CN111077307A

CN111077307A - Novel method for rapidly detecting sepsis by using gram-negative bacterial infection

Info

Publication number: CN111077307A
Application number: CN201811328316.8A
Authority: CN
Inventors: 吴海苹; 吴尚毅; 周燕英
Original assignee: Xiamen Bioendo Technology Co ltd
Current assignee: Xiamen Bioendo Technology Co ltd
Priority date: 2018-10-21
Filing date: 2018-10-21
Publication date: 2020-04-28
Anticipated expiration: 2038-10-21
Also published as: CN113302494B; CN113302494A; CN111077307B; WO2020083261A1

Abstract

Sepsis is a life-threatening systemic infection that requires appropriate and rapid treatment based on rapid pathogen determination. Gram-negative bacteria (GNB) are the major pathogenic pathogens with endotoxin (LPS) as its characteristic surrogate biomarker. In this study, a new method was first explored, first increasing LPS by GNB culture, and then detecting LPS in limulus amebocyte lysate (TAL) by dynamic nephelometry (KT-TALA). The sample preparation procedure was optimized. The results show that at high GNB concentrations LPS can be detected 3 hours after incubation, 6.5-7.5 hours earlier, whereas BD BACTEC positive reports are 9.5-10.5 hours; under low GNB load, the BD BACTEC system takes 22-26 hours to detect GNB, but the KT-TALA system only takes 9 hours to detect the LPS of GNB, 13-17 hours in advance. The new method of the present invention for detecting GNB-infected LPS is much faster than the traditional BD BACTEC system, especially in the early stages of sepsis where the bacterial load is low, which will help to make appropriate treatment decisions in an earlier time.

Description

Novel method for rapidly detecting sepsis by using gram-negative bacterial infection

Technical Field

The present invention generally relates to culturing blood suspected of sepsis and dynamically sampling endotoxin Lipopolysaccharides (LPS) produced in the blood by gram-negative bacteria (GNB) using a dynamic turbidity TAL assay (KT-TALA) system for rapid detection of LPS.

Background

Sepsis is the most dangerous situation and requires immediate dedicated treatment, since an hourly delay can increase mortality by 5-10%, resulting in up to-30% mortality. The high incidence of sepsis is mainly associated with trauma, infection, major organ dysfunction and the end stages of many diseases, such as cancer, aging, etc. Rapid detection of systemic invading pathogens is critical to life saving.

Currently, blood culture is still the gold standard for sepsis diagnosis, using metabolites that accumulate to a certain level to trigger positive reporter genes with the BDBACTEC system: (http：//www.bd.com/en-us/offerings/capabilities/ microbiology-solutions/blood-culture/bd-bactec-fx-blood-culture-system). This takes about 16-24 hours for-50% of septic patients and > 24 hours for the rest of patients, which is too long for rapid treatment decision making. Furthermore, not all GNBs in blood can be detected by the BD BACTEC system. Thomas et al reported that 12 samples were found to be endotoxin positive in culture without the corresponding GNB. In 7 of these 12 positive endotoxin tests, laboratory or clinical interpretation of these positive tests can be provided. This set of data indicates that a new assay is highly desirable to compensate for the "blind spot" of the BD BACTEC system in GNB infection.

It is well known that TAL prepared from amebocyte of horseshoe crab (Limulus polyphemus) is the most sensitive reagent for detecting LPS of GNB. The mechanism of action of the dynamic turbidity TAL assay (KT-TALA) is that endotoxin activates enzyme compound C, then activates compound B of the TAL, and triggers the cascade of coagulases and forms a gel, which can be recorded kinetically with a turbidimetric reader. Such sensitive detection methods have been widely used for detecting endotoxin in various biological samples, such as blood, urine, ascites, pleural effusion, cerebrospinal fluid, bronchoalveolar lavage, air and water. Compared with the traditional GNB culture test, the LPS-TAL test is more sensitive and more reliable.

