MODERN MEANS AND METHODS OF INDICATION OF BIOLOGICAL AGENTS IN THE ENVIRONMENT.

MODERN MEANS AND METHODS OF INDICATION OF BIOLOGICAL AGENTS IN THE ENVIRONMENT.

MODERN MEANS AND METHODS OF INDICATION OF BIOLOGICAL AGENTS IN THE ENVIRONMENT

Evgeny Nikolaevich KHRAMOV,
Doctor of Engineering Sciences, Professor, Deputy Director of the State Research Center «State Research Institute of Biological Instrumentation»

Lecture given on March 24, 2005 at the Moscow Institute of Physics and Technology
for students of the course Non-proliferation and Reduction of Weapons of Mass Destruction and National Security (http://armscontrol.ru/course/)

The problem of biomonitoring in the event of a terrorist threat determines the need to develop highly sensitive and specific (selective) methods for pathogen indication and to create, on the basis of these methods, advanced technical means suitable for organizing the necessary protective measures.

It is known that the reliability of population protection, all other things being equal, is directly dependent on the effectiveness of each of the links of anti-biological protection (ABP): biological control, emergency prevention (general and special), isolation and restrictive and other measures to eliminate the consequences of the use of bioagents, continuity, coherence of the actions of the units and parts of the NBC reconnaissance of the Ministry of Emergency Situations, the Ministry of Internal Affairs, the NBC protection troops, units of the medical service of the Main Military Medical Directorate of the Ministry of Defense of the Russian Federation, the Ministry of Health and others, implementing protection from biological objects.

The primary tasks facing biological control are to detect the fact of the use of a bioagent, to establish the type of pathogen used, the boundaries of infection, and the moment of application and removal of protective equipment.

In the current system of biological control, non-specific express detection is currently carried out to detect the fact of use by a number of signs (increased protein background levels, presence of enzymatic activity of the analyzed sample, etc.). At the same time, it is also assumed that samples will be taken to carry out the following task — establishing the type of pathogen used, which is assigned to the methods and means of specific indication.

In general, anti-biological protection includes:
  — biological control and assessment (forecasting) of the biological situation; use of individual and collective means of protection;
    — special treatment, including sanitization of personnel, disinfection of objects, terrain, roads, structures;
    — emergency (general and special) prevention of lesions and vaccination (revaccination);
    — isolation-restrictive and medical-evacuation measures;
    — maneuvering of mobile reserves.

Of the above, biological control is of decisive importance. The data obtained during control serve as the basis for assessing the biological situation, preparing proposals for protection against bioagents indicating the nature, volumes and timeframes for implementing measures in each specific case to eliminate the consequences of its use.

The use of individual and collective protective equipment, disinfection, general and special prevention, observation or quarantine are also carried out according to biological control data (non-specific biological indication and specific indication).

It is obvious that the sooner certain measures are put into effect, the more their effectiveness increases, including economic effectiveness.

On the other hand, the operational use of individual elements of the complex of measures for protection against bioagents should be carried out on the basis of reliable and complete information about the biological situation.

Hence, the essential feature of the PBZ process is that all subsequent activities are carried out based on the results of the analysis of the biological situation. At the same time, the biocontrol system, including NBI and SI, is the primary factor that implements the potential capabilities of the available means and methods of protection against bioagents.

When solving the problem of biological protection, it should be borne in mind that at present there remains a potential danger of the use of biological agents by some states, since there are no reliable mechanisms for monitoring compliance with the 1972 Convention and it is difficult to distinguish between permitted activities in the field of protection and research on improving biological objects. In addition, the problem of bioterrorism has recently become especially relevant. An analysis of the consequences of the use of biological agents, conducted by American specialists, shows that for a city with a population of 100 thousand people, the material damage can be from 470 million to 22 billion US dollars, depending on the type of biological agent used, the number of people affected with a fatal outcome can reach 35 thousand people. It should be especially emphasized that effective detection of biological agents and, accordingly, organization of protection is possible with the availability of information on possible methods and means of using biological agents, which in itself is the most difficult task.

It is believed that the most likely way of contamination of objects when using biological agents is the aerosol (aerogenic) route of spreading biological agents.

To ensure NBI and SI measures, the following sets of technical means should be included in the biocontrol system:
    — local and remote action NBI devices designed to establish the fact of use of biological agents;
    — kits (packages) for non-specific detection of biological agents in samples (this group of TS can also include devices for equipping laboratories);
    — means of sampling and sample delivery;
    — devices (automatic, semi-automatic and kits) for autonomous or laboratory determination of the species or group affiliation of BS in samples, including those allowing biological enrichment of the sample in order to provide a final answer on the identification of the pathogen.

