Clinical utility of combined butyrylcholinesterase activity measurements in assessing acute malathion intoxication severity: A case series

Abstract

Background

Accurate and prompt assessment of malathion intoxication severity remains a significant clinical challenge, often hampered by reliance on single diagnostic markers. This exploratory case series investigated the combined utility of rapid butyrylcholinesterase (BChE) activity measurements and gas chromatography-tandem mass spectrometry (GC-MS/MS) for quantifying urinary malathion to enhance diagnostic precision.

Methods

We investigated three independent patients admitted with acute malathion intoxication. BChE activity was measured using both a point-of-care (POCT) device and a laboratory-based enzyme-multiplied immunoassay technique (EMIT). Urinary malathion was quantified using a validated GC-MS/MS method.

Results

Malathion exposure was confirmed in all patients via urinary analysis. Strong per-case positive correlations (r ranging from 0.905 to 0.996) were observed between the two BChE measurement methods, though Bland-Altman analysis revealed noteworthy discrepancies (mean bias of 10%, limits of agreement ranging from −20% to 40%). Critically, statistically significant inverse correlations (p < 0.05) were identified between urinary malathion concentrations and both BChE activity measurements, underscoring the dynamic relationship between exposure and enzymatic inhibition.

Conclusion

These findings, derived from a small, exploratory case series, suggest the importance of an integrated diagnostic approach for malathion intoxication. This combined strategy may support improved assessment of severity and prognosis in individual cases, offering insights into the pesticide’s systemic impact and elimination kinetics, especially when exposure details are unclear. While rapid BChE tests are valuable for initial screening, their interpretation should occur within this multi-marker framework. The generalizability of these findings is limited by the small sample size, and no formal power calculation was performed.

Keywords

malathion organophosphate intoxication butyrylcholinesterase point-of-care testing GC-MS/MS biomarkers clinical toxicology diagnostic strategy

Introduction

Organophosphate (OP) insecticide poisonings represent a substantial global public health challenge, with malathion, a widely used OP, contributing significantly to this burden.^1,2 Despite its classification by the World Health Organization (WHO) as “slightly hazardous” and its inclusion in various pest control products, malathion’s extensive application in agriculture, public health initiatives, and domestic settings^3,4 frequently leads to both accidental exposures and deliberate self-poisonings worldwide.^5,6 Acute, high-concentration malathion self-poisoning often precipitates a rapid and severe cholinergic crisis, manifesting as excessive muscarinic (e.g., bronchospasm, bradycardia, miosis) and nicotinic (e.g., fasciculations, weakness, respiratory paralysis) receptor stimulation.^7–9 These symptoms can lead to severe and frequently fatal outcomes, further complicated by potential delayed effects such as intermediate syndrome and OP-induced delayed neuropathy (OPIDN).¹⁰ While prompt identification and emergency management are critical,¹¹ an accurate and timely diagnosis is often hampered by inherent limitations in current diagnostic methodologies.¹² Global epidemiological data highlight the gravity of this issue, with an estimated 740,000 OP insecticide poisonings and 7446 fatalities reported in 2020 alone, underscoring the persistent public health imperative for improved diagnostics.¹¹

Current diagnostic strategies for OP poisoning typically integrate clinical presentation with laboratory investigations. While clinical signs provide initial clues, laboratory confirmation is essential for a definitive diagnosis and for guiding treatment protocols. Traditional laboratory approaches involve quantifying the activity of cholinesterase enzymes, namely acetylcholinesterase (AChE)¹³ and butyrylcholinesterase (BChE),¹⁴ given their inhibition by OPs.^15,16 It is pertinent to note that while Butyrylcholinesterase (BChE) activity can be determined in whole blood (e.g., via rapid point-of-care tests), and Pseudocholinesterase (PsChE) activity is typically measured in plasma after centrifugation (e.g., via standard laboratory methods like EMIT), these enzymes are biochemically identical. However, sole reliance on these enzymatic methods presents several critical drawbacks: other compounds can also inhibit cholinesterases, potentially leading to false positives; the degree of inhibition does not consistently correlate with the severity of intoxication; and the methods may not precisely identify the specific OP involved, particularly in delayed presentations.¹⁷

