Exhaled gas VOCs (Volatile Organic Compounds) marker for early diagnosis of alveolar echinococcosis and application thereof

Abstract

The invention discloses an exhaled gas VOCs (Volatile Organic Compounds) marker for early diagnosis of alveolar echinococcosis and application of the exhaled gas VOCs marker. The ROC curve AUC of the biomarker A myristic acid is equal to 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 the biomarker B on methyl benzaldehyde is equal to 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 the biomarker C2-(hydroxymethyl)-benzoic acid is equal to 0.961, the sensitivity and the specificity can reach 96.4% and 76.9% respectively, and the accuracy reaches 86.7%. The levels of the three gases have statistical significance and can be used as a reference basis for diagnosing the AE. The detection system disclosed by the invention is low in cost and simple to operate, and can be used for early screening of alveolar echinococcosis diagnosis.

Description

Technical Field

[0001] The invention relates to an exhaled gas VOCs marker for early diagnosis of alveolar echinococcosis and application thereof, belonging to the technical field of detection. Background Art

[0002] Alveolar Echinococcosis (AE), also known as multilocular echinococcosis or alveolar echinococcosis, is a chronic parasitic disease caused by the alveolar cysts of the multilocular tapeworm Echinococcus multilocularis (E. multilocularis) parasitizing the human body. The disease is widely distributed around the world, especially in high-latitude cold regions or frozen land areas in the northern hemisphere.

[0003] Humans are accidental hosts of alveolar echinococcosis, which is mainly infected by accidentally ingesting vegetables or raw water containing worm eggs or contacting infected animal fur. The primary lesions of alveolar echinococcosis are almost always located in the liver, but they can also spread to organs such as the lungs and brain through blood circulation and other pathways to produce secondary or metastatic lesions. Alveolar cysts diffusely infiltrate and spread in the liver parenchyma, directly destroying and replacing liver tissue, and can form giant alveolar cysts, which can even affect the entire liver. Patients may have no clinical symptoms in the early stages, and are only discovered during a B-type ultrasound survey of the liver. As the disease progresses, patients may experience symptoms such as upper abdominal pain, lumps, fatigue, and weight loss. Diagnosis mainly relies on imaging examinations such as liver B-ultrasound examinations and liver CT scans, as well as immunological examinations such as echinococcosis intradermal tests and serum enzyme-linked immunosorbent assays (ELISA), so it has a 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 that needs to be solved. Summary of the invention

[0005] To solve the above problems, the present invention provides an exhaled gas VOCs marker for early diagnosis of alveolar echinococcosis and its application. Volatile organic compounds (VOCs) in human exhaled breath are analyzed by gas chromatography-mass spectrometry (GC-MS) technology to screen out effective biomarkers, and further provides a non-invasive, rapid, easy-to-operate and relatively inexpensive detection system for early diagnosis of alveolar echinococcosis.

[0006] The first object of the present invention is to provide an exhaled breath VOCs marker for early diagnosis of alveolar echinococcosis, which is one or more combinations of the following exhaled breath VOCs: myristic acid, p-methylbenzaldehyde, and 2-(hydroxymethyl)-benzoic acid.

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

[0008] An exhaled gas collection device for collecting volatile organic compounds in the exhaled gas of a subject; and

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

[0010] Exhaled gas detection device, used to detect the composition and content of markers in organic volatiles;

[0011] Among them, the marker is one or more combinations of the following exhaled gas 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: the myristic acid threshold is 1 ppbv, and / or the p-methylbenzaldehyde threshold is 1 ppbv, and / or the 2-(hydroxymethyl)-benzoic acid threshold is 0.5 ppbv.

[0013] In the present invention, when the group of biomarkers reaches a certain concentration (ie, a threshold value), it can be preliminarily determined that the patient has a risk of alveolar echinococcosis infection.

[0014] In one embodiment of the present invention, the exhaled gas collection device includes an air collection bag, a mouthpiece arranged on the air collection bag, and a sampling cap connected to 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 comprises a solid phase microextraction handle and an automatic extraction head coated with polydimethylsiloxane.

