Sverdlovsk Regional Scientific and Practical Enterprise “Phthisiopulmonology”

REPORT

On Research work “Investigations of the medical “Biofon” device effect on microbacteria and evaluation of its efficiency

Scientific supervisor, M.D. G.G.Mordovskoy

Ekaterinburg, 1999

Content

Introduction

1. Investigation of “Biofon” apparatus effect on Mycobacteria.
2. Investigations of “Biofon” apparatus action on experimental animals.
3. Investigation of Infra-red radiation effect of the “Phtisio-Biofon” apparatus on patients with urogenital tuberculosis.
4. Conclusion
5. References

Essay

Report 39 pp., 13 tables, 8 references.

“Biofon” apparatus, Mycobacteria, Experimental Animals, Patients with urogenital tuberculosis, Medical and Biological Activity, Safety.

The objects studied were: “Biofon” apparatus for medical purposes, microorganisms of Mycobacterium kind, experimental animals, patients with urogenital diseases.

The aim of the work was to evaluate medical and biological efficiency and harmlessness of the medical “Biofon” apparatus in treating tuberculosis infection.

In the course of the investigation microbiological, biochemical, clinical, immunological and biological methods were used.

Effect of the “Biofon” on Mycobacteria, action of the apparatus on experimental animals and human body (a limited group of patients suffering from urogenital tuberculosis) were examined.

As a result of the studies carried out it was estimated that:

1. Irradiation by the “Biofon” apparatus causes a suppressive action on the growth of the virulent Mycobacteria of Tuberculosis (MBT), stimulating avirulent Mycobacterium growth in the experiments in vitro. A direct dependence of the inhibiting growth effect of Mycobacteria on the degree of virulence has been found.
2. Irradiation by the “Biofon” apparatus increases tuberculostatic effect of Isoniazid, Streptomycin, and Ethambutol on the sensitive MBT test-strain. Besides, the “Biofon” apparatus causes a certain effect on a degree of resistance of tuberculosis mycobacteria to antituberculosis drugs. The most essential changes have been noted in the resistance to Streptomycin (an increased degree), and to Rifampicin and Kanamycin (a dicreased degree of resistance).
3. The exposure of experimental animals (white mice and guinea pigs) to the “Biofon” apparatus does not cause animal death, changes in their behaviour or in the condition of their hair coverage, food and water refusal, weight loss and does not affect the characteristics of immunity cell link.
4. Application of the "Biofon” apparatus for experimental tuberculosis treatment makes it possible to decrease specifical lesion manifestations. Combined use of irradiation and Isoniazid treatment improves chemotherapeutical activity of the preparation.
5. Application of “PhthisioBiofon” irradiation for treatment of patients with urogenital tuberculosis (a limited group, volunteers only) does not greatly influence the hematological and immunological features of blood.

The biochemical tests made in patients with urogenital tuberculosis after exposure to IR radiation of the “PhthisioBiofon” apparatus have not revealed statistically significant deviations from normal indices characterizing liver functions.

For complete and objective evaluation of apparatus efficiency and safey additional investigations should be carried on, involving a wider group of patients with urogenital tuberculosis.

Introduction

Social significance of the problem connected with tuberculosis is quite evident and it has been brightly manifesting itsef under the economic crises in Russia for the last decade of the leaving century. Tuberculosis is one of the most common and dangerous chronic infections for men . A causative agent of TB is referred to the vast group of mycobacteria, which are widely spread everywhere in the world and can multiply in the animal and human body, as well as accumulate in the environment. At present, more than 100 kinds of mycobacteria are known, Myc. tuberculosis and Myc. bovis reffering to the most pathogenic species, causing tuberculosis in men and farm animals.

During the last five years a sharp aggraviation in the epidemiological situation connected with tuberculosis has been noted in the Russian Federation, which is confirmed by the growing rate of mortality and mordibity in the country. Tuberculosis is certain to be one of the important problems of home and international Health in the 21^st century, as the rapid spread of the infection occurs not only in Russia, but in other countries as well. (A.Y.Khomenko, 1997). On this background an increase in the number of immunodeficient conditions of different genesis has been noted among the newly revealed patients with tuberculosis in recent years. (V.I.Litvinov, 1997). A sharp rise of TB incidence practically in all countries is due to the increased rate of patient affection by mycobacterium strains with multiple drug resistance. (N.M.Rudoy, 1996; J.S.Blanchard, 1996).