To address these diagnostic limitations, direct analytical methods, such as gas chromatography-mass spectrometry (GC-MS) and the more sensitive and selective gas chromatography-tandem mass spectrometry (GC-MS/MS),^18–20 have become indispensable for identifying and quantifying specific OPs and their metabolites in biological samples. This enables the unambiguous detection of malathion and its breakdown products.²¹ Nevertheless, the requirement for specialized equipment, highly trained personnel, and inherently longer turnaround times compared to enzymatic assays often restricts their immediate applicability in emergency settings.¹⁸ In Romania, specifically, the widespread availability of GC-MS/MS for routine toxicological screening in emergency departments is limited, making its application in acute OP poisoning cases a novel and less common practice.

Accurately assessing the severity of acute malathion poisoning is paramount for effective clinical management. While the precise identification and quantification of malathion in biological samples are foundational, relying solely on these measures often inadequately reflects the true physiological impact of the intoxication, especially in voluntary self-poisoning cases where ingestion details are frequently unreliable.

The present study directly addresses this complex diagnostic challenge by exploring the clinical utility of an integrated approach: combining measurements of BChE activity from whole blood and PsChE activity from plasma with urinary malathion quantification using GC-MS/MS. Despite the small number of subjects (n = 3), this ICU cohort was worth reporting due to the severe presentation of malathion intoxication in these cases. This provided a unique opportunity to evaluate the real-time clinical application and methodological innovation of combining rapid enzymatic assays with advanced analytical chemistry for comprehensive assessment. As a primary biological target of OPs,^14–16 BChE/PsChE serves as a direct biomarker reflecting the physiological impact of exposure. Furthermore, while direct OP identification is essential,^18–20 it may not fully capture the patient’s dynamic physiological response, as individual variations in metabolism and sensitivity can significantly influence the clinical picture irrespective of the initial dose. Therefore, enzyme activity, indicative of immediate functional impairment, provides a more direct metric of the patient’s current clinical status and potential for deterioration.

Analyzing urinary malathion provides valuable insight into systemic elimination, representing cumulative exposure and consequently offering a more robust marker of systemic burden than transient blood concentrations, particularly when considering the time since ingestion. In most clinical settings in Romania, GC-MS/MS for malathion quantification is not a standard diagnostic tool; therefore, its inclusion in this study represents a significant methodological innovation. From a practical diagnostic standpoint, urine is frequently the preferred matrix for initial broad-spectrum toxicological screening when the causative agent is unknown. This facilitates comprehensive preliminary analysis, and subsequently, the same urine sample can be directly subjected to more specific and highly sensitive malathion quantification via GC-MS/MS in Multiple Reaction Monitoring (MRM) mode, thereby preserving other critical admission samples (e.g., blood) for further essential medical investigations.²¹

Collectively, our findings demonstrate that integrating comprehensive BChE activity measurements with urinary malathion quantification provides a more nuanced and clinically pertinent approach to assessing the severity of acute voluntary malathion intoxication in humans. This combined strategy not only enhances diagnostic support but also underscores the inherent limitations of relying solely on OP measurement, thereby enabling a more informed understanding of the patient’s condition and guiding improved clinical decision-making.

Material and methods

Ethical considerations and patient samples

For this investigation, we utilized residual clinical samples that were obtained from individuals admitted to the Intensive Care Unit (Toxicology Department) at Bucharest Emergency Clinical Hospital between 2021 and 2023. These samples were originally collected for routine diagnostic and treatment monitoring purposes as part of standard clinical care for patients with suspected malathion intoxication. No additional sampling was performed for the sole purpose of this study. Our inclusion criteria stipulated a clinical suspicion of malathion exposure and the concurrent availability of both whole blood and urine samples. Patients were excluded if there was an inadequate sample volume, if the patient died before sample procurement, or if the requisite informed consent could not be documented.

The methodological framework of this study rigorously adhered to established ethical guidelines. It received endorsement from the institutional review board and upheld the principles articulated in the Declaration of Helsinki. Given that all three patients were incapacitated upon admission, direct informed consent could not be obtained at the time of sample collection. Retrospective informed consent was secured from the patients themselves or their legally authorized representatives (surrogates) prior to their discharge from the ICU. The consent specifically authorized the use of their anonymized, residual clinical data and samples for research and publication. The institutional review board approved this retrospective consent procedure, as it was not possible to obtain direct consent from the patients at the time of admission. All patient-related data underwent stringent anonymization procedures to preserve privacy and ensure confidentiality.

