Exhaled breath VOCs markers for early diagnosis of alveolar echinococcosis and use thereof

Abstract

The application discloses an exhaled gas VOCs marker for early diagnosis of alveolar echinococcosis and application thereof. The ROC curve AUC of biomarker A myristic acid is 0.965, the sensitivity and the specificity can reach 98.2% and 89.7% respectively, and the accuracy reaches 93.8%; the ROC curve AUC of biomarker B p-tolualdehyde is 0.964, the sensitivity and the specificity can reach 94.5% and 92.3% respectively, and the accuracy reaches 93.3%; the ROC curve AUC of biomarker C 2-(hydroxymethyl)-benzoic acid is 0.961, the sensitivity and the specificity can reach 96.4% and 76.9% respectively, and the accuracy reaches 86.7%. The three kinds of gas levels all have statistical significance, and can be used as a reference basis for diagnosis of AE. The detection system is low in cost, simple in operation and capable of being used for an early screening diagnosis method of alveolar echinococcosis.

Description

Technical Field

[0001] This invention relates to exhaled VOCs biomarkers for early diagnosis of alveolar echinococcosis and their applications, belonging to the field of detection technology. Background Technology

[0002] Alveolar echinococcosis (AE), also known as multilocular echinococcosis, is a chronic parasitic disease caused by the larvae of the Echinococcus multilocularis tapeworm, the alveolar echinococcosis larva. This disease is widespread globally, especially prevalent in high-latitude, cold regions or permafrost areas of the Northern Hemisphere.

[0003] Humans are incidental hosts of alveolar echinococcosis, primarily contracted through ingestion of vegetables or untreated water containing echinococcosis eggs, or contact with infected animal fur. The primary lesion of alveolar echinococcosis is almost always located in the liver, but it can also spread to organs such as the lungs and brain via the bloodstream, causing secondary or metastatic lesions. The cysticercus larvae diffusely infiltrate and grow within the liver parenchyma, directly destroying and replacing liver tissue, forming giant cysticercus larvae, and potentially affecting the entire liver. Early-stage patients may be asymptomatic, only being diagnosed during routine liver ultrasound screening. As the disease progresses, patients may experience symptoms such as upper abdominal pain, a mass, fatigue, and weight loss. Diagnosis relies mainly on imaging examinations such as liver ultrasound and liver CT scans, as well as immunological tests such as the intradermal echinococcosis test and serum enzyme-linked immunosorbent assay (ELISA), thus exhibiting a time lag.

[0004] In addition, medical conditions in nomadic areas are relatively limited, and there is a lack of means for early screening. Therefore, providing a low-cost, simple-to-operate diagnostic method for alveolar echinococcosis that can be used for early screening is an urgent problem to be solved. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a biomarker for the early diagnosis of alveolar echinococcosis and its application. It analyzes volatile organic compounds (VOCs) in human exhaled breath using gas chromatography-mass spectrometry (GC-MS) to screen for effective biomarkers. Furthermore, it provides a non-invasive, rapid, easy-to-operate, and relatively inexpensive detection system for the early diagnosis of alveolar echinococcosis.

[0006] The first objective of this invention is to provide a biomarker of exhaled VOCs for the early diagnosis of alveolar echinococcosis, comprising one or more of the following exhaled VOCs: myristic acid, p-methylbenzaldehyde, and 2-(hydroxymethyl)-benzoic acid.

[0007] A second objective of this invention is to provide a detection system for the early diagnosis of alveolar echinococcosis, the detection system comprising:

[0008] An exhaled breath collection device for collecting volatile organic compounds from the exhaled breath of a subject; and,

[0009] An exhaled gas separation device for separating and extracting volatile organic compounds from exhaled gas; and,

[0010] Exhaled breath detection device, used to detect the composition and content of markers in volatile organic compounds;

[0011] The biomarkers are one or more combinations of the following exhaled VOCs: myristic acid (biomarker A), p-methylbenzaldehyde (biomarker B), and 2-(hydroxymethyl)-benzoic acid (biomarker C).

