Field of the Invention

Chemical science and agriculture with regard to a screening kit for risk of insecticide exposure

Background of the Invention

Insecticide exposure is deemed to be an incident that can occur in daily lives, not exclusively to agriculturists, where there may be exposure to insecticides in the quantity exceeding standards. Frequent insecticide use in households or heavy use may, in turn, results in accumulated residuals in the body in the quantity exceeding standards as well. Data from the Department of Disease Control revealed survey findings that there were people at risk of illnesses from poisons of insecticides and pesticides, who had a chance of contracting infectious skin diseases as well as a higher risk of chronic diseases in groups of those with low immunity or having blood vessel diseases. It has been found that the morbidity from poisons of insecticidal and pesticidal chemicals is increasing every year. Considering the severity of the said chemicals, it has found that their toxicity can be manifested in two forms, namely the acute one, where signs are present immediately upon chemical exposure, such as nausea, vomiting, headache, myalgia, muscle rigidity and spasm, diarrhea, difficulty breathing, blurred vision or burning eyes; and the chronic one which is caused by chronic chemical exposure and caused by accumulated poisons that lead to diseases or health problems, such as cancer, diabetes mellitus, paresis, paralysis, skin diseases, sterility, congenital deformities, hearing loss, impotence, life-threatening conditions and so on. For example, organophosphates-carbamates have inhibitory activities toward nervous system functions.

As such, risk level assessment of insecticide exposure is another important thing in reducing risk potentials that may lead to physical detriment or cause diseases. Should such risk be detected quickly, lifestyles can be modified, or safe products can be chosen to reduce physical risk. Risk level assessment of exposure to insecticides or chemicals varies in its format so that it is right for the operation, right for the specimen and right for the type of substance that needs assessed. Based on exploring, work that has applied for the protection and has similarities to the said work has been found as follows.

A Thai petty patent application number 1703002151 related to a portable quantitative screening kit for risk of insecticide exposure consists of a reactive unit serving to show a reaction between the blood to be screened and a chemical that is a cholinesterase substrate containing thiocholine, a reaction meter unit with spectrophotometry serving to measure ferricyanide changes of the product from the reactive unit, and an information-processing unit to convert signals from the reaction meter unit so that the chemical reaction changes are visualized. The said invention uses an analytical tool with detectable specificity, hence limitation in the measurement.

A United States patent under the publication number US20110166043A1 is related to a composite method in detecting a marker of exposure of mammals to organophosphate compounds. Organophosphate compounds consist of pesticides and metabolites, and include highly reactive organophosphate compounds. The biomarker represents the interaction of organophosphate compounds and polypeptides. The biomarker interaction detected by an optical sensor, which consists of a receptor linked to a biopolymer, results in optical reading for detection of change values. The said invention is different with regard to the process and the reagent used in the detection.

A United States patent under the publication number US 10913967B2 is related to a system capable of assessing red blood cell AChE function for alerting of exposure and probably suggesting treatment under medical standards, a portable system to oversee in vitro diagnostic points that can detect red blood cell acetylcholinesterase (AChE) from undiluted blood samples as an alert prior to signs of exposure to organophosphorus compound poisons or other poisons. The said invention uses different red blood cells as test samples and results in a limitation in that laboratory is required.

The above-mentioned inventions reveal different assessment processes as per objectives of chemicals that need testing and have a limitation in that laboratory is required for their use, hence the research and development of enzyme-derived assessment kit with accuracy in indicating insecticide exposure levels. The developed technique was designed for easy, quick and convenient use with sufficient accuracy in indicating risk levels of insecticide exposure to be used in the daily life of each individual.