The initial whole blood sample for BChE activity assessment was collected upon admission, prior to any antidote administration. Clinicians routinely awaited these initial BChE results, interpreting the degree of BChE depression below the lower reference limit as an indicator of poisoning severity, to guide therapy; atropine was the sole antidote, with BChE levels also guiding subsequent dosing and patient discharge. Due to the residual nature of specimens, precise ingestion-to-sampling times and comprehensive concurrent medication data were not consistently available; general clinical information is presented in the results where relevant. Whole blood specimens were collected into EDTA-anticoagulated tubes, while urine samples were retrieved using sterile, chemically inert containers. For every patient, an initial whole blood specimen was drawn upon admission. This sample was immediately processed for BChE activity assessment using both a point-of-care test (POCT) and an enzyme-multiplied immunoassay technique (EMIT). Additional whole blood samples were subsequently collected during the patient’s hospitalization, with the total number of samples varying per case: 10 for Case 1, nine for Case 2, and nine for Case 3. All specimens were maintained at 4°C and processed for biochemical analysis within 1 hour of collection to mitigate potential analyte degradation.

Participant Flow: A total of 14 patients were initially suspected of organophosphate (OP) ingestion. Of these, eight were confirmed by GC-MS/MS to have OP exposure. From this confirmed cohort, the 3 patients included in the study were specifically selected due to confirmed malathion presence and documented self-ingestion.

Butyrylcholinesterase (BChE) activity measurement

We determined Butyrylcholinesterase (BChE) activity using two distinct methods: a point-of-care testing (POCT) device and an enzyme-multiplied immunoassay technique (EMIT) analyzer.

For POCT, whole blood BChE activity was quantified with the Securetec Chek® Mobile System (Detektions-Systeme AG, Neubiberg, Germany). This portable instrument uses an Ellman-based spectrophotometric assay. After an initial absorbance reading, whole blood was introduced via a glass capillary. The device automatically computed hemoglobin-corrected BChE activity in U/L within 4 min, using pre-calibrated cartridges that eliminated the need for user calibration. The manufacturer-provided reference interval for whole blood BChE with this system is 1623–3861 U/L, based on a healthy population and provided with the device’s documentation.

Plasma BChE activity, also known as pseudocholinesterase (PsChE), was determined using the Siemens Viva ProE System (Siemens Healthcare Diagnostics Inc., Forchheim, Germany). This assay utilized ready-to-use liquid ELITechGroup Clinical Chemistry Cholinesterase reagents. Calibration was performed with ELITech Clinical Systems ELICAL2 (a lyophilized human serum calibrator), and quality control was maintained with ELITech Clinical Systems ELITROL I and ELITROL II (lyophilized human serum controls with predetermined constituent levels). The manufacturer-provided reference interval for plasma PsChE with this system is 5320–12920 U/L, also based on a healthy reference population and provided with the assay documentation.

For reporting, BChE and PsChE activity levels were exclusively expressed as percentages. These percentages were derived by calculating the measured value relative to the lower limit of the respective manufacturer-provided reference intervals (i.e., (Measured Value/Lower Reference Limit) × 100%). We explicitly defined the lower limit of the reference interval as our 100% reference point. The reference interval for the point-of-care (POCT) BChE assay is 1623–3861 U/L, while the reference interval for the laboratory-based EMIT PsChE assay is 5320–12920 U/L. We acknowledge that these manufacturer-provided reference values, based on a healthy reference population, were not age-or sex-adjusted; this is a limitation of our study.

Urinary malathion quantification (GC-MS/MS)

Reagents and solutions

We obtained certified malathion reference material from LGC (Teddington, UK). Cypermethrin Pestanal analytical standard, along with analytical-grade chloroform, dichloromethane, and 1,2-dichloroethane, were procured from Supelco (Merck, Darmstadt, Germany). Methanol, utilized as a solvent, was sourced from Sigma-Aldrich (Merck, Darmstadt, Germany).