However, in many cases of sepsis, especially during the onset of sepsis, endotoxin levels in the blood are undetectable even with the most sensitive KT-TALA method. It was suggested that only when plasma LPS > 0.5EU/ml, it could be established that GNB-induced endotoxin occurs only in patients with septic shock or severe sepsis, which is late for effective treatment. There is an urgent need to develop more sensitive TAL-based methods for the detection of LPS.

Blood samples from GNB sepsis were cultured for short periods (several hours) at 37 ℃ in nutrient rich media, with rapid doubling of GNB every 20 minutes, producing/releasing LPS in large amounts that were easily detectable by the KT-TALA method and reported to be LPS + GNB + sepsis much earlier than BDBACTEC system blood cultures.

The development of new methods for sensitive LPS detection not only aids in the early diagnosis of GNB sepsis, but also in monitoring the efficacy of treatment, which may be welcomed by clinicians and researchers.

Disclosure of Invention

The object of the present invention is to provide a new method for developing sensitive LPS detection that is not only useful for early diagnosis of GNB sepsis, but also for monitoring the therapeutic effect.

In a first aspect, the present invention provides a novel method for detecting endotoxin produced by gram-negative bacteria (GNB) in blood of a septic patient, wherein the method comprises blood culture, dynamic sampling, dilution, heating, rotation, further dilution, and measurement using a dynamic turbidity TAL assay (KT-TALA) system and a limulus amebocyte lysate (TAL), the dynamic turbidity TAL assay (KT-TALA) system comprising:

(A) techniques and devices for blood culture, sampling, dilution, heating, rotation, further dilution and testing in optimal conditions;

(B) special limulus amebocyte lysate (TAL) reagents;

(C) reader for dynamic turbidity TAL assay (KT-TALA).

In another preferred embodiment, the subject is a human or an animal.

In another preferred embodiment, the test target is endotoxin Lipopolysaccharide (LPS) produced in blood by gram-negative bacteria (GNB).

In another preferred embodiment, the test agent is a deformed cell lysate comprising TAL.

In another preferred embodiment, the dynamic sampling is every 1-2 hours.

In another preferred example, the dynamic turbidity readings are used in the gelation process of LPS-targeted TAL.

In another preferred embodiment, the entire process for blood culture, sampling and testing is fully automated by the device.

In another preferred example, the method can be used in all fields related to LPS detection.

In a second aspect, the present invention provides a detection system comprising:

(b) special limulus amebocyte lysate (TAL) reagents;

(c) a reader (KT-TALA) for dynamic turbidity TAL assay; and

(d) limulus amoebocyte lysate (TAL).

In a third aspect, the present invention provides a use of the detection system according to the second aspect of the present invention for preparing a reagent or a kit for detecting endotoxin produced by Gram Negative Bacteria (GNB) in blood of a patient having sepsis.

In another preferred embodiment, the reagent or kit is also used for detecting endotoxin Lipopolysaccharide (LPS) produced by gram-negative bacteria (GNB) in blood of a patient with sepsis.

Drawings

Figure 1 shows the new steps of LPS detection in blood suspected of being infected with GNB.

Figure 2 shows the results compared to LPS standard curves to determine if LPS increases in GNB in suspected blood over time.

Detailed Description

After extensive and intensive research, the invention unexpectedly discovers a novel method for detecting LPS in GNB infected blood for the first time, and particularly, compared with a BD BACTEC system, the KT-TALA system adopted by the invention for detecting the LPS of the GNB can greatly shorten the detection time, namely the detection time of the LPS of the GNB only needs 9 hours, and the method has very high sensitivity. On this basis, the present inventors have completed the present invention.

The main advantages of the invention include:

(1) the method of the invention can greatly shorten the time for detecting the LPS of the GNB.

(2) The method of the invention has very high sensitivity.

(3) Compared with the BD BACTEC system, the KT-TALA system adopted by the invention can greatly shorten the detection time, namely the detection of LPS of GNB only needs 9 hours, and the detection of GNB sepsis is much earlier than that of the traditional BD BACTEC system.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

The materials and reagents used in the examples were all commercially available products unless otherwise specified.

