CN1386863A - Fast diagnosis method for anaerobe infection - Google Patents
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- CN1386863A CN1386863A CN 02115903 CN02115903A CN1386863A CN 1386863 A CN1386863 A CN 1386863A CN 02115903 CN02115903 CN 02115903 CN 02115903 A CN02115903 A CN 02115903A CN 1386863 A CN1386863 A CN 1386863A
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Abstract
A fast diagnosis method for anaerobe infection includes collecting specimen, pretreating, solid-phase microextracting (SPME), gas-phase chromatographic analysis, and determining the existance of anaerobe according to the volatile short-chain fatty acid peak generated by anaerobe metabolism. Its advantages are short time (30-40 min) and high reliability.
Description
Technical field the present invention relates to a kind of diagnostic method of anaerobic infection, is in particular to use routine sampling, and without cultivation, the direct method of quick diagnosis.
The background technology anerobe be a class can only be at anaerobic or low oxygen partial pressure, suboxide reduction potential (Eh), contain certain C O
2The bacterium that could grow in the environment, it can only obtain energy from anaerobic glycolysis.The kind of anerobe is very complicated, and more than 30 genus, hundreds of arranged, and with the fast development of anaerobism cultivation, separation, authenticate technology, is also constantly finding novel species.Infecting the common anerobe of causing a disease clinically has 10 to belong to kind more than 30.
Anerobe extensively is present in human body skin deep and cavity deep mucous membrane surface as normal microflora.Mouth, pharyngeal cavity and enteron aisle are " the storage bacterium storehouses " of the anerobe of maximum in the human body, are that it is a kind of very common conditioned pathogen because the flora imbalance that a variety of causes causes, body's immunity decline or bacterial translocation cause infecting.The anerobe separation rate of clinical samples is reported as 51～78% abroad, and domestic is 30～67%.So, do not do the anerobe diagnosis almost with the pathogenic bacteria of omitting more than 50%.A lot of anerobes are insensitive to common antimicrobial agents (as sulfamido, aminoglycosides, Macrolide, quinolones and many cephalosporins medicines).So, do not do the anerobe diagnosis and will cause the state of an illness to be incured loss through delay and drug waste.
The diagnostic method of anaerobic infection includes following several at present---
1, bacteriodiagnosis
It is a classical method.Its diagnosis algorithm should comprise three grades---(1) clinical samples anaerobism cultivation+G of former generation
+The tentative diagnosis of dyeing anerobe; (2) separate anaerobism cultivation+G
+Dyeing+some biochemical test; (3) separate anaerobism cultivations+special biochemical test gentle-liquid chromatography is to thanking to product analysis its last reign of a dynasty at end.Clinical detection stays in one-level basically.In order to guarantee diagnostic reliability, following each link all should fully ensure:
(1) collection of sample: all samples that may be polluted by normal microflora, as the phlegm of expectoration, Bronchofiberscope sucking-off, Bronchofiberscope cytobrush secretory product, Bronchio-lung-douching fluid, effusive purulence, nose-mouth-pharynx-anus-vagina-sinus swab, midstream urine or urethral catheterization, the stomach-intestinal contents etc. got, all do not have censorship and be worth, promptly the routine clinical sampling sample more than 90% can not be used for carrying out the bacteriodiagnosis of anerobe.With clinical the most common lower respiratory tract anaerobic infection is example; the secretory product or the tracheotomy extraction thing that require sample to adopt the Bronchofiberscope cytobrush of lung puncture, thyrocricocentesis, band special protection cover to get are made sample; this not only brings misery, risk to the patient; but also need certain facility condition, carry out so can not extensively popularize.