[0012] In one embodiment of the present invention, the detection thresholds are: myristic acid threshold of 1 ppbv, and / or p-methylbenzaldehyde threshold of 1 ppbv, and / or 2-(hydroxymethyl)-benzoic acid threshold of 0.5 ppbv.

[0013] In this invention, once the group of biomarkers reaches a certain concentration (i.e., a threshold), it can be preliminarily determined that the individual has a risk of echinococcosis infection.

[0014] In one embodiment of the present invention, the exhaled gas collection device includes a gas collection bag, a mouthpiece disposed on the gas collection bag, and a sampling cap fitted onto the mouthpiece.

[0015] In one embodiment of the present invention, the gas separation device is a solid-phase microextraction device.

[0016] In one embodiment of the present invention, the solid-phase microextraction device includes a solid-phase microextraction handle and an automated extraction head coated with polydimethylsiloxane.

[0017] In one embodiment of the present invention, the exhaled gas detection device is a gas chromatography-mass spectrometry (GC-MS) instrument.

[0018] In one embodiment of the present invention, the detection parameters of the gas chromatography-mass spectrometry (GC-MS) instrument are set as follows: carrier gas is He, flow rate is 1 ml / min, injection temperature is 250°C, and injection time is 2 minutes; column oven temperature is increased from 40°C to 250°C at a rate of 5°C / min; chromatographic-mass spectrometry interface temperature is 250°C, MS ion source temperature is 200°C, and scan range is 40-450 amu.

[0019] In one embodiment of the present invention, the chromatographic column used in the gas chromatography-mass spectrometry (GC-MS) instrument is a DB-1 gas chromatographic column.

[0020] A third object of the present invention is to provide the application of the exhaled VOCs markers for early diagnosis of alveolar echinococcosis in the preparation of products for early diagnosis of alveolar echinococcosis.

[0021] Beneficial effects

[0022] The present invention provides exhaled VOCs biomarkers for the early diagnosis of alveolar echinococcosis. Biomarker A has an AUC of 0.965, with sensitivity and specificity of 98.2% and 89.7%, respectively, and accuracy of 93.8%. Biomarker B has an AUC of 0.964, with sensitivity and specificity of 94.5% and 92.3%, respectively, and accuracy of 93.3%. Biomarker C has an AUC of 0.961, with sensitivity and specificity of 96.4% and 76.9%, respectively, and accuracy of 86.7%. All three gas levels are statistically significant and can be used as a reference for diagnosing echinococcosis (AE). The detection system of this invention is low-cost, simple to operate, and can be used for the early screening and diagnosis of alveolar echinococcosis. Attached Figure Description

[0023] Figure 1 Sampling device: A consists of a gas collection bag, nozzle, and sampling cap; B is valve open; C is valve closed.

[0024] Figure 2 This study compares the differences in the expression levels of tetradecanoic acid between the two groups.

[0025] Figure 3 This study compares the differences in expression levels of 4-methyl-Benzaldehyde (p-methylbenzaldehyde) between the two groups.

[0026] Figure 4 This study compares the differences in expression levels of 2-(hydroxymethyl)-Benzoic acid between the two groups.

[0027] Figure 5 ROC curve for the expression level of Tetradecanoic acid.

[0028] Figure 6 ROC curve for the expression level of 4-methyl-Benzaldehyde.

[0029] Figure 7 ROC curve for the expression level of 2-(hydroxymethyl)-Benzoic acid.

[0030] Figure 8The ROC curve for clinical validation of Tetradecanoic acid.

[0031] Figure 9 ROC curve for clinical validation of 4-methyl-Benzaldehyde.

[0032] Figure 10 The ROC curve for clinical validation of 2-(hydroxymethyl)-Benzoic acid. Detailed Implementation

[0033] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0034] Materials involved in the embodiments:

[0035] The Teflon FEP membrane gas collection bag was purchased from Dalian Haide Technology Co., Ltd.