Currenlty it is known that drug-resistance of TB mycobacteria develops to all existing antituberculosis preparations. Difficulties in the modern chemotherapy of TB are in many respects connected with the increased role of mycobacteria possessing natural or artifical resistance to the majority of the drugs used. In spite of the application of different combinations of tuberculostatic means, one does not always manage to prevent the development of drug resistance in the long-term treatment of TB patients, especially with the so widely spread destructive forms of the lesion. At the same time, as the number of antituberculosis drugs increases and their intake lasts for a long period of time, a problem of the secondary toxico-allergic effect of the drugs on the patient organism causes a deep concern. The risk of side-effect development grows in the current conditions when a great deal of preparations is used.

Thus, TB morbidity rate growth, as well as the appearance of polyresistant forms of mycobacteria make chemotherapy efficiency low. The resulting situation requires searching out new approaches to the TB infection treatment. In this connection, the Biofon” apparatus for medical purposes is of great interest as it is designed for TB treatment in combination with chemotherapy.

The aim of the present investigation was to evaluate medical and biological efficiency and safety of the “Biofon” device for TB infection treatment.

To achieve the above goal the following main tasks were set forth:

1. to study “Biofon” apparatus effect on mycobacteria in vitro;
2. to study “Biofon” apparatus action by experiments on animals;
3. to study “Biofon” apparatus effect on the human body (a limited patient group with urogenital tuberculosis).

During the work microbiological, biomedical, clinical, immunologic and biological methods have been used.

The methods and literature used are given in the appropriate parts of the report. Researches in this field were carried out according to the Contract No. 1364 of 19.01.99 (continued to the contracts No. 1364 of 13.04.99 and No. 1714 of 23.06.99).

1. Effect in the "Biofon" apparatus on Mycobacteria in vitro.

The aim of the work: to examine the effect of the low-frequency electromagnetic radiation of the "Biofon" apparatus on the growing properties and drug sensitivity of mycobacteria.

Materials and methods: Five (5) laboratory tester strains of mycobacteria, obtained from the State Research Institute for Standartization and Control of Medical Biological Preparations after L.A.Tarasevich (YISK) and 72 strains of MTB (mycobacteria of tuberculosis), isolated from the patients, being treated in the antituberculosis medical establishments of Sverdlovsky region were used in the research work. MTB strains of the human kind H37 Rv (virulent), H33 Ra (avirulent), of the bovin kind Bovinus 8 (virulent), of the paultry kind Avium (virulent), as well as a saprophytic strain Myc. Smegmatis were included into a group of tester strains. The second group comprised MBT, obtained from the patients, sensitive to antituberculosis drugs – 37 strains, and bacteria possessing resistance of various degree – 35 strains. To cultivate these strains of mycobacteria laboratory serieses of Levenstein-Yensen and "New" media, prepared in accordance with the recommended prescription and technology (Y.Y.Mordovsky, 1972; V.N.Basilyev, 1974), were used in parallel experiments.

To examine growth activity, mycobacterium suspension was prepared according to the turbidity standard GCI 10^-1, 10^-2, 10^-3, 10^-4, 10^-5, 10^-6, as well as in a higher concentration of 100 units. Then, 0.1 ml of prepared suspension was seeded on the surface of Levenstein-Yensen medium. Growth rate and microbiological mass yield were determined by counting the colonies seen by the naked eye on each of the media.

Drug-resistance studies were carried on using routine techniques (N.I.Kozulitsina et al., 1978).

1. Investigations of "Biofon" apparatus effect on mycobacteria growing properties.

The technique of the apparatus effect on TB Mycobacteria has been tried and optimized. H37 Rv, H37 Ra laboratory strains have been used for this purpose. The main investigations were carried out according to the pattern given in the "Instruction Manual". Additional studies were made using the same mycobacterium strains with 10, 20, 50, 100 – fold increase in the number of radiation sessions, and, also, with the variation of the radiation scheme.

Results

Suppresion of growth of H37 Rv, Bovinus 8 and Avinum virulent strains has been revealed in all serieses independently of the number of sessions and duration of the radiation. The greatest differences in mycobacteria, exposed to radiation and control (unexposed) ones were noted on the 12-15^th days and 28-34^th days. The results are represented in Tables 1 and 2.

On the average, the slow down of the growth rate of the exposed laboratory virulent strains amounted to 60-85% compared to the control group, and the microbial mass yield decreased by 35-47%

Investigations of the "Biofon" apparatus on the virulent MTB cultures, obtained from the patients (37 cultures) also resulted in prolonged periods of Mycobacterium growth by 75% and decrease of microbial mass yield by 15-70%

Table 1

Investigations of "PhthisioBiofon" apparatus radiation effect on the growth rate and MBT microbial yield depending on the microbial load.