[0017] In one embodiment of the present invention, the exhaled gas detection device is a gas chromatograph-mass spectrometer.

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

[0019] In one embodiment of the present invention, the chromatographic column used in the gas chromatography-mass spectrometer is a DB-1 gas chromatography column.

[0020] The third object of the present invention is to provide the use of the exhaled gas VOCs marker for early diagnosis of alveolar echinococcosis in the preparation of a product for early diagnosis of alveolar echinococcosis.

[0021] Beneficial Effects

[0022] The exhaled gas VOCs markers for early diagnosis of alveolar echinococcosis provided by the present invention have an ROC curve AUC of 0.965 for biomarker A, a sensitivity and specificity of 98.2% and 89.7%, respectively, and an accuracy of 93.8%; an ROC curve AUC of biomarker B of 0.964, a sensitivity and specificity of 94.5% and 92.3%, respectively, and an accuracy of 93.3%; an ROC curve AUC of biomarker C of 0.961, a sensitivity and specificity of 96.4% and 76.9%, respectively, and an accuracy of 86.7%. The three gas levels are statistically significant and can be used as a reference for diagnosing AE. The detection system of the present invention has low cost and simple operation, and can be used for the diagnosis of alveolar echinococcosis for early screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The sampling device: A is the air collecting bag, mouthpiece and sampling cap; B is the valve open; C is the valve closed.

[0024] Figure 2 This is a comparison of the difference in expression of Tetradecanoic acid (myristic acid) levels between the two groups.

[0025] Figure 3 This is a comparison of the difference in expression levels of 4-methyl-Benzaldehyde (p-methylbenzaldehyde) between the two groups.

[0026] Figure 4 This is a comparison of the difference in expression levels of 2-(hydroxymethyl)-Benzoic acid (2-(hydroxymethyl)-benzoic acid) between the two groups.

[0027] Figure 5 This is the ROC curve of Tetradecanoic acid expression.

[0028] Figure 6 This is the ROC curve of 4-methyl-Benzaldehyde expression.

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

[0030] Figure 8This is the ROC curve for the clinical validation of Tetradecanoic acid.

[0031] Fig. 9 This is the ROC curve for the clinical validation of 4-methyl-Benzaldehyde.

[0032] Fig.10 This is the ROC curve for the clinical validation of 2-(hydroxymethyl)-Benzoic acid. DETAILED DESCRIPTION

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

[0034] Materials involved in the examples:

[0035] Teflon FEP film air collection bag was purchased from Dalian Haide Technology Co., Ltd.

[0036] Experimental methods:

[0037] (1) Breath collection

[0038] Medical staff informed the subjects and distributed gas collection bags. Each subject received a Teflon EFP film gas collection bag, a mouthpiece, and a sampling cap ( Figure 1 In the A), the subjects fasted and stopped smoking for 12 hours before sampling, and did not eat foods with strong pungent smells (such as garlic, stinky tofu, leeks, etc.) within 24 hours. They brushed their teeth and fasted in the morning on the day of sampling. The subjects ensured indoor air circulation and exhaled gas into the sampling bag.

[0039] The subject remains at rest and maintains normal breathing. Connect a disposable Teflon mouthpiece to the air outlet of the sampling bag, hold the mouthpiece in the subject's mouth, and slowly open the airtight valve of the sampling bag, which is a plug-through valve ( Figure 1 The subject breathes calmly and blows air slowly into the air bag. After collecting 100 mL of gas, the airtight valve is quickly closed and the sampling cap is tightly closed ( Figure 1 C), store in a sealed container, making sure the air collecting bag is not damaged to prevent air leakage.

[0040] After collecting the gas, the subject should store it in a cool, dry, dark place until it is recovered. To ensure accuracy, the gas collection bag should be recovered in the afternoon of the same day.