To coordinate PBZ activities, there should also be communication channels for transmission to automated control systems, and the TS should have the necessary interfaces for transmitting and receiving signals. The TS should be located on the appropriate vehicles that ensure biocontrol, sample delivery, and analysis (RCHB reconnaissance vehicles, helicopters, medical and sanitary-epidemiological laboratories).

At present, the standard PBZ tools (the ASP alarm and the MLD microscope), which are the material basis of the existing biocontrol system, cannot sufficiently solve the tasks facing the PBZ. Based on this, the creation of new, more modern technical tools is an important, urgent task.

To solve the problems of biomonitoring, it is necessary to develop highly sensitive and specific (selective) methods for indicating pathogens, ecopathogens and to create modern technical means based on these methods.

At the same time, at present there are no regulatory and technical documents (RTD) regulating the requirements for the biological monitoring system and its components.

At the same time, in the late 90s, specialists from interested ministries and departments of the country, together with the State Research Institute of Biotechnology, developed on their own initiative a draft NTD “System of General Technical Requirements. Means of the System of Biological and Ecological Monitoring of the Environment.”

In accordance with this document, depending on the purpose and operating principle, means of the system of biological and ecological monitoring of the environment must include the following classification groups:
    — automatic biological aerosol alarms;
    — automatic analyzers of biological agents;
    — high-performance sampling devices for biological aerosols;
    — automatic (semi-automatic) devices for indicating biological objects;
    — equipment, devices, kits, sets and devices for detecting and identifying biological objects;
    — equipment for control, data collection and processing.

In the proposed system, automatic biological aerosol alarms must constantly monitor the contamination of the ground layer of air, ensuring the detection of biological aerosol in the cloud as it passes through the controlled point.

Automatic analyzers of biological agents in the periodic operation mode must ensure differentiation of biological agents in the aerosol state into the following conditionally taxonomic groups: viruses, rickettsia, bacteria (separately vegetative and spore forms), bacterial toxins.

High-performance devices for biological aerosols are designed to collect representative samples of biological agents from the air and store them under conditions that ensure maximum preservation of viability.

Automatic (semi-automatic) devices for indicating biological agents should provide express indication (establishment of the presence within conditional taxonomic groups) of biological agents and products of microbiological synthesis in native samples collected by high-performance sampling devices for biological aerosols.

Equipment, devices, kits, sets and devices for detection and identification of biological agents should ensure the establishment of the species (type) affiliation of pathogens of infectious diseases and bacterial toxins in native and enriched samples of air, soil, water and other environmental objects.

The primary task for the proposed system and its elements is to establish the fact of biological contamination.

To solve this problem, non-specific detection is carried out at the first stage, which also includes sampling for specific indication. Subsequently, specific indication is carried out at the second stage.

In our country, for the first time in the world, a technical means for detecting BS in an aerosol state was developed. The created ASP alarm is based on the chemiluminescent luminol method of analysis. The decision on the presence of a bioaerosol in the surface layer of the atmosphere is made by the ASP alarm based on a comparison of the electronic response threshold with the analytical signal recorded using a photomultiplier during the chemiluminescent luminol reaction catalyzed by aerosol particles with peroxidase activity. Exceeding the amplitude of the analytical signal over the electronic threshold actually serves as the basis for alerting about the use of BA.

Being the most widespread device for non-specific indication of biological objects, the ASP alarm has a number of the following significant drawbacks that reduce its effectiveness in detecting the fact of bioaerosol use:
    — limited range of detectable bioaerosols due to low level of peroxidase activity or its absence in a number of BA;
    — deterioration of the sensitivity threshold when detecting BA bioaerosol in a dusty atmosphere.

It should also be noted that for continuous operation of the alarm as intended during the day, four liters of indicator reagent from the indicator kit (KISA) included in the device are required.

Further improvement of these devices should be carried out in the direction of reducing their weight and size characteristics and energy consumption by using the latest technologies for detecting luminescence and light scattering of particles and a modern element base (photodetector, compact semiconductor laser systems for optical probing and microprocessor technology).

The alarms are equipped with sampling devices designed to collect a sample of aerosol of biological agents for subsequent analysis by methods of specific indication and implementation of biological protection measures.

For the ASP alarm, this device is made in the form of a built-in cyclone, air is supplied to which after detection of BA. The volumetric air flow through the cyclone is 160-220 l/min. The particles being collected have an average diameter of more than 2.5 μm.

