Effects of acute organophosphate ingestion on cognitive function, assessed with the mini mental state examinationSS Jayasinghe
Department of Pharmacology, University of Ruhuna, and South Asian Clinical Toxicology Research Collaboration, University of Peradeniya, Sri Lanka
Correspondence Address: Source of Support: Wellcome Trust and Australia's National Health and Medical Research Council International Collaborative Research Grant (GR071669MA) through the South Asian Clinical Toxicology Research Collaboration, Conflict of Interest: None DOI: 10.4103/0022-3859.101374
Source of Support: Wellcome Trust and Australia's National Health and Medical Research Council International Collaborative Research Grant (GR071669MA) through the South Asian Clinical Toxicology Research Collaboration, Conflict of Interest: None
Background : Chronic damage to the central nervous system resulting in cognitive impairment has been shown with repeated, low doses of organophosphorus (OP) exposure over month or years. Aim: The study aimed to find out whether there is any cognitive impairment following acute OP exposure that could be detected by a simple screening instrument, the Mini Mental State Examination (MMSE), in clinical settings. Settings and Design: A cohort study. Materials and Methods: The study was conducted with matched controls. Consecutive patients admitted to the hospital with acute ingestion of OP were recruited. Cognitive function was assessed with the MMSE, digit span test, test of long-term memory function and concentration. Patients were assessed twice: at 1 and 6 weeks of exposure. Statistical Analysis: Continuous variables were analyzed with the paired and unpaired T-tests. Non-normally distributed data were analyzed with the Mann-Whitney U test and Wilcoxon Signed Rank test. Discrete variables were analyzed with the Chi-square test. Results: There were 60 patients and 61 controls. The mean age (SD) of the patients and controls was 31.5 (11.6) and 31.3 (11.8) years, respectively. Forty-two patients turned up for the second assessment. Significant impairment of cognitive function was seen in the total score of MMSE (95% CI -2.5 to -0.3), orientation (95% CI -1 to -0.2) and language (95% CI -0.9 to -0.1) domains of MMSE, digit span test (95% CI 0.1-1.7) and test of long-term memory function (95% CI 0.3-2.3) in the first assessment compared with the controls. When the results of the second assessment were compared with the controls, no significant differences were seen. Conclusion: Although there was a slight transient cognitive impairment detected with the screening tests following acute OP ingestion, no long-term cognitive defects was detected.
Keywords: Cognition, organophosphorus compounds, suicide
Organophosphorus (OP) and carbamate insecticides are the most common groups of chemicals involved in poisoning in Sri Lanka, accounting for almost 54% of all pesticide poisonings. 
Chronic damage to the central nervous system resulting in cognitive impairment has been shown in some studies with single acute or repeated low dose of OP exposure over months or years. ,,,, There are some inconsistencies among these studies. Hence, the level of evidence is controversial.  Delay in cognitive processing with acute OP ingestion has been reported even after recovery from the acute cholinergic phase of intoxication, and this did not improve even 6 months after the exposure.  Studies have been performed to determine the effects on cognitive function in acute and chronic occupational exposure.  However, information on cognitive function following acute self-ingestion is scant. We were interested in looking at whether the cognitive impairment could be detected by simple screening test/s in the clinical settings following acute OP ingestion.
We carried out a cohort study with matched controls to determine whether there is any cognitive impairment with acute OP exposure that could be detected by simple screening instruments, such as the Mini Mental State Examination (MMSE), the digit span test, a simple questionnaire to assess long-term memory and a test of concentration that can be used in clinical settings.
A cohort study was conducted with age, gender and occupation-matched healthy controls. Patients with acute OP poisoning were recruited to the study from a tertiary care hospital and a secondary care hospital in the Southern Province of Sri Lanka from June 2008 to April 2010. Ethical clearance was granted by the institutional ethical review committee. An informed written consent was obtained from the patients and the controls after explaining the study to them.
At the time of recruitment to the study, these patents either had features of the cholinergic syndrome or had been given atropine in the peripheral units and were then transferred to the collaborating hospitals.
Age (±3 years), gender, educational level and occupation (according to the International Standard Classification of Occupation; ISCO-88 by the International Labor Organization) matched controls were recruited from persons accompanying patients to the hospital.
Participants who had neuropathies, diabetes mellitus, dementia and psychiatric illness and who were on medication that had the potential to alter cognitive function (anticonvulsants; vigabatrin, gabapentin and lamotrigine, minor tranquilizers; diazepam and antihypertensive like methyldopa), visual impairment and head injury were excluded from the study.
