines can lead to renal impairment, characterized by elevated serum creatinine levels and a decreased glomerular filtration rate (GFR).In contrast, cannabis abuse has shown a limited impact on the kidney profile.Studies have been inconclusive, with some reporting a potential association between long-term cannabis use and mild kidney dysfunction, while others have found no significant effects 31 .Further validate our finding that neither creatinine nor blood urea nitrogen (BUN) levels were significantly affected among abusers (Table 3).
It is alarming to see that, in a fungus culture test using sputum samples from amphetamine and cannabis users, the majority of the samples (35 cultures) showed positive growth while just 15 cultures showed negative growth.Additionally, a few of the positive colonies are later stained with "Crystal Violet" and put under a microscope for inspection.A single or group of oval-shaped cells with budding features is seen in Fig. 2d; this is an example of a unicellular fungus that may suggest that due to the suppressed immunity, observed with blood results in www.nature.com/scientificreports/opportunistic pathogens like yeast and mold inhabiting the upper respiratory tract of these chronic immunocompromised patients of drug abuse.However, further studies should be performed to identify the specific type.Our finding further validates the concept of "fungal priority pathogens", published by WHO recently (2022) which includes 19 groups of fungal pathogens that cause high-risk morbidity and mortality and aims to direct research to increase public health measures against invasive fungal diseases (IFDs) 21 .
The research aims to improve drug abuse research, diagnostics, treatment, and public health.It highlights that chronic amphetamine abuse has more severe effects on blood indices, liver profile, and kidney profile than cannabis abuse.However, individual variations in drug abuse patterns and susceptibility may influence these effects.

Conclusion
This study explores the immune system and hematological disturbances linked to amphetamine and cannabis abuse, providing insights into health risks and targeted interventions.The findings complement previous research on drug users' hematological abnormalities, particularly neutrophil cytotoxicity in differential white blood counts.Routine hematological tests (CBC) help identify alterations in homeostatic conditions, improving patient knowledge and preventing major issues, may suggest specific screening protocols or monitoring strategies for individuals known to abuse these substances.The multifaceted approach needed to address substance abuse effectively in future research targeted multi-drug abuse prevention, early detection, and intervention.Addressing the limitation of the present study with a larger sample size with the addition of a control group in the study could further enhance the study's outcomes.
At present this cross-sectional design allows for a snapshot of the immune system and hematological status among abusers, laying the groundwork for future longitudinal research studies, that would allow for the assessment of long-term effects of substance abuse on the immune system and hematological parameters, providing insights into the progression of potential health issues and the effectiveness of the intervention.Besides the potential policy implications and efforts to combat drug abuse by the Saudi Government, there is a need to have more stress on drug education programs and support services for substance abusers.

Figure 1 .
Figure 1.The microscopic view of blood film for patients who abuse the mixing drugs "Amphetamine and Cannabis".(A) Normal neutrophils.(B-C) Neutrophils with toxic change.

Table 2 .
Complete Blood Count (CBC) analyzed results for Cannabis and Amphetamine abusers.
*Significance, if present, is in favor of the category with the highest arithmetic mean, **Correlation is significant at p < 0.001.

Table 3 .
Simple linear regression analysis test of the effect of the number of years of Amphetamines and Cannabis abuse on different parameters of LFTs, and KFTs profile.ALT-GPT alanine aminotransferaseglutamic pyruvic transaminase, AST-GOT aspartate aminotransferase-glutamic oxaloacetic transaminase, ALP alkaline phosphatase, BUN blood urea nitrogen.*Correlation is significant at p < 0.001.

Table 4 .
Correlation analysis of Age variables with CBC, LFTs, and KFTs in Cannabis and Amphetamine abusers.
*Significance, if present, is in favor of the category with the highest arithmetic mean, *Correlation is significant at p < 0.001.

Table 5 .
Fungal culture test of Cannabis and amphetamine abuser's sputum samples on Sabouraud dextrose agar (SDA).