Table 2

Effect of the "Biofon" apparatus on the mycobacterium growth properties.

1.2. Examination of "Biofon" apparatus effect on the mycobacterium drug sensitivity.

1.2.1. Examination of combined effect of IR-radiation and antituberculosis drugs on TB mycobacteria by the vertical diffusion technique has been conducted. H37 Rv laboratory strain has been used in the experiment. Mycobacterium suspension with the microbial mass load of 100 units and 19 units was seededon the medium. Then 0.3 ml of antituberculosis drug dilutions were added to the tubes: Isoniazid – 0.1-1 μg/ml, Streptomycin – 5-50 μg/ml; Rifampicin – 20-50 μg/ml; Kanomycin – 30-50 μg/ml; ethambutol – 2-5 μg/ml; pyrazinamide – 20-50 μg/ml. The tuberculostatic effect was determined by the mycobacterium growth low-down occurred. Data are given in Table 3.

Table 3

Tuberculostatic efficiency of antituberculosis drugs in combination with radiation

It has been determined that IR-radiation augments the tuberculostatic action of Isoniazine, Streptomycin and Ethambutol: zones of Mycobacterium growth inhibition became larger for Tubazide by 20.7%, Streptomycin by 21.1%, and Ethambutol by 20.3% compared to the unexposed control group.

1.2.2. Studies of "Biofon" IR-radiation effect on the degree of MBT resistance to antituberculosis frugs have also been carried out.

During the work 35 MBT cultures, obtained from the pulmonary tuberculoses patients sensitive to the main antituberculosis preparations have been used. According to the current criteria the cultures were considered to be resistant in case of their growth on the media, containing > 1 μg/ml of Tubazide, > 5 μg/ml of Streptomycin, > 20 μg/ml of Rifampicin, > 20 μg/ml of Kanamycin, > 2 μg/ml of Ethanbutol, > 2 μg/ml of Tibol. Of the number of cultures stadied 17 showed resistance to one (5 cultures), two (10 cultures) or three drugs (2 cultures). The rest were sensitive to all the mentioned preparations.

The degree of resistance to minimum doses of antituberculosis drugs has been studied. The concentrations used were as follows: Tubazide 0.1; 0.2 and 0.5 μg/ml; Streptomycin – 0.5; 1.0 μg/ml; Rifampicin – 2.0; 5.0; 10.0 μg/ml; Kanamycin – 2.0; 5.0; 1.0 μg/ml; Ethambutol – 0.2; 0.5; 2.0 μg/ml; Tibon – 0.2; 0.5; 1.0 μg/ml.

The degree of resistance was determined by the absolute concentration method on the dense "New" medium. MBT suspension was prepared according to turbidity standard of 10 units GCI diluted 1:10 and seeded by 0.1 ml into the tubes with the medium, containing different drug concentrations. Cultures were incubated in the horizontal position at the ºC for 12 days.

In the experiment incubated tubes were radiated by the "Biofon" apparatus according to the known scheme. Findings are given in Tables 4,5, and 6.

Table 4

Effect of IR-radiation on the degree of resistance of mycobacteria sensitive to drug microdoses.

Table 5

IR effect on the level of MBT resistance to antituberculosis preparations

Table 6

Effect of IR-radiation on MBT resistance degree

Results

Cultures sensitive to all antituberculosis drugs did not show resistance to tubazide microdoses in the majority of cases (16 cultures of 18). It was noted only in 2 cultures (to 0.5 μg/ml). Of 13 cultures resistant to other drugs, but sensitive to 1 and more mg/ml of Tubazide, the development of the initial resistance was observed in 11 cultures (mainly in the dose of 0.5 μg/ml). Both exposed and unexposed cultures had the same degree of resistance to small Tubazide concentrations, and only in one culture it increased from 0.2 to 0.5 μg/ml after radiation. In 4 cultures resistant to the higher Tubazide concentrations (> 1 μg/ml) differences in the degree of resistance were not revealed, as well as in exposed and unexposed cultures.

Half of the cultures resistant to the main antituberculosis drugs showed resistance to minimal concentrations of Streptomycin (9 of 18).