Summary of the Invention

Screening kit for risk of insecticide exposure is a kit that serves to assess the risk of exposure to health-threatening substances with enzyme in assessing insecticide exposure levels in the body. Assessment steps consist of blood sample collection, solvent preparation, sample separation and reaction, and then the solution is tested in a color meter so that exposure risk levels of insecticides accumulated in the body for the past 12-day period can be determined accurately. The intent of this invention has been to develop screening kit for risk of insecticide exposure that can assess the exposure risk of health-threatening substances, which may include chemicals, pesticides and insecticides in the field because the said kit has been developed for easy use with accuracy that yields results quickly; and to develop the use at an organization level to facilitate safe work mode as well as contributing to further efficient health check-up welfare.

Brief Description of the Drawings

Figure 1 shows a part of a sample color meter.

Figure 2 shows another part of a sample color meter.

Detailed Description of the Invention

Indication of insecticide exposure risk using enzyme: The said enzyme is re-generated every 12 days, and the screening kit for risk of insecticide exposure thereby helps indicate insecticide exposure in the past 12-day period. The reaction was designed for easy, quick and convenient use with sufficient accuracy in indicating risk levels of insecticide exposure, suitable for field use and use for the assessment of insecticide exposure risk in households, communities, organizations and people at risk of insecticide exposure, as a tool in monitoring insecticide exposure levels of individuals that can provide assessment results within one to two hours following blood drawing, thereby enabling the use of the risk assessment results in conjunction with risk factor assessment in individuals to assist medical and public health personnel in promoting health or as suggestion in insecticide exposure control and minimize the risk in individuals efficiently.

The assessment process of the screening kit for risk of insecticide exposure consists of the following steps.

A. Blood sample collection

- Prick a fingertip for blood with a finger pricking device and fill two blood collection tubes.

- Keep the blood collection tubes with blood collection apparatus to allow the blood to separate into compartments for a duration of 30 minutes.

- Centrifuge the blood collection tubes in a hematocrit centrifuge for a duration of five minutes.

B. Solvent preparation - Prepare 3 ml solvent at the substance concentration of 0.1-1.5% weight by volume, where the solvent can be chosen from calcium chloride (CaCk), potassium chloride (KC1), dipotassium phosphate (KH2PO4), sodium chloride (NaCl), magnesium chloride (MgCk) or 5,5-dithiobis(2- nitrobenzoic acid).

- Adjust the pH to a range of 7.0-8.50 with sodium hydroxide (NaOH) or hydrochloric (HC1) in reaction tube.

- Add 1 drop of substrate being acetylthiocholine iodide at the concentration of 2.50-7.50% weight by volume and butyrylcholine iodide at the concentration of 2.50-7.50% weight by volume and an enzyme cofactor, namely magnesium chloride (MgCk) or zinc chloride (ZnCk), at the concentration of 0.2- 1.5% weight by volume.

C. Sample separation

- Blow the blood collecting tubes using a dropper to separate the serum.

- Add 1 drop of the serum into the reaction tube.

D. Result examination

- Shake the reaction tube to mix.

- Check with the color meter for the reaction of the solution between the blood sample and the solvent for a duration of 30 seconds to 3 minutes.

Figures 1 and 2 show a sample color meter, where the color meter serves to check colors specific to the screening kit for risk of insecticide exposure. The color meter consists of the body 100 provided as a sample tube receptacle with an arranged aperture 101 that supports sample tube insertion, and one side of the body 100 is arranged with a display 102 for showing readings of risk level assessment that can be shown as color bands, numerals, letters or symbols, and one side of the body 100 is arranged with a slot 103 used for connecting with another equipment cable or power supply charging,

The body 100 is assembled with the base 104 that makes a test tube aperture 105 for receiving a test tube 106 align with or close to the sample tube aperture 101. The color measurement consists of at least one bulb 107 as lighting in metering color intensity levels of the test tube 106. Also, there is a socket 108 used for connecting with another equipment cable or power supply charging, and the socket 108 is arranged with a part that connects with battery 109 as power supply when using the color meter outdoor. The assessment of insecticide exposure risk is the color level measurement of solution through measuring only the increase or decrease of the red color and/or the blue color. Subsequently, results from the color meter are shown on the display and interpreted per the instructions in Table 1. This color meter is not limited to measurement in a specified wavelength range and is thereby deemed to have an advantage when compared with a spectrophotometer.