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

[0042] The VOCs in the samples were separated and enriched by solid phase microextraction. According to the properties of the test substances, the solid phase microextraction (SPME) handle of Supelco and the automatic extraction head coated with 100um polydimethylsiloxane (PDMS) were used. 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 the extraction was carried out for 1 hour at a constant temperature of 60℃; the fiber head was retracted and the needle was pulled out, and the samples were transferred to GC-MS for analysis. Qualitative analysis was performed by searching the NIST mass spectrometry database, and the peak area was calculated by calculus for quantitative analysis.

[0043] The gas chromatograph-mass spectrometer was a Shimadzu GC-MS-QP2020, the capillary column was a DB-1 gas chromatograph column (30 m × 0.25 mm × 0.25 um) produced by Agilent, and the instrument parameters were set as follows: the carrier gas was He, the flow rate was 1 ml / min, the injection temperature was 250 °C, and the injection time was 2 min; the column box temperature was increased from 40 °C to 250 °C at a rate of 5 °C / min; the chromatographic and mass spectrometric interface temperature was 250 °C, the MS ion source temperature was 200 °C, and the scanning range was 40-450 amu.

[0044] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0045] Example 1: Differential analysis of biomarker levels in exhaled breath

[0046] Study subjects: A total of 60 AE patients and 60 control group CAE patients were selected.

[0047] (1) AE group: patients from all parts of Qinghai Province who were 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 infection and without alveolar echinococcosis infection, matched 1:1 by 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 allergy;

[0052] ③ Patients with liver and kidney dysfunction or abnormality caused by other reasons;

[0053] ④ Those who cannot cooperate with the exhaled gas collection.

[0054] The exhaled air collection method of the present invention was used to collect exhaled VOCs, and the exhaled VOCs solid phase microextraction and GC-MS detection methods were used to detect the collected gas. SPSS23.0 was used to complete the statistical analysis. The difference in the VOCs peak area between the AE group and the CAE group was compared.

[0055] GC-MS results showed that there were more than 400 compounds in each breath sample, and there were statistical differences in the concentrations of multiple VOCs in the exhaled VOCs of the AE group and the control group (CAE group, non-echinococcosis), and the concentrations of three biomarkers increased (Tetradecanoic acid (myristic acid, biomarker A), 4-methyl-Benzaldehyde (p-methylbenzaldehyde, biomarker B), 2-(hydroxymethyl)-Benzoic acid (2-(hydroxymethyl)-benzoic acid, biomarker C)). According to GC-MS analysis, it can be found that all three biomarkers are statistically different compared with healthy people.

[0056] Among them, the difference analysis of Tetradecanoic acid (myristic acid) levels is as follows Figure 2 As shown, the average peak area of ​​AE group was 3.5×10 8 The average peak area of ​​CAE group was 1.9×10 7 The peak areas of the two groups were statistically significant by non-parametric test (P<0.0001).

[0057] Differential analysis of 4-methyl-Benzaldehyde levels Figure 3 As shown, the average peak area of ​​AE group was 1.01×10 10 The average peak area of ​​CAE group was 7.4×10 8 The peak areas of the two groups were statistically significant by non-parametric test (P<0.0001).

[0058] Differential analysis of 2-(hydroxymethyl)-Benzoic acid levels Figure 4 As shown, the average peak area of ​​AE group was 3.2×10 8 The average peak area of ​​CAE group was 1.6×10 7 The peak areas of the two groups were statistically significant by non-parametric test (P<0.0001).

[0059] Example 2: Reliability of biomarkers in AE diagnosis

[0060] According to the above statistical analysis, the ROC curve AUC of biomarker A is 0.965, the sensitivity and specificity can reach 98.2% and 89.7% respectively, and the accuracy is 93.8%. Figure 5 ; The ROC curve of biomarker B has an AUC of 0.964, with a sensitivity and specificity of 94.5% and 92.3% respectively, and an accuracy of 93.3%. Figure 6 ; The ROC curve of biomarker C has an AUC of 0.961, with a sensitivity and specificity of 96.4% and 76.9% respectively, and an accuracy of 86.7%. Figure 7 The levels of the three gases were statistically significant and could be used as a reference for diagnosing AE.