[0036] Experimental methods:

[0037] (1) Exhaled breath sampling

[0038] Medical staff informed the subjects and distributed gas collection bags. Each subject received one Teflon EFP membrane gas collection bag, one mouthpiece, and one sampling cap. Figure 1 (A) Subjects were required to fast and abstain from smoking for 12 hours prior to sampling, and refrain from consuming foods with strong, pungent odors (such as garlic, stinky tofu, and leeks) for 24 hours prior to sampling. On the morning of sampling, subjects were required to brush their teeth and fast. Indoor air circulation was ensured, and subjects exhaled into the sampling bag.

[0039] The subject remained at rest and maintained normal breathing. A disposable Teflon mouthpiece was attached to the outlet of the sampling bag. The subject held the mouthpiece in their mouth and slowly opened the airtight valve of the sampling bag. This valve was a stopcock direct-flow valve. Figure 1 (Medium B). The subject breathes calmly and slowly blows air into the air bag. After collecting 100 mL of gas, quickly close the airtight valve and tighten the sampling cap. Figure 1 (C) Store in a sealed container, taking care not to damage the gas collection bag to prevent air leakage.

[0040] After the gas is collected from the subject, it should be stored in a cool, dry, and dark place until recovery. To ensure accuracy, the gas collection bag should be recovered on the afternoon of the same day.

[0041] (2) Solid-phase microextraction and GC-MS detection of exhaled VOCs

[0042] VOCs in the sample were separated and enriched using solid-phase microextraction (SPME). Based on the properties of the analytes, an automated extraction head coated with 100µm polydimethylsiloxane (PDMS) using a SPME handle from Supelco (USA) was employed. The needle was inserted into the gas collection bag through the sampling cap septum. The handle piston was pushed to place the fiber head in the gas bag, and extraction was performed at a constant temperature of 60°C for 1 hour. The fiber head was then retracted, the needle removed, and the sample transferred to a GC-MS for analysis. Qualitative analysis was performed using the NIST mass spectrometry database, and quantitative analysis was performed by calculating the peak area using the integral calculus method.

[0043] The gas chromatography-mass spectrometry (GC-MS) system was a Shimadzu GC-MS-QP2020, and the capillary column was an Agilent DB-1 gas chromatograph column (30m×0.25mm×0.25um). The instrument parameters were set as follows: carrier gas was He, flow rate was 1ml / min, injection temperature was 250℃, and injection time was 2 minutes; the column oven temperature was increased from 40℃ to 250℃ at a rate of 5℃ / min; the GC-MS interface temperature was 250℃; the MS ion source temperature was 200℃; and the scan range was 40-450 amu.

[0044] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0045] Example 1: Differential Analysis of Biomarker Levels in Exhaled Breath

[0046] Study subjects: 60 patients with acute exacerbations (AE) and 60 patients in the control group with chronic anemia (CAE) were selected.

[0047] (1) AE group: Patients from various parts of Qinghai Province who have been diagnosed with AE within the past year, regardless of gender or age, with a gender ratio of 1:1.

[0048] (2) CAE group: 60 patients without echinococcosis and no vesicular echinococcosis infection. They were matched for enrollment in a 1:1 ratio according to age and sex.

[0049] (3) Exclusion criteria:

[0050] ① Suffering from hepatitis A, hepatitis B, hepatitis C, hepatitis D, or hepatitis E;

[0051] ② History of alcoholism and drug allergies;

[0052] ③ Other causes of liver and kidney dysfunction or abnormalities;

[0053] ④ Cannot cooperate with the person collecting exhaled breath.

[0054] Exhaled VOCs were collected using the exhaled breath sampling method of this invention, and the collected gases were detected using exhaled VOCs solid-phase microextraction and GC-MS. Statistical analysis was performed using SPSS 23.0. The differences in VOCs peak area between the AE group and the CAE group were compared.