(2) sample recommendation for admission to school: the not ingress of air of should trying one's best after the collection of specimens.May cause facultative anaerobe hypertrophy or sample drying because of in air, exposing, and influence the existence of anerobe, even cause death.Usually the employing method is: or the syringe needle rubber shutoff of syringe extraction back, in 20～30min, inspect by ready samples; Or the special sealing of injection of puncturing immediately, be full of pure N in advance
2The oxygen-free seal bottle in, tissue sample is inserted special anaerobism bag or jar is inspected by ready samples in 1～2h; Or bedside is inoculated immediately.This not only needs special device, and careless slightlyly promptly false negative will occur, is difficult to reach requirement clinically.
(3) anaerobism is cultivated: adopt suboxide reduction potential substratum (requiring fresh preparation) and strict oxygen-free environment (70%N
2+ 20%CO
2+ 10%H
2).What often adopt is " anaerobic jar ", " biological (anaerobism bag) bag ", " anaerobic room or anaerobism glove box ", promptly needs special device.Former being commissioned to train supported 2～3 days at least, and getting rid of diagnosis then needed for 2 weeks.Interval between diagnosis is long, easily affects the treatment of disease adversely.
From as can be known above-mentioned, all quite difficult for the diagnosis of most anaerobic infections from specimen sampling, sample recommendation for admission to school and anaerobism cultivation, so domestic most of hospitals all do not have to classify it as conventional sense.
2, immunologic test
Fluorescent antibody staining method, enzyme linked immunological group method etc. all have been successfully applied to the diagnosis of anaerobic infection, and itself and bacteriodiagnosis coincidence rate are very high.But their high-specificities, different anerobes requires different diagnosis boxes, and the section of anerobe, genus, kind are so many, bring certain difficulty to preparation, so value for clinical application is little.
3, the diagnosis of molecular biology of anerobe
Dna probe, gene amplification (PCR) technology also are used for the anerobe diagnosis research.Identical with the immunology detection reason, its value for clinical application is little.
4, the special meta-bolites of anerobe detects
The anerobe metabolism produces multiple volatilization or non-volatility short chain fatty acid, as acetate, propionic acid, butyric acid, isopropylformic acid, valeric acid, isovaleric acid, caproic acid, lactic acid, succsinic acid etc.The anerobe that does not belong to together, plants produces acid spectrum difference, is mainly used in the anaerobism Pseudomonas in the past, plants and identify.The minority aerophil also can produce acetate, propionic acid and lactic acid, but does not produce other short chain fatty acids, particularly butyric acid, isopropylformic acid, valeric acid, isovaleric acid, succsinic acid.Above-mentioned short chain fatty acid can be directly or the laggard promoting the circulation of qi phase of derivatize chromatogram (GC) analyzing and diagnosing.Adopt gas chromatography analysis method: to after measuring big sample (as chest-ascites, bile duct drainage liquid etc.) and directly adopting organic solvent (as ether, chloroform) to extract repeatedly and concentrate, or the further laggard promoting the circulation of qi analysis of hplc of derivatization treatment (as esterification); To measure little sample (as a small amount of fester of Bronchio-pulmonary secretions, extraction etc.) in advance short distance (1～3 day) anaerobism cultivate, after then substratum being adopted organic solvent to extract, concentrate also further derivatization treatment (as esterification) repeatedly, carry out gas chromatographic analysis again, if analyze the multiple short chain fatty acid of suitable concentration, particularly isopropylformic acid, valeric acid, isovaleric acid, succsinic acid can judge that it is an anaerobic infection.Adopt organic solvent to extract repeatedly, concentrate and carry out derivatization treatment owing to adopt before this method (1) sample to handle, thus still find sth. annoying or trying trivial, and because of having adopted a large amount of organic solvent extractions, concentratedly can having polluted environment; (2) adopt the quantitative Diagnosis standard, but organic solvent extraction, concentration process Short-Chain Fatty Acids also will volatilize with solvent, from quantitative angle, its rate of recovery is undesirable, poor repeatability; (3) clinical most sample is in a small amount, still needs anaerobism cultivation in advance.In view of the foregoing, its clinical value is little, so really be not used for clinical.