[0061] Example 3: Clinical Validation of Biomarker Sensitivity, Specificity and Accuracy

[0062] Study subjects: In addition, 30 AE patients and 30 control group CAE group were selected.

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

[0064] (2) CAE group: 30 patients without echinococcosis infection and without alveolar echinococcosis infection, matched 1:1 by 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 allergy;

[0068] ③ Patients with liver and kidney dysfunction or abnormality caused by other reasons;

[0069] ④ Those who cannot cooperate with the exhaled gas collection.

[0070] The exhaled breath collection method of the present invention is used to collect exhaled VOCs, and the exhaled VOCs solid phase microextraction and GC-MS detection methods are used to detect the peak areas of the collected gas markers A, B, and C. Standard products are used to perform quantitative concentration detection through the external standard method to determine the threshold, and statistical analysis is performed. The sensitivity, specificity, and accuracy are calculated through the ROC curve.

[0071] Table 1 Pathological reference concentration thresholds of three VOCs markers:

[0072]

[0073]

[0074] The ROC curve of biomarker A has an AUC of 0.950, with a sensitivity and specificity of 96.7% and 90.0% respectively, and an accuracy of 93.4%. Figure 8 ; The ROC curve of biomarker B has an AUC of 0.976, with a sensitivity and specificity of 97.0% and 88.0% respectively, and an accuracy of 92.5%. Fig. 9 ; The ROC curve AUC of biomarker C was 0.975, the sensitivity and specificity could reach 93.1% and 95.0% respectively, and the accuracy was 94.0%. Fig.10 . Levels of all three gases were clinically significant.

[0075] Parallel testing of three biomarkers:

[0076] Combined 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] The accuracy is about 87.3%

[0079] Tandem testing of three biomarkers:

[0080] Combined 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] The accuracy is about 94.0%

[0083] It can be seen that the combined diagnosis method of three biomarkers in series has 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 one or more embodiments in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments and features of the different embodiments described in this specification without contradicting each other.

Claims

1. An exhaled gas VOCs marker for early diagnosis of alveolar echinococcosis, characterized in that: It is one or more combinations of the following exhaled breath VOCs: myristic acid, p-tolualdehyde, and 2-(hydroxymethyl)-benzoic acid.

2. A detection system for early diagnosis of alveolar echinococcosis, characterized in that: The detection system comprises: An exhaled gas collection device for collecting volatile organic compounds in the exhaled gas 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 organic volatiles; Among them, the marker is one or more combinations of the following exhaled gas VOCs: myristic acid, p-methylbenzaldehyde, and 2-(hydroxymethyl)-benzoic acid.

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

4. The detection system according to claim 2, characterized in that: The exhaled gas collection device comprises an air collection bag, a mouthpiece arranged on the air collection bag, and a sampling cap sleeved with the mouthpiece.

5. The detection system according to claim 2, characterized in that: The gas separation device is a solid phase microextraction device.

6. The detection system according to claim 5, characterized in that: The solid phase microextraction device comprises a solid phase microextraction handle and an automatic extraction head coated with polydimethylsiloxane.

7. The detection system according to claim 2, characterized in that: The exhaled gas detection device is a gas chromatograph-mass spectrometer.

8. The detection system according to claim 7, characterized in that: The detection parameters of the gas chromatography-mass spectrometer 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 box temperature was increased from 35-45°C to 240-260°C at a rate of 4-6°C / min; chromatographic and mass spectrometric interface temperature was 240-260°C, MS ion source temperature was 190-210°C, and scanning range was 40-450 amu.

9. The detection system according to claim 7, characterized in that: The chromatographic column used in the gas chromatography-mass spectrometry instrument is a DB-1 gas chromatography column.

10. Use of the exhaled gas VOCs marker for early diagnosis of alveolar echinococcosis according to claim 1 in the preparation of a product for early diagnosis of alveolar echinococcosis.

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