Remarks:

1. It is recommended that each test be duplicated for accurate result.

2. In the event of one round of test, if the test result falls in criteria 3-5, another test should be performed to confirm the result, and if the test result falls in criteria 3-5, it is recommended that the suggestion at the end of Table 1 be followed.

Additional Details for Consideration of Use

1. This test kit is for field use for research or study on changes of insecticide exposure levels/insecticide exposure risk only. 2. The enzyme used in this assessment has interfering factors being 1. Hepatic diseases, 2.

Hyperlipidemia, 3. Diabetes mellitus, 4. Hypertension and 5. Renal diseases. Group of population with pathology related to the said factors induce abnormal enzyme levels, and assessment findings may fall in levels 3-5. Therefore, the assessment is not recommended in this population group.

3. Test result confirmation: it is recommended that results be interpreted in conjunction with risk- screening questionnaire, history of underlying diseases, history of insecticide use both in occupation like farm spraying and in households, such as substances to kill ants, termites, flies, weevils and annoying insects.

4. Should subjects have test results that fall in levels 3-5 who are found to have no underlying diseases as per 2. And to have history of insecticide use, recommendations for detoxification and health promotion are as follows.

- Reduce insecticide use in their daily lives and stay away from areas with insecticide use and have insecticide exposure levels re-assessed in 12 days.

- Consider using herbs and health supplements used in detoxification, such as Thunbergia tea and liver health-promoting food, to detoxify existing poisons and promote liver health. Subsequently, have insecticide exposure levels re-assessed every 12 days to monitor changes in insecticide exposure levels in the body.

An efficiency test of the reagent in the screening kit for risk of insecticide exposure consisted of:

Test 1. Two hundred and nine serum samples were selected from a population group with history of insecticide exposure (positive) and a group without history of insecticide exposure (negative). 2. The samples were tested in a spectrophotometer to test the efficiency of the reagent in the screening kit for risk of insecticide exposure in comparison with Ellman reaction, ^{1 2} which is a standard laboratory technique (spectrophotometer, 405 nm).

3. A cut-off value was used that indicated enzyme inhibition based on a study by Rao et al. 2016. ³ Test Results

Efficiency Assessment of Reaction Selected for Use in Product Design

Sensitivity or true positive rate:

= true positive/(tme positive + false negative) = (87/(87+l))X100 = 98.86%

Specificity or true negative rate:

= true negative/(tme negative + false positive)

= (116/(116+5)) X 100 = 95.87%

Accuracy (ACC): = (true positive + true negative)/(true positive + false positive + true negative + false negative)

= ((87 + 116)/(87 + 1 + 116 + 5)) X 100 = 97.13%

The test results demonstrated the efficiency or accuracy at 97.13% while the sensitivity was 98.86%, and the specificity was 95.87%; that is, greater than 95% for all variables tested, leading to a conclusion that they are sufficient to support efficient use of the screening kit for risk of insecticide exposure in classifying insecticide exposure.

References 1. Ellman GL, Courtney KD, Andres V, Featherstone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol. 1961;7:88-95.

2. Komersova A, Komers K, Cegan A. New Findings about Ellman’ s Method to Determine Cholinesterase Activity. Zeitschrift fur Naturforsch - Sect C J Biosci. 2007;62(1-2):150-154. doi: 10.1515/znc-2007 - 1 -225

3. Rao G, Jyothsna M. Serum Cholinesterase Fevels in Organophosphorous Poisoning Patients on Ventilatory Support. Orig Res Artie Indian J Clin Anaesth. 2016;3(l):52-55. doi: 10.5958/2394- 4994.2016.00012.3

Best Mode of the Invention

As described in Detailed Description of the Invention