Sample size was calculated using the following formula:
(u = 1.64, ν = 1.96, σ1 = population standard deviation, σ2 = sample standard deviation, μ1 - μ2 = expected mean difference between the population and the sample). 
Cognitive function was assessed only if the educational level of the participant was at least up to year five. Assessments were done once the patient had recovered from the acute cholinergic crisis (the first assessment) and at 6 weeks following the exposure (the second assessment). Assessments were carried out by trained Clinical Research Assistants who were blind to the study hypothesis.
Mini mental state examination
A validated Sinhalese translation of the MMSE was used with a few modifications.  [Table 1] illustrates the modifications made in the MMSE administered in our study, which reflected our need to use this in the community. A total score of less than 24 out of 30 was considered as cognitive impairment. No response or refusal to perform any item was scored as zero.
Digit span test
Digit span is a common measure of short-term memory. In this test, the ability of a person to absorb and recall a number of digits in the correct serial order after hearing them is tested. Auditory digit span was tested by saying numbers (7, 6, 9, 5, 1, 4, 0 and 3) slowly at 1-second interval in a monotonous voice. Then, the participant was asked to repeat in the correct order.  The average adult has an auditory digit span of 6-7. A digit span of less than 6 is considered as indicating impairment of short-term memory.
Long-term memory function
An interviewer-administered simple questionnaire was used to assess long-term memory function [Table 2]. The maximum score was 10.
The participant was asked to say the months of the year backwards in the correct order, and the number of months that were in the correct order was counted.  The maximum score was 12.
Estimation of plasma cholinesterase levels
Plasma cholinesterase (ChE) levels were quantified within the first 12 h after the exposure using the modified Ellman method developed by Worek et al. 
The data were analyzed using Graphpad Prism 4. Continuous variables were analyzed with paired and unpaired T-test. Non-normally distributed data was analyzed with the Mann-Whitney U test and Wilcoxon Signed Rank test. Discrete variables were analyzed with the Chi-square test. Spearman's correlation was used to analyze the severity of poisoning (plasma ChE) with the measures of cognitive function.
There were 60 patients and 61 controls. All the patients had acute OP exposure as a result of suicide ingestion. The mean age (SD) of the patients and controls was 31.5 (11.6) and 31.3 (11.8) years, respectively. None of the patients or controls had previous acute exposure to pesticides. [Table 3] provides the descriptive data of the patients and the controls. Three patients and one control were farmers in occupation. Forty-two patients turned-up for the follow-up assessment at 6 weeks following the exposure. Three of 60 patients required Intensive Care Unit (ICU) admission for ventilation due to the development of intermediate syndrome.
Plasma ChE levels were available in 28 patients. The median (interquartile range) plasma ChE level was 745 (138-3341) μmol/L/min.
Seventeen of 60 patients in the first assessment and nine of 61 in the controls scored less than 24 in the MMSE (χ2 =3.3, P=0.06). Seven patients out of 42 showed MMSE score less than 24 in the second assessment. There was no significant difference between the number of patients with impaired MMSE in the second assessment versus controls (χ2 =0.07, P=0.7) and the second assessment versus the first assessment (χ2 =1.8, P=0.17). All three patients who had ICU admissions had an MMSE score more than 26.
The mean (SD) scores of the digit span test, long-term memory function and concentration are shown in the [Table 4].
[Table 5] indicates the mean (SD) score of individual domains of MMSE in the patients and the controls. The total score of MMSE, orientation and language domains of the MMSE showed significant impairment in the first assessment of the patients compared with the controls. There was no significant impairment when scores in the second assessment were compared with the controls.
In the first assessment of the patients, 56/60 and in the second assessment, 33/42 had impaired (<6) digit span. In the controls, 44/61 had impaired digit span. The number of patients who had impaired digit span was significantly high in the first assessment compared with the controls (χ2 =4.8, P=0.02). When it was compared with the second assessment versus controls, no significant difference was seen (χ2 =0.5, P=0.5).
Correlation coefficient (P-value) of plasma ChE with measures of cognitive function is shown in [Table 6].
Although there was a slight transient cognitive impairment detected with the screening tests following acute OP exposure, no long-term cognitive defects were detected by MMSE in clinical settings.