[0055] GC-MS results showed that each breath sample contained over 400 compounds. Statistically significant differences were found in the concentrations of several VOCs in the exhaled breath between the AE group and the control group (CAE group, non-echinococcosis). The concentrations of three biomarkers were increased (Tetradecanoic acid (biomarker A), 4-methyl-Benzaldehyde (biomarker B), and 2-(hydroxymethyl)-Benzoic acid (biomarker C)). GC-MS analysis revealed statistically significant differences in all three biomarkers compared to healthy individuals.

[0056] Among them, the differential analysis of Tetradecanoic acid levels is as follows: Figure 2 As shown, the average peak area of ​​the AE group is 3.5 × 10⁻⁶. 8 The average peak area of ​​the CAE group was 1.9 × 10⁻⁶. 7 The peak areas of the two groups were statistically significant after nonparametric testing (P<0.0001).

[0057] Differences in 4-methyl-Benzaldehyde (p-methylbenzaldehyde) levels, such as Figure 3 As shown, the average peak area of ​​the AE group is 1.01 × 10⁻⁶. 10 The average peak area of ​​the CAE group was 7.4 × 10⁻⁶. 8 The peak areas of the two groups were statistically significant after nonparametric testing (P<0.0001).

[0058] Differences in 2-(hydroxymethyl)-benzoic acid levels, such as... Figure 4 As shown, the average peak area of ​​the AE group is 3.2 × 10⁻⁶. 8 The average peak area of ​​the CAE group was 1.6 × 10⁻⁶. 7 The peak areas of the two groups were statistically significant after nonparametric testing (P<0.0001).

[0059] Example 2: Reliability of Biomarkers in the Diagnosis of Acute Respiratory Syndrome (AE)

[0060] Based on the above statistical analysis, the ROC curve AUC of biomarker A is 0.965, with sensitivity and specificity reaching 98.2% and 89.7%, respectively, and accuracy reaching 93.8%. (See attached data.) Figure 5 The ROC curve for biomarker B showed an AUC of 0.964, with sensitivity and specificity reaching 94.5% and 92.3%, respectively, and accuracy of 93.3%. (See attached data.) Figure 6 The ROC curve of biomarker C showed an AUC of 0.961, with sensitivity and specificity reaching 96.4% and 76.9%, respectively, and accuracy of 86.7%. (See attached data.) Figure 7 The levels of all three gases were statistically significant and can be used as a reference for diagnosing acute exacerbations (AEs).

[0061] Example 3: Clinical validation of the sensitivity, specificity and accuracy of biomarkers

[0062] Study subjects: In addition, 30 patients with acute exacerbations (AE) and 30 patients in the control group with CAE were selected.

[0063] (1) AE group: Patients diagnosed with AE within the past year from various parts of Qinghai Province, regardless of gender or age, with a gender ratio of 1:1.

[0064] (2) CAE group: 30 patients without echinococcosis and no vesicular echinococcosis infection. They were matched for enrollment in a 1:1 ratio according to age and sex.

[0065] (3) Exclusion criteria:

[0066] ① Suffering from hepatitis A, hepatitis B, hepatitis C, hepatitis D, or hepatitis E;

[0067] ② History of alcoholism and drug allergies;

[0068] ③ Other causes of liver and kidney dysfunction or abnormalities;

[0069] ④ Cannot cooperate with the person collecting exhaled breath.

[0070] The exhaled VOCs were collected using the exhaled VOCs collection method of the present invention, and the peak areas of the collected gaseous markers A, B, and C were detected by exhaled VOCs solid-phase microextraction and GC-MS detection methods. The threshold was determined by quantitative concentration detection using external standard method with standard samples, and statistical analysis was performed. The sensitivity, specificity, and accuracy were calculated by ROC curve.