Currently, a high-performance sampling device is being developed, the design of which includes a respirable aerosol fraction concentrator with a dispersion of 1 to 10 microns as a mandatory element. Its performance should be at least 1.5-2 m /min.

The main technical characteristics of existing and developed sampling devices are given in Table 1.

SAMPLING MEANS FOR SPECIFIC INDICATION

Along with the development of automatic alarms in the 80-90s, the State Research Institute of Biochemicals developed remote means of indicating aerosols of biological BS. The lidar ground complexes were based on the luminescent method of recording biological aerosol at a distance of up to several kilometers, which was successfully used to monitor emissions of BVK Kirishi and Svetlogorsk biochemical plants.

For non-specific detection of biological objects in samples, field portable kits are used, designed for analyzing samples for the purpose of their primary sorting. The main task when using the kits is to obtain a quick, within 10-15 minutes, preliminary answer about the presence and group affiliation of biological materials.

For this purpose, a set of colorimetric, enzymatic reactions and biochemical tests is used, characteristic of various groups of biological objects.

Using the above reactions and tests, the State Research Institute of Biotechnology developed the KSP-11 and KSAP sample analysis kits. The kits are designed for detection and group indication of microorganisms — pathogens of infectious diseases in field conditions.

The indicator papers, wetting solutions, reagents, solvents, instruments and materials included in the kits ensure the selection, preparation and analysis of unknown samples and microorganisms. The classification of the microorganism to be determined into taxonomic groups can be carried out using the corresponding set of positive and negative reactions of the indicator means included in the kits.

The KSAP kit ensures partial group indication of microorganisms (with the classification of the detected microorganisms into the viral-rickettsial and bacterial vegetative groups) in environmental objects in the temperature range from minus 20 to 30°C. The kit is serviced by one operator.

Currently, the KSAP kit is being modernized in the direction of expanding its functional capabilities for classifying the identified microorganisms in addition to the viral and bacterial spore taxonomic groups.

Currently, the following methods and means of microbiological express analysis are used to solve problems of specific indication: the fluorescent antibody method (FAM), the indirect hemagglutination reaction (IHR), and the solid-phase enzyme immunoassay (SIE).

The sensitivity of MFA and RNGA is 105.. 106 mt/ml.

The specific indication scheme also involves biological enrichment and research of MFA and RNGA methods, laboratory animals are also used, the research takes up to 2-3 days. If a negative answer is received, a full microbiological analysis is carried out. It can take up to 36 days or more. Such terms of analysis are no longer acceptable at present.

The basis for almost all methods used for specific indication are some modifications of immunological (immunochemical) methods. It was the insufficient efficiency of classical biological analysis that served as the driving force for the development of immunochemical and amplification (hybridization) methods of specific indication and diagnostics of infectious disease pathogens.

In particular, the State Research Institute of Biotechnology further developed methods of immunofluorescence analysis of biomaterials in the sample volume. One of them is the method of universal polyphase concentration of complexes of bioagents (viruses, microorganisms, toxins) with fluorescein isothiocyanate (FITC)-labeled antibodies. The main disadvantages of this method are low resistance to the effects of high concentrations of interfering impurities and a strong influence of the degree of purification of antibodies on the analysis results. These disadvantages were overcome by using solutions with different specific densities as a two-phase system. In this case, a luminescence peak is observed in the equilibrium density zone, caused by the formation of an immune complex of the bioagent with luminescent immunoglobulins. At the same time, high concentrations of impurities are distributed in one of the phases in accordance with their density and do not interfere with the determination.

Improvement of methods and technical means of express indication based on solid-phase luminescent immunoassay is aimed at searching for new luminescent labels and detection methods that provide a significant increase in the signal/background ratio. A high sensitivity level of 10 10 M (in terms of label concentration) was achieved by using labels with abnormally long luminescence and a special method for its isolation in the time-resolved mode with cutoff of the short-lived background component. This approach was implemented in lanthanide immunofluorescence analysis, the so-called ELISA method, or dissociation-enhanced lanthanide fluorescence immunoassay. Lanthanide chelates (Eu, Tb, Dy, Sm) covalently bound to antibodies were used as labels. This method has proven itself in the diagnostics of viral infections. At the same time, the IFA method turned out to be insufficiently effective for conducting highly sensitive multicomponent analysis of infectious agents. This is due to the decrease in the sensitivity of the method when using other rare earth labels with weaker luminescence instead of Eu.