A malathion stock solution was prepared in methanol at a concentration of 1000 µg/L. Calibration standards, ranging from 5 to 1000 ng/mL, were then generated by serial dilution of the stock solution in methanol. Similarly, precision standards were prepared from the stock solution at concentrations of 5, 50, and 500 ng/mL. To maintain method validity, all solutions were freshly prepared on the day of analysis. For the determination of the limit of quantification (LOQ), a 5 ng/mL solution was used.

Sample preparation and extraction

Urine samples were processed within 1 h of collection. To each 30 mL urine sample, a 50 µL aliquot of a 100 µg/L cypermethrin internal standard solution was added. Liquid-liquid extraction (LLE) used 15 mL of a chloroform/dichloromethane/1,2-dichloroethane solvent mixture (1:1:1, v/v/v). The mixture was stirred using a digital magnetic stirrer, centrifuged (EBA 200 Hettich, Tuttlingen, Germany), and the organic phase was transferred. The organic phase was then evaporated to dryness (Memmert UN30 oven, Schwabach, Germany) and reconstituted in 1 mL of methanol. A 1 µL aliquot of the reconstituted solution was subsequently injected into a GC-MS/MS system for analyte identification and quantification.

GC-MS/MS analysis

Malathion detection and quantification were carried out using a gas chromatography-tandem mass spectrometry (GC-MS/MS) system, comprising an Agilent 8890 gas chromatograph, a 7010B triple quadrupole mass spectrometer, and a 7693 A autosampler (Agilent Technologies, USA). Chromatographic separation was achieved using an HP-5 ms column (15 m × 250 µm × 0.25 µm, Agilent Technologies, USA). Helium (99.999%) served as the mobile phase, delivered at a constant flow rate of 1.1 mL/min.

The oven temperature program, lasting 42 min, initiated at 60°C, followed by a rapid temperature increase to 120°C at 40°C/min. Subsequently, the temperature was ramped to 310°C at 5°C/min and maintained for 1 min. The injector and transfer line temperatures were both held at 280°C. A wool liner was utilized for splitless injection of 1 µL sample aliquots. The ion source temperature was set to 300°C. For initial screening, the mass spectrometer operated in full scan mode, scanning a mass range of 40 to 600 m/z with a scan time of 100 ms, employing electron impact ionization at 70 eV.

For confirmatory analysis and quantification, multiple reaction monitoring (MRM) was employed, utilizing electron impact ionization. The mass spectrometer operating parameters were optimized to maintain a cycle rate of 11.5 cycles per second throughout the chromatographic run, ensuring adequate peak shape, detection limits, and data point density. Instrument control, data acquisition, and data processing were conducted using MassHunter Software (Agilent Technologies, USA).

Analytical method validation

A robust gas chromatography–tandem mass spectrometry (GC-MS/MS) method was developed and rigorously validated for the quantification of malathion in urine samples, in accordance with the comprehensive guidelines set forth by the Scientific Working Group for Forensic Toxicology (SWGTOX).²²

The optimization of the triple quadrupole MS/MS parameters was initiated by direct injection of a malathion methanol solution. This meticulous process focused on maximizing analytical sensitivity through the careful selection of appropriate precursor and product ions, along with fine-tuning collision energies. Our optimized method established a malathion retention time of 19.91 min. For quantification, we utilized the transition from the precursor ion m/z 173 to the product ion m/z 99, applying a collision energy (CE) of 15 V. For confirmation, a second distinct transition from m/z 127 to m/z 99 was incorporated, with a collision energy of 5 V. This optimized configuration, comprising two distinct multiple reaction monitoring (MRM) transitions, thereby provides four identification points, fully satisfying the pesticide confirmation criteria stipulated in European Commission Decision 2002/657/EC.²³ These parameters were meticulously chosen following a review of published methodologies, which show variability in malathion detection. For instance, reported retention times for malathion range from 13.5 min²⁴ to 23.39 min.²⁵ While m/z 173 is a commonly selected precursor ion across studies, the chosen product ions and corresponding collision energies often vary. For example, some studies use m/z 173 transitioning to m/z 127 with collision energies of 0.45 V²⁵ or 10 V,²⁶ or to m/z 99 with collision energies of 10 V.^24,27 Our selection of m/z 173 → m/z 99 at 15 V for quantification and m/z 127 → m/z 99 at 5 V for confirmation specifically aimed to provide high specificity and performance for malathion in human urine. For the internal standard, cypermethrin, MRM analysis employed the following transitions: 181 → 152.1, 164.9 → 91, 163 → 91, and 163 → 127 (with a retention time of 32.14 min and collision energies of 30, 15, 15, and 5 V, respectively).