Solid-phase microextraction (SPME) technology that grow up the nineties in 20th century is easy, quick more than the liquid-phase extraction method, selectivity is good, extraction efficiency is high, and pollution-free.The SPME+ chromatographic technique has been widely used in the monitoring of environment, food, customs's drugs and minority medicine, does not still have the clinical application report so far.
Summary of the invention the objective of the invention is at above-mentioned present situation, aiming to provide does not a kind ofly have any requirement to sample, uses routine sampling, without cultivation, can pass through the existence of the various features volatility short chain fatty acid of anerobe metabolism generation, rapid detection goes out the method for the quick diagnosis of anaerobic infection.
The implementation of purpose of the present invention is, the method for a kind of quick diagnosis of anaerobic infection, and its concrete steps are:
1) collection of specimens: with clinical normal conventional method collect specimen
2) pre-treatment of sample:
(1) liquid sample directly adds and saltouts after saturated sodium-chloride becomes saturated solution,
(2) thickness sample as phlegm, purulence class, adds acid as 2～10% hydrochloric acid+hypochlorous acid (1: 2～6) or enzyme concussion digestion several minutes, fully after the dissolving, adds saturated sodium-chloride again and saltouts, and enzyme comprises trypsinase, Chymotrypsin, stomach en-, bromeline,
(3) solid sample as tissue block, shreds earlier and grinds to form homogenate, add the concussion of acid or enzyme again and be digested to solution, after add saturated sodium-chloride and saltout, acid and enzyme as above,
3) solid-phase microextraction of sample (SPME): the extraction syringe needle adopts the syringe needle that polyethylene glycol/divinylbenzene, polyethylene glycol/resin anion(R.A) or polydimethylsiloxane/divinylbenzene compound coating is arranged, the extraction mode adopts air-tight bottle from 1 centimeter left and right sides of sampling liquid level headspace extraction method, extraction is more than 1 minute, or extract more than 5 minutes in the liquid
6) gas chromatographic analysis: chromatographic column---elastic quartz capillary tube chromatographic column or packed column, stationary phase are the reactant of carbowax-20M and 2-nitro terephthalic acid, are called for short FFAP,
Carrier gas---N
2,
Detector---hydrogen flame detector or thermal conductivity cell class detector,
Temperature of vaporization chamber---200～250 ℃,
Desorption time---second more than 5～10,
Column temperature---90～150 ℃ of following constant temperature, for desired separated respectively becomes swarming, or in 60～220 ℃ of branch second orders or multistage temperature programming,
Sensitivity---10
2～3, decay 1/1～8,
7) diagnosis: adopt etiologic diagnosis, to detect following one-tenth swarming positive with clear and definite, clear, is anaerobic infection
(1) clearly, clear one of detected in isopropylformic acid, valeric acid, the isovaleric acid peak person,
(2) clearly, clear one of detected in acetate, propionic acid and another the short chain fatty acid peak person,
(3) clear and definite, the clear short-chain fat peak person who detects two kinds of non-isopropylformic acids, valeric acid, isovaleric acid, acetate, propionic acid.
The solid-phase extraction device shown in Fig. 1 is adopted in extraction of the present invention, and 1 is extracting head among the figure, and 2 is handle.When adopting headspace extraction, be to improve positive rate, should ultrasonic concussion, heat or stir under carry out.
Owing to the anerobe density and the breeding-metabolic rate of normally living away from home with focus of infection have very big-difference, anerobe density and breeding-metabolic rate that mouth-pharyngeal cavity-skin surface is normally lived away from home are low, the normal short chain fatty acid amount that produces is well below focus of infection, the present invention selects the homemade of Domestic Gas Chromatograph SP-520 type or other models, the inlet gas chromatography, its sensitivity be not subjected to when being enough to various short chain fatty acid in measuring clinical various sample that normal microflora pollutes influence, so, the solid-phase microextraction of clinical infection sample+gas chromatographic analysis SPME+GC detects, needn't worried normally the live away from home pollution of flora, again because SPME+GC detects is anerobe meta-bolites but not viable bacteria, so employing the present invention, collection of specimens there is not particular requirement, the no special conditions of sample recommendation for admission to school requires and the time restriction, need not cultivate.