[0071] Table 1. Recommended pathological reference concentration thresholds for biomarkers of the three VOCs:

[0072]

[0073]

[0074] The ROC curve for biomarker A showed an AUC of 0.950, with sensitivity and specificity reaching 96.7% and 90.0%, respectively, and accuracy of 93.4%. (See attached image.) Figure 8 The ROC curve of biomarker B showed an AUC of 0.976, with sensitivity and specificity reaching 97.0% and 88.0%, respectively, and accuracy of 92.5%. (See attached data.) Figure 9 The ROC curve of biomarker C showed an AUC of 0.975, with sensitivity and specificity reaching 93.1% and 95.0%, respectively, and accuracy of 94.0%. (See attached data.) Figure 10 All three gas levels are clinically significant.

[0075] Parallel testing of three biomarkers:

[0076] Joint sensitivity = [1 - (1 - 0.967) × (1 - 0.970) × (1 - 0.931)] × 100% ≈ 99.7%

[0077] Combined specificity = (0.900 × 0.880 × 0.950) × 100% ≈ 75.2%

[0078] Accuracy approximately 87.3%

[0079] Tandem testing of three biomarkers:

[0080] Joint sensitivity = (0.967 × 0.970 × 0.931) × 100% ≈ 88.5%

[0081] Combined specificity = [1 - (1 - 0.900) × (1 - 0.880) × (1 - 0.950)] × 100% ≈ 99.6%

[0082] Accuracy approximately 94.0%

[0083] It is evident that using a tandem approach for diagnosis of the three biomarkers yields higher accuracy and greater clinical diagnostic value.

[0084] It should be noted that the specific features, structures, materials, or characteristics described in this specification may be combined in any suitable manner in one or more embodiments. Furthermore, those skilled in the art can combine and integrate the different embodiments described in this specification and the features of those embodiments without contradiction.

Claims

1. The application of exhaled VOCs markers for early diagnosis of alveolar echinococcosis in the preparation of products for early diagnosis of alveolar echinococcosis, characterized in that, The markers are myristic acid, p-methylbenzaldehyde, and 2-(hydroxymethyl)-benzoic acid.

2. The application according to claim 1, characterized in that, The product is a detection system for the early diagnosis of alveolar echinococcosis, the detection system comprising: An exhaled breath collection device for collecting volatile organic compounds from the exhaled breath of a subject; and, An exhaled gas separation device for separating and extracting volatile organic compounds from exhaled gas; and, Exhaled gas detection device, used to detect the composition and content of markers in volatile organic compounds.

3. The application according to claim 2, characterized in that, The detection thresholds are: myristic acid threshold of 1 ppbv, and / or p-methylbenzaldehyde threshold of 1 ppbv, and / or 2-(hydroxymethyl)-benzoic acid threshold of 0.5 ppbv.

4. The application according to claim 2, characterized in that, The exhaled gas collection device includes a gas collection bag, a mouthpiece set on the gas collection bag, and a sampling cap that is attached to the mouthpiece.

5. The application according to claim 2, characterized in that, The gas separation device is a solid-phase microextraction device.

6. The application according to claim 5, characterized in that, The solid phase microextraction device includes a solid phase microextraction handle and an automated extraction head coated with polydimethylsiloxane.

7. The application according to claim 2, characterized in that, The exhaled gas detection device is a gas chromatography-mass spectrometry (GC-MS) instrument.

8. The application according to claim 7, characterized in that, The detection parameters for the gas chromatography-mass spectrometry (GC-MS) instrument were set as follows: carrier gas was He, flow rate was 0.8–1.2 ml / min, injection temperature was 240–260 °C, and injection time was 1–3 minutes; column oven temperature was increased from 35–45 °C to 240–260 °C at a rate of 4–6 °C / min; chromatographic-mass spectrometry interface temperature was 240–260 °C; MS ion source temperature was 190–210 °C; and the scan range was 40–450 amu.

9. The application according to claim 7, characterized in that, The chromatographic column used in the gas chromatography-mass spectrometry (GC-MS) system is a DB-1 gas chromatographic column.

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