Example 1 method for rapidly detecting LPS in cultured blood of suspected sepsis patient

A new step of LPS detection in blood suspected to suffer from GNB infection. Briefly, 3-5ml of blood was added to the culture flask. After 2 hours, 0.5ml samples were taken every hour for dynamic testing of LPS. The samples were diluted, heated, spun and diluted again before LPS quantification with the KT-TAL system.

EXAMPLE 2 reference Standard Generation

The lyophilized endotoxin standard stock solution was reconstituted with endotoxin-free water, diluted to final concentrations of 50, 10, 1, 0.1, 0.01 and 0EU/ml and subjected to three independent experiments on microplates. Into each holeAfter addition of 100. mu.l TAL reagent, the plate was placed in a dynamic culture reader (BioTek)^TMELx808IULALXH) and the reader immediately begins to measure endotoxin levels using a kinetic assay procedure. Gel formation was recorded every 30 seconds for 30-60 minutes at a wavelength of 630 nm. For this set of studies, the formula generated was log (Y) ═ a × log (X) + B, where Y ═ reaction time (onset time), a ═ Y intercept, X ═ endotoxin concentration, B ═ slope of the regression curve. In this study, A was-0.279, B was 5.95, and R was²(associated efficiency) was 0.994. If the standard curve is completed using the same batch of reagents and the same procedure, it can be stored in a reader as a reference for the following analysis. However, if the reagent or procedure changes, new criteria need to be created anew as a new reference. The results of the unknown samples were compared to the LPS standard curve to determine if GNB in the suspected blood increased over time and LPS increased with it.

Example 3 Effect of heating on LPS Release from GNB samples and assay specificity

Based on the following knowledge: (1) LPS has two forms, free LPS and LPS associated with intact cell walls (Jorgensen et al, 1973); and (2) heat not only promotes the release of LPS but also denatures/precipitates interfering substances and facilitates more sensitive quantification of LPS, using heat as an essential step in sample preparation. As shown in Table 1, a series of cultured E.coli samples were collected at 4, 5, 6, 7, 8 and 9 hours and compared to the detected LPS levels for pairs of samples prepared simultaneously, with or without heating. At 4 hours, the LPS of the unheated sample was 0.144EU/ml, while that of the heated sample was 6.689 EU/ml. Similarly, at hours 5, 6, 7, 8, the unheated sample rose relatively slowly from 0.326, 0.999, 4.237 to 10.706EU/ml, and at hours 9 > 14.125EU/ml, while at hour 5, the heating time reached 10.854, corresponding to the 8 th unheated sample. Later, at 6 hours, the heated sample reached > 14.125, corresponding to the unheated sample at 9 hours, indicating that the heating step can significantly increase LPS from an undetectable form to a detectable form and reduce assay interference factors, thereby significantly facilitating the detection of LPS in early GNB infections.

TABLE 1 Effect of heating on LPS released from GNB samples and assay specificity^#

^#The sample was diluted 1: 4 with LPS-free water, heated at 100 ℃ for 10 minutes, spun at 450g for 3 minutes, and then the supernatant was further diluted 1: 10 and subjected to LPS assay using KT-TAL system. The experiment was repeated three times with the same trend, i.e. in the GNB E.coli group, the LPS of the heated sample was much higher than that of the unheated sample: (^*P < 0.05), whereas in the gram-positive s.aureus group there was no difference in ET between the heated and unheated samples.

Example 4 Effect of centrifugation on ET recovery

Because the KT-TALA assay gels, whereas LPS reacts with TAL reagents, sample cleaning and transparency are required. However, after heating and denaturation, the blood sample becomes turbid and the turbid material has to be spun. Table 2 shows that the speed and time of centrifugation may affect the recovery of LPS. The turbid material was spun using a 1.5ml tube in an Allegra 21R centrifuge and it was found that LPS loss rate was 14.97% when spun at 10,000rpm for 3min and 8.75% at 3,000rpm for 3min, similar to 6.55% without spinning. This data indicates that LPS can co-precipitate during spinning. The high speed rotation pulls down more LPS than the low speed rotation. The optimum spin speed was 3,000rpm for 3 minutes. If the sample is clean and transparent, rotation may not be required. The speed and time of rotation is largely dependent on the transparency of the sample.