Adopt the inventive method to containing 10
-5Acetate, propionic acid, isopropylformic acid, butyric acid, isovaleric acid, valeric acid, caproic acid, isocaproic acid, the hybrid standard short chain fatty acid solution of enanthic acid detect, adopt the ultrasonic concussion of head space to extract 1 minute down, desorption 10 seconds, 60～200 ℃ of second order temperature programmings, heavy caliber elasticity quartz capillary FFAP chromatographic column, the detected result (see figure 2), A among the figure, P, IB, B, IV, V, C, IC, E peak are the peak of above-mentioned each short chain fatty acid, and visible the present invention can detect the volatility short chain fatty acid that the anerobe metabolism produces really.
To clinical common aerophil, streptococcus aureus, Staphylococcus albus, suis, Neisseria, Listeria, dysentery bacterium, Salmonellas, dust Xi Shi dysentery bacterium, citrobacter, Cray Bai Shi pneumobacillus, Bacillus proteus, enterobacteria, Alcaligenes, acinetobacter calcoaceticus, morganella morganii, bloodthirsty hemophilus influenza, pseudomonas aeruginosa, catarrhalis, actinomycetes, Nocardia bacteria etc. 19 belong to 47 kind of 52 strain with above-mentioned same method; Clinical common anerobe, the nutrient solution that has genera bacillus, general Salmonella, fusobacterium, Wei Rong Salmonella, peptostreptococcus, dyspepsiacoccus, excellent bacillus, clostridium, cilium bacterium, actinomycetes 10 to belong to 23 kinds reference culture detects.The result shows: in the aerophil nutrient solution only the nutrient solution of streptococcus aureus, intestinal bacteria, the false single luxuriant bacillus of verdigris, proteus vulgaris, Bacillus subtilus, actinomycetes, morgan's bacillus can detect the acetate and the propionic acid (see figure 4) of trace, all the aerophil nutrient solutions all do not detect other volatility short chain fatty acids except that acetate, propionic acid; And all clear in the nutrient solution of anerobes, clearly detected (see figure 3)s such as multiple volatility short chain fatty acid, particularly isopropylformic acid, butyric acid, isovaleric acid, valeric acid.Detected result and authority's " anerobe laboratory manual " be basically identical (seeing Table 1) (VPI).
The GC analytical results of the clinical common anerobe reference culture nutrient solution Short-Chain Fatty Acids of table 1
Annotate: A-acetate, P-propionic acid, IB-isopropylformic acid, B-butyric acid, IV-isovaleric acid, V-valeric acid, C-caproic acid, IC-isocaproic acid, E-enanthic acid; Lowercase is expressed as trace ingredients.
| Reference culture | Extracted with diethyl ether/GC | ?SPME/GC | ?VPI |
| The bacteroides fragilis bacteroides oralis produces black Fusobacterium bacteroides vulgatus aerogenesis Fusobacterium peptostreptococcus anaerobic cocci Wei Rong Shi coccus bacteroides thetaiotaomicron clostridium botulinum clostridium tetani | ????A、p、ib、iv ????A、p、iv ????A、p、iv ????p ????A、B ????A、P、iv ????a、B ????A、p、iv ????p、iv ????A、P、b ????A、p、b | ?A、p、ib、iv ?A、p、ib、iv ?A、p、B、IV ?A、p、IV ?A、B、IV ?A、P、IB、iv ?A、ib、B、 ?A、p、IV ?A、p、IV ?A、P、B、IV ?A、P、B | ?A、p、ib、iv ?A、p、iv ?A、p、IV ?A、p、IB、IV ?A、IV ?a、p、ib、b、iv、ic ?A、ib、b ?a、p、iv ?A、p、ib、iv ?A、ib、P、IV ?A、P、B |
Therefore aerophil does not have influence to the present invention.