The analytical method’s selectivity for malathion in urine was rigorously assessed through the analysis of 10 blank urine samples. This evaluation confirmed the absence of any interfering peaks at the characteristic retention times of malathion, thereby affirming the assay’s specificity. To further investigate potential matrix effects and carryover contamination, six blank samples were injected following each calibration standard. This systematic assessment revealed no significant matrix-derived interference or carryover, underscoring the method’s robustness for urine analysis.

The linearity of the malathion quantification method was meticulously established by analyzing six distinct malathion concentration levels, ranging from 5 to 1000 ng/mL. Each concentration was prepared in triplicate and processed across five independent analytical runs. The resulting calibration data demonstrated excellent linearity, characterized by a high coefficient of determination (R²) of 0.9998. The regression model derived from this data exhibited a statistically significant slope of 40.31 (standard error = 0.06, p < 0.0001) and an intercept of −69.99 (standard error = 25.73, p = 0.05).

Overall method performance, encompassing both accuracy and precision, was comprehensively evaluated by analyzing fortified urine samples. These samples were prepared at low (5 ng/mL), medium (50 ng/mL), and high (500 ng/mL) concentrations, analyzed in triplicate over multiple days. The results consistently demonstrated the method’s reliability and precision across the tested range. At the low concentration level (5 ng/mL), the calculated mean concentration was 5.0 ng/mL, showing a minimal bias of 0.3%, with within-run and between-run coefficients of variation (CV) of 11.03% and 14.18%, respectively. For the medium concentration (50 ng/mL), the mean was 51.7 ng/mL, reflecting a bias of 3.4%, alongside low within-run (1.47%) and between-run (1.56%) CVs. At the high concentration (500 ng/mL), the mean measured was 502.4 ng/mL, with a bias of 0.5%, and remarkably low within-run (0.67%) and between-run (0.82%) CVs.

The limit of detection (LOD) for malathion was determined to be 0.33 ng/mL, derived from the standard deviation of the y-intercepts obtained from multiple calibration curves. The limit of quantification (LOQ) was established at 5 ng/mL, confirmed through replicate analyses of spiked urine samples on three separate days. At this LOQ, the observed bias ranged from −17% to 19%, and the CV ranged from 3.50% to 14.95%, both well within acceptable quantitative performance criteria.

Statistical analysis

Statistical analyses were performed using Microsoft Excel (Version 16.92). Pearson’s correlation coefficient was used to evaluate the linear relationship between point-of-care (POCT) and EMIT butyrylcholinesterase (BChE) activity, and between admission malathion concentration and cholinesterase (BChE, PsChE) activities. Agreement between POCT and EMIT BChE measurements was assessed using Bland-Altman analysis. A p-value of less than 0.05 (p < 0.05) was considered statistically significant.

Results

Patient clinical profile and initial findings

Our study included three adult male patients, aged approximately 50–65 years, who were admitted to the Intensive Care Unit in a comatose state following voluntary acute malathion intoxication. Upon admission, malathion was detected in the urine of all three patients, unequivocally confirming exposure. Initial malathion concentrations in urine and cholinesterase activities are presented in Table 1.

Table 1.

Initial clinical and laboratory data of malathion-intoxicated patients upon ICU admission.

Parameter	Case 1	Case 2	Case 3
Age (years)	50-65	50-65	50-65
Sex	Male	Male	Male
Mode of ingestion	Voluntary	Voluntary	Voluntary
Consciousness at admission	Comatose	Comatose	Comatose
Malathion, µg/L (urine)	311	465	399
BChE activity, %^a	18	16	17
PsChE activity, %^b	18	14	16

^aBChE for point-of-care testing (POCT) device.

^bPsChE for enzyme-multiplied immunoassay technique (EMIT) analyzer.