Cognitive function was tested with the simple and quick assessment tool of MMSE. The MMSE provides a measure of cognition that covers a broad set of cognitive domains: Orientation, registration, short-term memory, attention, calculation, visuo-spatial skills and apraxia.  The sensitivity and specificity of MMSE were 100% and 69% to the cut-off level of less than 24 evaluated against the performances at the Cambridge Cognitive Score. 
The current study cross-checked some domains in the MMSE with other simple tests. The registration and recall domains of the MMSE were cross-checked with the digit span test, and attention and calculation domain in the MMSE was crossed-checked with concentration ability.
The results of the patients being compared with matched controls were a strength in the current study.
Stephens et al.  reported impaired cognitive function in sheep farmers who were exposed to OP through sheep dipping, which involves immersing each animal in a pesticide solution to control parasitic infections. Exposure to OP appeared to be splashing on to the skin where protective cloths were seldom worn. The speed of performance of simple reaction time, symbol-digit substitution and syntactic reasoning were significantly slower in the farmers than in the controls. No impairment was found in short-term memory (by digit span test and visual spatial memory) and long-term memory (by serial word learning, category search classification, category search recognitions) in the cases compared with the controls. ,
The study done by Starks et al. enrolled licensed pesticide applicators and showed an adverse association between high pesticide exposure events and a test of visual scanning and processing (Digit symbol), and visual scanning and motor speed (Sequence A), of the nine neurobehavioral tests that they had used to assess the central nervous system function. ,
Steenland et al.  investigated the chronic squeal of acute occupational OP poisoning. , They studied cognitive function via finger tapping (motor speed test), sustained visual attention, visuomotor accuracy, visuomotor speed, symbol digit, visual memory and serial digit learning tests. Significant impairment was found in sustained visual attention and symbol digit tests in the cases (proportion) compared with the non-exposed reference. ,
A study done on volunteers with varying degrees of occupational exposure to OP showed significant impairment on the Bender Visual Motor Gestalt test (used to evaluate visual motor maturity) and part B of the Trail Making test (Trail Making test is a neuropsychological test of visual attention). 
The study done by Fiedler et al.  recruiting tree fruit farmers who had occupational exposure to OP demonstrated significantly slow simple reaction time for the dominant and non-dominant hands of the exposed than the controls. No significant impairment was seen in the visuomotor coordination, verbal memory, verbal ability and expressive and receptive language. ,
Long-term neurobehavioral effects of mild poisonings with OP or carbamate pesticides among banana workers were explored by Wesseling et al. They observed marked impairment of the performance of the measures of cognitive function in the OP-poisoned subjects compared with controls. 
The review article by Kamel et al. looked into the association of pesticide exposure with neurological dysfunction. They found inconsistencies among the studies.  Most studies found deficits in one or more tests of cognitive function, but different tests were affected in different studies.  The review article by Colosio et al. described positive, uncertain and negative effects on cognitive function as a consequence of a prolonged, low-dose exposure to OP and/or carbamate pesticides. 
In most of the previous studies, the severity of the poisoning was not explored or the exposure state and the effect were impossible to correlate due to previous exposures.  The current study enables to show negative correlation of the measures of cognitive function with severity of poisoning (plasma ChE levels).
Not all patients who were assessed at 1 week turned up for the follow-up assessment at 6 weeks following the exposure. The dropout rate was 15%. Patients were not followed-up after 6 weeks of exposure. It would be better if we could follow them up further, but the dropout rate may be high if the follow-up duration is long.
The current study did not show persistent impairment of cognitive function following acute OP exposure. The methods we used may not be sensitive enough to detect mild cognitive impairment. Further studies are required with more sophisticated neurobehavioral tests to assess cognitive function following acute OP poisoning.
The study was funded by the Wellcome Trust and Australia's National Health and Medical Research Council International Collaborative Research Grant (GR071669MA) through the South Asian Clinical Toxicology Research Collaboration.
The author thanks all participants, consultants who gave their patients for the study at the Teaching Hospital, Galle and the General Hospital, Matara, Sri Lanka, and administrative staff and health care professionals at the Teaching Hospital, Galle and the General Hospital, Matara. Heads and the staff members of the Department of Pharmacology and the Department of Medicine, Faculty of Medicine, University of Ruhuna, Sri Lanka and members of the South Asian Clinical Toxicology Research Collaboration are greatly acknowledged. A special word of thank is extended to Professors NA Buckley, AH Dawson, Anoja Fernando, KD Pathirana, PL Ariyananda and Dr. Bilesha Perera.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]