Adopt the inventive method to clinical 220 routine lower respiratory infection inpatient (gerontal patient's 155 examples; Young and middle-aged patient's 65 examples) phlegm of gathering in morning detects, wherein 108 routine gerontal patients, 28 routine young and middle-aged patient's inspections have anaerobic infection, its anaerobic infection rate is respectively 69.68% and 43.08%, and is similar to reported literature, but its Diagnostic Time only needs 30～40 minutes.To wherein 80 routine patients' phlegm in morning, mouthwash carry out anaerobism cultivation, solvent extraction+chromatogram GC, gas-chromatography SPME/GC detection, positive rate 97.5% (see figure 5) that the positive rate 100% that result---phlegm is cultivated, mouthwash are cultivated synchronously; The solvent extraction of phlegm+GC positive rate 18.75%; The SPME/GC of phlegm detects positive rate 63.75%, and the detection positive rate of mouthwash is 0% (seeing Table 2).
Table 2 80 routine lower respiratory infection patients carry out phlegm and mouthwash anaerobic bacteria culture, extracted with diethyl ether/GC, SPME/GC detected result synchronously
| Detection method | Expectoration sample example number | Contain the water of coughing |
| Positive negative positive rate | Positive negative positive rate | |
| Anaerobism is cultivated | ???80??????0??????100％ | ???78?????2???97.5％ |
| Extracted with diethyl ether/GC | ???15??????65?????18.75％ | ???0??????80???0％ |
| SPME/GC | ???51??????29?????63.75％ | ???0??????80???0％ |
This shows that the phlegm anaerobism of common collection is cultivated and had very high false positive rate, so can not be used for the anerobe diagnosis; Direct solvent extraction+GC the positive rate of phlegm is too low, and this method can not be used for the anerobe diagnosis; The SPME/GC of phlegm detects the positive rate height, and the influence of the bacterium that is not subjected to normally to live away from home this shows diagnostic value height of the present invention.
Adopt the inventive method to the lower respiratory infection inpatient of clinical 20 examples with wide spectrum potent antibiotics result of treatment difference, the phlegm of gathering in morning is detected, 17 examples detect and are associated with the anaerobic infection (see figure 6), the patient adds with the state of an illness after the tinidazole in treatment significantly to be alleviated, the SPME/GC monitoring shows that the kind of the short chain fatty acid that detects and peak area reduce (little) with state of an illness alleviation, even the disappearance (see figure 7).This shows that not only the present invention diagnoses reliably, and can be used for state of illness monitoring, and the diagnosis and treatment of anaerobic infection disease are had very high value.
Adopt the inventive method that clinical other are suspected the sample of anaerobic infection, the fester, urine etc. that comprise the purulence of purulence, the fistula of infectious ascites, hydrothorax, bile duct drainage liquid, ecphyaditis perforation, purulence that abscess is extracted out, burn infection detect, and also all can successfully detect.
Adopt the present invention, collection of specimens does not have particular requirement, and the no special conditions of sample recommendation for admission to school requires and the time restriction, need not cultivate, and whether can detect fast patient soon has the anaerobic infection situation, and Diagnostic Time only needs 30～40 minutes.The present invention's influence of bacterium that is not subjected to normally to live away from home detects the positive rate height, and diagnosis is reliable.Not only can be used for the diagnosis of anaerobic infection, and can be used for state of illness monitoring.