Confirmation of malathion exposure

Consistent with the clinical suspicion, malathion was detected in the urine samples of all three patients included in the study. The admission malathion concentrations were determined to be 311 µg/L (Case 1), 465 µg/L (Case 2), and 399 µg/L (Case 3). Overall, the concentration of urinary malathion ranged from 311 µg/L to 465 µg/L, with a mean concentration of 391.7 ± 77.3 µg/L. The definitive identification of malathion in every patient’s urine confirmed exposure to the organophosphate, thereby establishing a crucial link between the observed clinical presentation and the causative toxic agent.

Figure 1 illustrates the full scan chromatogram obtained from the urinary sample of Case 1, a patient diagnosed with acute malathion intoxication. This chromatogram provides a comprehensive overview of the volatile and semi-volatile compounds present, allowing for the initial identification of malathion based on its characteristic mass spectral fragmentation pattern. Additionally, the figure presents the mass spectra of malathion (shown in blue) and its primary metabolite, malaoxon (shown in green), further confirming their presence in the sample.

Figure 1.

Full scan chromatogram and corresponding mass spectra of malathion (blue) and malaoxon (green) from a patient with acute malathion intoxication.

Comparison of BChE measurement methods

We assessed butyrylcholinesterase (BChE) activity using two distinct methodologies in our three patients with acute malathion intoxication: a colorimetric point-of-care device (ChE Check Mobile System, Securetec, Neubiberg, Germany) for BChE, and an enzyme-multiplied immunoassay technique (EMIT) on a Viva ProE System (Siemens, Forchheim, Germany) for pseudocholinesterase (PsChE).

Figure 2 illustrates the dynamic changes in BChE and PsChE levels, reported as a percentage of the lower limit of the reference interval, as measured by each method for each patient throughout their hospital stay.

Figure 2.

Comparison of BChE (colorimetric measurements by ChE Check Mobile System) and PsChE (EMIT Immunoassay measurements by Viva ProE System) activity levels during hospitalization after malathion exposure. All data is reported as a percentage (%). For this analysis, the low limit of the reference interval (LLRI) for each assay was defined as 100%. The LLRI for the POCT BChE assay is 1623 U/L, and the LLRI for the EMIT PsChE assay is 5320 U/L.

For the three Malathion intoxication patients (n = 3), BChE and PsChE activities demonstrated a strong linear relationship. Individual Pearson correlation coefficients (r) varied from 0.905 to 0.986, consistently showing robust statistical significance (t-values 6.75–25.40; p < 10^-4). A scatter plot of percentage butyrylcholinesterase (%BChE) activity against percentage pseudocholinesterase (%PsChE) activity (Figure 3) further revealed a strong positive linear relationship between the two methods. Correlation analysis confirmed this association (r = 0.8599, t = 11.55, p < 0.0001). While both methods captured the general trend of increasing %BChE activity with increasing %PsChE activity, indicating a positive correlation, direct comparison revealed discrepancies in absolute %BChE values, as evidenced by the scatter of points around the trend line.

Figure 3.

Relationship between percentage butyrylcholinesterase and percentage pseudocholinesterase activities.

A Bland-Altman plot (Figure 4) was constructed to assess the agreement between the two enzyme activity measurement methods. The plot revealed a positive bias of approximately 10%, indicating that the point-of-care (POCT) method tended to yield systematically higher values compared to the laboratory-based enzyme-multiplied immunoassay technique (EMIT). While a consistent bias might be considered clinically acceptable for simply tracking trends, the 95% limits of agreement (LOA) were wide, ranging from −20% to +40%. This suggests substantial and unpredictable individual variability between the methods.

Figure 4.

Bland-altman analysis of enzyme activity measurement methods in malathion intoxication.

This degree of disagreement is considered clinically significant and demonstrates that the two methods are not interchangeable for precise malathion intoxication severity assessment. Such wide variations could lead to misinterpretation of a patient’s true BChE activity, which could directly impact critical therapeutic decisions. For example, relying on a single POCT measurement could lead to an incorrect assessment of a patient’s condition, potentially affecting the timing and level of atropine dosing, the frequency of patient monitoring, or the criteria for ICU discharge, where even small changes in enzyme activity are used to guide management. The POCT is therefore best suited for rapid, initial screening and tracking trends in an integrated, multi-marker framework, rather than for making precise, quantitative decisions in isolation.