TABLE 2 Effect of centrifugation on LPS loss^＊

^*The negative medium from the same BD BACTEC bottle, cultured for 5 days, was divided into several tubes, each tube was added with standard 1EU/ml LPS at different ratesCentrifugation was performed, LPS was measured and LPS loss rate was calculated as (1-measured LPS/1.045). times.100%.

Example 5 Effect of sample dilution on LPS assay results

BD BACTEC culture flasks with 3-10ml whole blood contained a large number of components that could bind TAL and prevent its gelation with LPS, producing negative results even with large amounts of ET. While the thermal/rotational process can remove some interfering species with large molecular weights, optimized dilution prior to KT-TALA assays will help reduce interfering species with small molecular weights. To determine the optimized dilution factor, 4 samples (1 normal blood, designated M1; 3 patient blood, designated CN1, 2, 3) were cultured in BD BACTEC bottles, each divided into two tubes: one as background and the other with the addition of standard 1EU LPS. The paired samples were heated, spun at 3000rpm for 3 minutes and diluted at different ratios (1: 10, 1: 20, 1: 40 or 1: 100) prior to the KT-TALA assay. The results (Table 3) show that LPS was not detected (< 0.007EU/ml) in all 4 pairs of samples in the 1: 10 dilution group, indicating that high levels of interferents block LPS-TAL gelation. In the 1: 20 dilution group, 4 spiked solutions also showed very low levels of LPS (0.01-0.025 EU/ml). In the 1: 40 and 1: 100 dilution groups, all spiked samples showed LPS levels near 1EU with high recovery of 97.66% -118.99%. The data support that TAL reagents can only begin to react with LPS and form a gel after the interfering substance is diluted to a certain low level. With this new process, the optimal dilution factor is 1: 40 to 1: 100.

TABLE 3 optimization of sample dilution for spiked sample recovery test^#

#: 1EU/ml LPS was added to all tubes labelled with-S and the LPS detected should be-1, < 1EU/ml, indicating the presence of some strong factor inhibiting the assay system.

M1^*: media supplemented with 3ml of blood was used as background control.

M1-S^*: to M1^*To this was added standard LPS (1EU/ml) to perform LPS recovery (%) at different dilutions.

CN1^*，CN2^*，CN3^*: clinical blood culture negative samples from BD flasks without LPS addition were used as background controls.

CN1-S^*，CN2-S^*，CN3-S^*: at different dilutions to CN1^*，CN2^*，CN3^*Standard LPS (1EU/ml) was added for calculation of LPS recovery (%) at different dilutions.

Example 6 comparison of GNB detection time between KT-TALA System and BD BACTEC System

The aim of this study was to combine blood culture with the KT-TALA assay to detect LPS of GNB much earlier than the traditional BDBACTEC system. To demonstrate this, 0.5McF of E.coli, Klebsiella pneumoniae (as a test) and Staphylococcus aureus (as a negative control) were used at 1: 10^-8、1∶10^-9、1∶10^-11、1∶10^-12The same number of bacteria were injected into two BD BACTEC bottles, one for the BD BACTEC detector reporting positive detection time and the other for a 37 ℃ bacteria shaking incubator (functioning similarly to the BD BACTEC detector), with LPS sampling being performed every hour, starting 3 hours after incubation, to determine the first time point at which LPS detection was positive. The results of the independent experiments (Table 4) show that in experiment 1, when the ratio is 1: 10^-8LPS positive reporting times for KT-TALA systems were 3 hours, 6.5 to 7.5 hours, at the time of GNB inoculation (starting from 0.5 McF). Earlier than the bacterial positive reporting time of the BD BACTEC system. This early detection of LPS and specificity for GNB was further confirmed in experiment 2, indicating that LPS reporting time was 6.5 to 8.5 hours earlier than the bacterial positive reporting time and that LPS was not detected in gram positive staphylococcus aureus medium. When the number of bacteria in the culture flask is smallThe advantage of LPS early reporting time is more obvious, and experiment 3 proves that GNB is 1: 10^-9，1∶10^-11，1∶10^-12At concentrations (starting from 0.5 McF), ET positive reporting times were 13h, 16h or 17h earlier than bacterial positive reporting times, respectively. This may mean that the BD BACTEC system takes 22-26 hours to detect GNB in the early stages of sepsis with low load bacterial infection, whereas the KT-TALA system only takes 9 hours to detect the LPS of GNB, which will help the physician to make appropriate treatment decisions at an earlier time.