Molecular genetic analysis has the highest sensitivity of all known methods of express indication. The use of traditional fluorescent labels for detection of bioagents using DNA probes, as a rule, allows to identify hundreds to thousands of cells in a sample. Amplification of genetic material by the polymerase chain reaction (PCR) of nucleic acids reduces this threshold to single cells. The development of PCR methodology is currently considered one of the most important areas for the creation of sensitive and specific methods for the indication and identification of pathogens that have no equal among laboratory diagnostic methods in terms of sensitivity and specificity of analysis. At the same time, as the experience of practical application of PCR in the KPBC-1U kit for express indication of pathogens created in 2001 has shown, significant limitations of the method are its duration and low noise immunity from contamination by reaction products. In this regard, one of the areas in improving the PCR methodology for specific indication is the development of methodological techniques and the creation of new-generation luminescent probes that can significantly reduce the number of amplification cycles by increasing the sensitivity of label detection. As in the case of solid-phase immunoassay, the most promising is the use of long-luminescent labels based on lanthanide or metalloporphyrin chelates. Nucleic acid amplification methods can increase the sensitivity of immunochemical methods for detecting toxins and antigens by creating hybrid antibody molecules labeled with oligonucleotides. Solid-phase analysis goes through two stages. At the first stage, a complex of the desired analyte and the associated molecules of hybrid antibodies is adsorbed on the solid phase; at the second stage, nucleic acid amplification and its detection are carried out using various, including luminescent, DNA probes.

At present, traditional methods of immunofluorescence analysis with the FITC antibody label still dominate in practical work. At the same time, the modern base of luminescence microscopy allows the use of several fluorochromes that differ in spectral-luminescence characteristics, which can provide much more reliable identification of pathogens. The functionality of immunoluminescence microscopy methods is expanded by using long-luminescent labels and recording the luminescence signal in the phosphorimetric mode or two-photon excitation with luminescence background cutoff.

Apparently, the combination of modern ultramicroanalysis technologies used in luminescence microscopy, such as laser excitation of DNA probe luminescence with registration in phosphorescence or two-photon excitation mode, with computer pattern recognition will eliminate amplification stages and control the presence of bioagents in the sample directly by the level of the luminescence signal. However, the complexity and high cost of the equipment required to carry out this type of analysis determine the possibility of its implementation only in large diagnostic centers.

One of the areas in the development of means for rapid indication of pathogens is the creation of luminescent immunosensors. In this area, methodological approaches using long-luminescent labels with registration in the time-resolution mode prevail. At the same time, an analysis of scientific and technical literature data shows that, unlike semiconductor electrochemical immunosensors, the creation of capillary and fiber-optic immunoluminescent sensors has not yet left the stage of scientific, methodological and technological development.

The presented results indicate the possibility of improving express-indication means based on a combination of modern methods of immunochemical and molecular-genetic analysis with the latest developments in the field of creating luminescent labels, technology for recording ultra-weak light fluxes in the photon counting mode and laser spectroscopy methods. The tasks of express-indication of biological agents, neither at present nor in the near future, apparently, can be solved using any universal method.

For studies that require rapid detection of pathogens and toxins, immunosensor technologies and technologies that implement the principles of homogeneous immunoassay are optimal.

For stationary laboratories, such characteristics of methods as sensitivity, specificity, productivity are of primary importance, while the analysis time factor is less important. In this situation, the methods of molecular hybridization and multicomponent solid-phase immunoassay with registration of biospecific binding products in the luminescence time-resolved mode, mutually complementing each other, will apparently dominate over the others.

As a promising simple means of indicating BA in the environment, the method of analysis on immunochromatographic test strips using colloidal gold or graphite as labels should certainly be recognized. Intensive research is being conducted in this direction at the institute, especially since the production and technological base for “dry” chemistry was created when solving the problem of diagnostic tools for patients with diabetes.

In conclusion, it should be noted that the complexity of the tasks and the integrated approach to bioindication problems require continuous improvement of the organizational structure, which in turn can be reformed based on real successes in the development of methods and technical means of indication. Taking into account the existing organizational structure and specific tasks of various departments, a number of key areas in the development of indication tools can be identified:
    — personal bio- and immunosensors;
    — field installations and kits for sample processing;
    — tools for equipping auto laboratories, including devices for fully automated sampling and analysis of biosamples;
    — means for continuous and periodic automatic monitoring of water and air;
    — devices and diagnostic kits for stationary laboratories.

It should be constantly remembered that despite the need to develop methods for diagnosing infectious diseases, where a person acts as a detector, a significant reduction in the consequences of the possible use of biological agents, including in bioterrorism, is possible only through the timely detection of these biological materials, which is achieved through the widespread use of technical indication means.