Relationship between BChE activity and urinary malathion concentration

A statistically significant inverse correlation was demonstrated between admission urinary malathion concentrations and both percentage butyrylcholinesterase (%BChE) activity (r = −0.9971, t = 13.01, p = 0.0488) and percentage pseudocholinesterase (%PsChE) activity (r = −0.9970, t = 6.42, p = 0.0496) at admission.

Patients with elevated admission malathion concentrations consistently tended to exhibit a greater degree of %BChE and %PsChE inhibition. For example, Case 2, who presented with the highest admission malathion concentration (465 µg/L), displayed the lowest %BChE (16%) and %PsChE (14%) activities. In contrast, Case 1, with a malathion concentration of 311 µg/L, showed comparatively higher %BChE and %PsChE activities of 18%. These observed inverse correlations suggest that measurements of BChE and PsChE activity can serve as valuable indices of malathion exposure levels and subsequent cholinesterase inhibition in this patient cohort. While the extent of enzyme inhibition varied among patients, likely reflecting differences in exposure levels and individual patient characteristics, the limited sample size (n = 3) restricts the statistical power of this investigation. Consequently, further research involving larger patient cohorts is warranted to explore the relationship between malathion exposure and cholinesterase inhibition more comprehensively.

Clinical course and biomarker trends

For each patient, we observed their clinical progression in relation to the dynamic trends of BChE activity and urinary malathion concentrations over time. Table 2 summarizes these key clinical and biomarker data from admission to discharge from the Intensive Care Unit (ICU).

Table 2.

GC-MS/MS analysis of malathion in urine samples from malathion-intoxicated patients.

	Admission to the ICU			Discharge from the ICU			ICU hospitalization, days
	Malathion, µg/L	BchE^a activity, %	PschE^b activity, %	Malathion, µg/L	BchE^a, activity, %	PschE^b activity, %	ICU hospitalization, days
Case 1	311	18	18	< LOD	92	61	20
Case 2	465	16	14	< LOD	45	46	12
Case 3	399	17	16	< LOD	78	41	17

^aBChE for point-of-care testing (POCT) device.

^bPsChE for enzyme-multiplied immunoassay technique (EMIT) analyzer.

Upon admission, all three patients exhibited significant malathion exposure with detectable urinary concentrations and markedly inhibited BChE and PsChE activities, consistent with severe acute organophosphate intoxication. For instance, Case 2, who presented with the highest admission malathion concentration (465 µg/L), also showed the lowest initial %BChE (16%) and %PsChE (14%) activities, reflecting the most severe enzyme inhibition.

As the clinical course progressed and patients received treatment, urinary malathion concentrations declined, reaching below the limit of detection (LOD) by the time of hospital discharge for all cases. This decrease in urinary malathion corresponded with a crucial recovery of BChE and PsChE activity. For example, Case 1, admitted with 311 µg/L malathion and 18% BChE/PsChE activity, showed a substantial recovery to 92% BChE and 61% PsChE activity upon discharge after 20 days in the ICU. Similarly, Case 3, despite a longer ICU stay of 17 days, demonstrated recovery from 17% BChE/16% PsChE to 78% BChE/41% PsChE as urinary malathion cleared. Even Case 2, with the most severe initial presentation, showed a notable recovery to 45% BChE and 46% PsChE activity by discharge after 12 days.

These trends highlight the integrated utility of simultaneously monitoring both BChE activity (reflecting the toxicodynamic effect) and urinary malathion concentration (reflecting exposure and elimination). The observed increase in BChE/PsChE activity concomitant with the clearance of urinary malathion provides valuable insights into the resolution of the toxic effect and the patient’s recovery trajectory, demonstrating the combined markers’ ability to track the ongoing toxicological process.

Discussions

This study suggests an integrated diagnostic approach for acute malathion intoxication by combining rapid butyrylcholinesterase (BChE) measurements with urinary malathion quantification. Our findings are consistent with malathion exposure in all patients, hint at a strong correlation but potential clinical discrepancies between point-of-care (POCT) and laboratory BChE assays and identify a statistically significant inverse correlation between urinary malathion concentrations and BChE activity in this small case series. Patient clinical improvement consistently paralleled these dynamic biomarker trends.