Description of drawings
Fig. 1 solid-phase extraction device structural representation
Fig. 2 hybrid standard short chain fatty acid solution color atlas
Fig. 3 anerobe reference culture color atlas
Fig. 4 aerophil reference culture color atlas
The same anaerobic infection patient's of Fig. 5 mouthwash color atlas
The color atlas of the same anaerobic infection patient of Fig. 6 expectoration in morning
The color atlas of the morning expectoration of the same anaerobic infection patient of Fig. 7 after the anaerobe resistant treatment
Claims (2)
1, the method for a kind of quick diagnosis of anaerobic infection is characterized in that its concrete steps are:
1) collection of specimens: with clinical normal conventional method collect specimen
2) pre-treatment of sample:
(1) liquid sample directly adds and saltouts after saturated sodium-chloride becomes saturated solution,
(2) thickness sample as phlegm, purulence class, adds acid as 2～10% hydrochloric acid+hypochlorous acid (1: 2～6) or enzyme concussion digestion several minutes, fully after the dissolving, adds saturated sodium-chloride again and saltouts, and enzyme comprises trypsinase, Chymotrypsin, stomach en-, bromeline,
(3) solid sample as tissue block, shreds earlier and grinds to form homogenate, add the concussion of acid or enzyme again and be digested to solution, after add saturated sodium-chloride and saltout, acid and enzyme as above,
3) solid-phase microextraction of sample (SPME): the extraction syringe needle adopts the syringe needle that polyethylene glycol/divinylbenzene, polyethylene glycol/resin anion(R.A) or polydimethylsiloxane/divinylbenzene compound coating is arranged, the extraction mode adopts air-tight bottle from 1 centimeter left and right sides of sampling liquid level headspace extraction method, extraction is more than 1 minute, or extract more than 5 minutes in the liquid
4) gas chromatographic analysis: chromatographic column---elastic quartz capillary tube chromatographic column or packed column, stationary phase are the reactant of carbowax-20M and 2-nitro terephthalic acid, are called for short FFAP,
Carrier gas---N
2,
Detector---hydrogen flame detector or thermal conductivity cell class detector,
Temperature of vaporization chamber---200～250 ℃,
Desorption time---second more than 5～10,
Column temperature---90～150 ℃ of following constant temperature, for desired separated respectively becomes swarming, or in 60～220 ℃ of branch second orders or multistage temperature programming,
Sensitivity---10
2～3, decay 1/1～8,
5) diagnosis: adopt etiologic diagnosis, to detect following one-tenth swarming positive with clear and definite, clear, is anaerobic infection
(1) clearly, clear one of detected in isopropylformic acid, valeric acid, the isovaleric acid peak person,
(2) clearly, clear one of detected in acetate, propionic acid and another the short chain fatty acid peak person,
(3) clear and definite, the clear short-chain fat peak person who detects two kinds of non-isopropylformic acids, valeric acid, isovaleric acid, acetate, propionic acid.
2, the method for quick diagnosis according to claim 1, it is characterized in that headspace extraction should ultrasonic concussion, heat or stir under carry out.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1320355C (en) * | 2003-04-22 | 2007-06-06 | 江南大学 | Method for analyzing fragrancer matter in apple wine |
| CN104807933A (en) * | 2015-04-30 | 2015-07-29 | 天津中医药大学 | Method for detecting short-chain fatty acid in milk product |
| CN105277402A (en) * | 2014-07-14 | 2016-01-27 | 汪振宏 | Rapid liquefaction agent of sputamentum beneficial to isolated culture of bacteria |
-
2002
- 2002-05-30 CN CNB021159033A patent/CN1184327C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1320355C (en) * | 2003-04-22 | 2007-06-06 | 江南大学 | Method for analyzing fragrancer matter in apple wine |
| CN105277402A (en) * | 2014-07-14 | 2016-01-27 | 汪振宏 | Rapid liquefaction agent of sputamentum beneficial to isolated culture of bacteria |
| CN104807933A (en) * | 2015-04-30 | 2015-07-29 | 天津中医药大学 | Method for detecting short-chain fatty acid in milk product |
| CN104807933B (en) * | 2015-04-30 | 2016-05-25 | 天津中医药大学 | The detection method of dairy products Short-Chain Fatty Acids |
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