TABLE 4 KT-TAL detects GNB in culture flasks faster than BD BACTEC system^*

^*Coli, Klebsiella pneumoniae (test) and Staphylococcus aureus (negative control) were adjusted to 0.5McF at 1: 10⁸，1∶10⁹，1∶10¹¹Or 1: 10¹²Further dilution series of ratios (v). Two equal amounts of bacteria were injected into two BD BACTEC bottles, one placed in the BD BACTEC detector for GNB positive detection reporting time and the other in the laboratory 37 ℃ bacterial shaker for LPS sampling per hour, beginning 3 hours after incubation. The KT-TAL assay was performed to detect LPS of GNB at different time points. The difference in time of positive detection between the two assays was determined by subtracting the two reporting times.

Example 7 the novel method for detecting GNB sepsis much earlier than the conventional BD BACTEC system

To demonstrate that our new method can detect sepsis with GNB infection earlier than the traditional BD BACTEC system, 10ml of blood from a patient suspected of sepsis was injected into two BD BACTEC bottles, 5ml each, one for the BD BACTEC system to report pathogens and the other for LPS assay as samples at different times after incubation. BD BACTEC seriesThe system reported positive for GNB pathogen at 28.5 hours, whereas the KT-TALA system detected positive for LPS at 12 hours post-culture, indicating that the novel method of the invention detected GNB 16.5 hours earlier than the conventional BD BACTEC system. The etiologic agent of the infection was identified as Acinetobacter baumannii (acinetobacter bau)mannii)。

TABLE 5 dynamic testing of LPS in culture samples from patients with Acinetobacter baumannii sepsis^*

^*10ml of blood from a patient suspected of sepsis were injected into 2 BD BACTEC bottles, 5ml each, one for the BD BACTEC system reporting pathogens and the other for the LPS assay sampling at the indicated time points. At 28.5 hours, the BDBACTEC system reported pathogen positivity, and LPS was detected 12 hours after culture, with a difference of 16.5 hours. The etiologic agent of the infection was identified as Acinetobacter baumannii (acinetobacter bau)mannii)。

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A novel method for detecting endotoxin produced by Gram Negative Bacteria (GNB) in blood of a septic patient, said method comprising culturing the blood, dynamically sampling, diluting, heating, spinning, further diluting and measuring with a dynamic turbidity reader and a limulus amoebocyte lysate (TAL), said dynamic turbidity TAL assay (KT-TALA) system comprising:

(B) special limulus amebocyte lysate (TAL) reagents;

(C) reader for dynamic turbidity TAL assay (KT-TALA).

2. The method of claim 1, wherein the subject is a human or an animal.

3. The method of claim 1, wherein the test target is endotoxin Lipopolysaccharide (LPS) produced in blood by gram-negative bacteria (GNB).

4. The method of claim 1, wherein the test agent is a amoebocyte lysate comprising a TAL.

5. The method of claim 1, wherein the dynamic sampling is every 1-2 hours.

6. The method of claim 1, wherein the dynamic turbidity readings are used in the gelation process of LPS-targeted TAL.

7. The method of claim 1, wherein the entire process for blood culture, sampling and testing is fully automated by the device.

8. The method of claim 1, which can be used in all areas related to LPS detection.

9. A detection system, comprising:

(b) special limulus amebocyte lysate (TAL) reagents;

(c) a reader (KT-TALA) for dynamic turbidity TAL assay; and

(d) limulus amoebocyte lysate (TAL).

10. Use of a test system according to claim 9 for the preparation of a reagent or kit for the detection of endotoxin produced by gram-negative bacteria (GNB) in the blood of a patient with sepsis.