Per-case Pearson correlations (r = 0.905–0.986) suggested a strong agreement between POCT and EMIT BChE, indicating their potential utility for tracking cholinesterase inhibition and rapid initial screening. However, the Bland-Altman analysis revealed a 10% positive bias, along with wide 95% limits of agreement (−20% to +40%). In a clinical context, a 10% bias, while present, may be acceptable for rapid, initial trending. However, the wide limits of agreement suggest that the two methods are not clinically interchangeable for precise quantitative measurements. This degree of disagreement could potentially impact precise dosing decisions if used in isolation. Nevertheless, the rapid turnaround time of POCT is crucial for immediate clinical decisions, as it can provide critical trending information. Unlike traditional symptom-based management, which relies on subjective clinical signs, POCT results offer an objective metric to guide the timing and level of initial atropine administration and to monitor treatment response. For example, a continued or worsening drop in BChE activity, as indicated by POCT, could signal the need for an increased atropine dose, while a plateau or a gradual rise could suggest that the patient is responding, allowing for a cautious dose reduction.

The significant inverse correlations between admission urinary malathion and both %BChE (r = −0.9971, p = 0.0488) and %PsChE (r = −0.9970, p = 0.0496) in this cohort underscore the mechanistic interplay in malathion intoxication. Urinary malathion quantifies the absorbed dose and elimination, while BChE reflects the systemic anticholinesterase effects. This integrated approach may offer a more nuanced understanding of poisoning severity, systemic impact, and elimination kinetics than relying on a single marker alone. The ability to monitor both the xenobiotic’s clearance (via GC-MS/MS) and the enzyme’s recovery (via POCT BChE) could have significant clinical implications for managing acute malathion intoxication, enabling more informed decisions on antidote administration, observation, and supportive care. This combined data set may also provide a more objective basis for ICU discharge criteria, as a patient with both a stable BChE level and a significantly reduced malathion load may be considered at lower risk of delayed complications or relapse. Tracking malathion clearance and BChE recovery could also guide prognosis and help optimize the timing of discharge.

Limitations

The limitations of this study should be explicitly acknowledged. This is a small, single-center case series with a sample size of only three patients. Given the rarity of malathion intoxication cases at our institution, no formal sample size or power calculation was performed. As such, the results are exploratory, not generalizable to a broader patient population, and our conclusions are limited to descriptive observations. Any statistical inferences should be viewed with considerable caution.

Future directions

Scaling this approach to larger cohorts holds promise. The rapid turnaround and portability of POCT devices make them feasible for more widespread assessment, particularly in areas with limited laboratory infrastructure. Larger, multi-center studies would be necessary to validate our preliminary findings, develop more robust prognostic indicators, and establish definitive clinical cut-off values for precise treatment guidance. Such studies could also evaluate the potential for cost-effectiveness by demonstrating how this integrated approach might shorten hospital stays and reduce overall healthcare resource utilization.

Conclusions

In conclusion, this exploratory study suggests that an integrated diagnostic approach, combining rapid butyrylcholinesterase activity measurements with direct urinary malathion quantification, may offer a more nuanced and comprehensive assessment of acute malathion intoxication. The observed inverse correlation between urinary malathion levels and BChE activity provides preliminary insights into the dynamic interplay of exposure and physiological recovery. This multi-marker strategy holds potential to enhance clinical decision-making, refine prognosis assessment, and ultimately improve patient care in cases of organophosphate poisoning. However, due to the small sample size (n = 3), these findings are exploratory and not generalizable, and therefore their clinical utility should be suggested cautiously.

Footnotes

ORCID iD

Andreea-Camelia Hîrjău

Ethical considerations

The study was conducted in accordance with the Declaration of Helsinki.

Consent to participate

Informed consent was obtained from all subjects involved in the study.

Author contributions

A.-C.H.: Conceptualization; Formal Analysis; Writing—original draft preparation. I.-M.M.: Data curation; Writing—review and editing. G.-L.R.: Methodology; Supervision; Writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Nucleu Program within the National Research Development and Innovation Plan 2022-2027, carried out with the support of MCID, project no. 23 44 02 01.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Data Availability Statement

Data are contained within this article.*

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