After the "Biofon" application in 3 of these cultures resistance to 0.5 μg/ml of the drug appeared, while unexposed cultures remained sensitive. In two other cultures raise of resistance degree (from 0.5 to 1 and from 0.5 to 2 μg/ml) was observed under the influence of IR-radiation. In one case radiation resulted in the decrease of resistance (from 1 to 0.5 μg/ml), while for 3 cultures it did not influence the level of the latter.

Of 17 resistant cultures 14 were resistant to > 5 μg/ml of Streptomycin, the resistance degree not changing in them under the action of radiation excluding 1 culture, in which the reduction in the degree was from 10 to 7.5 μg/ml.

Three cultures turned out to be resistant to other antituberculosis drugs, but sensitive to > 5 μg/ml of streptomycin, in one of them the resistance to 0.5 μg/ml of the preparation appeared after the apparatus application. In two cultures differences in the levels of initial resistance to Streptomycin among exposed and unexposed cultures were not revealed.

Of 18 sensitive cultures only three developed resistance to Rifampicin (to 2 mg/ml), two of them recovering sensitivity to the drug after IR application. Four cultures of 17 resistant ones were resistant to 20-50 μg/ml of Rifampicin and radiation did not influence the degree of their resistance. Of 13 cultures resistant to other antituberculosis drugs, but sensitive to > 20 mg/ml of Rifampicin, the decrease in the initial sensitivity to this drug in concentrations 2-10 μg/ml was noted in 3 cultures. After radiation the degree of resistance decreased in 2 cultures (from 10 to 5 μg/ml), and remained without changes in 1 (2 μg/ml).

One third of cultures (6) showed the decrease in sensitivity to Kanamycin in the concentrations of 2-5 μg/ml, application of the "Biofon" contributing to the appearance of initial resistance to 2 μg/ml in 1 case, while in the other case the radiation resulted in sensitivity recovery. Radiation did not cause any action upon the resistance of 4 cultures.

Only 3 of 7 resistant cultures were resistant to Kanamycin in concentrations of > 30 μg/ml, radiation did not change the level of resistance. Eleven cultures, resistant to other drugs, showed resistance to small concentrations of Kanamycin, resistance degree decreasing in 2 of them after IR-radiation, while recovering the resistance in 2. Only one culture was noted to have a reverse reaction, i.e. decrease in the resistance degree from 2 to 10 μg/ml.

The majority cultures (15 of 18) appeared to be resistant to small concentrations of Ethambutol. In 6 of them radiation by the "Biofon" resulted in futher decrease of sensitivity, and in two, on the contrary, in its decrease. The rest 7 cultures after IR-radiation did not change the degree of resistance to Ethambutol.

Six cultures of 17 resistant ones were resistant to high concentrations of Ethambutol, and only one of them showed an increase in resistance degree caused by radiation. Of 11 cultures resistant to other antituberculosis preparations, but sensitive to Ethambutol in concentrations > 2 μg/ml resistance to this drug (to 0.5-1 μg/ml) was revealed in 9. In 4 cultures decrease in resistance degree to Ethambutol was noted after radiation, and in one culture there was a rise.

All 35 cultures used in the experiment showed some degree of initial resistance to Tibon. Four of 18 resistant cultures reduced their sensitivity to this drug (in small concentrations 0.2-1 μg/ml) and 3, on the contrary, increased it.

Resistant cultures were also resistant to Tibon in low concentrations and only in 3 cultures the raise in resistance was noted after radiation, in the rest 17 cultures no changes were revealed.

Thus, IR-radiation of the "Biofon" caused a certain effect on degree of MBT resistance to antituberculosis drugs.

The most essential changes occurred in the resistance to Streptomycin – in 50% of cultures. Cultures with initial resistance to the drug increased their resistance on radiation exposure. In 66.7% of the cultures with initial resistance to Rifampicin and in 47.1% to Kanamycin, on the contrary, decrease in resistance level occurred after IR-radiation. In 56% cultures resistant to low concentrations of Ethambutol there were some changes in the resistance level after "Biofon" appplication, but both rises and decreases were in equal proportion. It is also worthy to mention that radiation did not practically influence MBT resistance to Tubazide and slightly changed this value to Tibon. IR-radiation did not cause any effect on MBT resistance to high concentrations of the drugs.

Conclusion

It is estimated that "Biofon" apparatus causes a suppressive action on the growth properties of MBT virulent strains, stimulating avirulent mycobacterium growth in the experiments in vitro. Direct relations of the inhibiting effect of mycobacterium development with virulence degree has been revealed, and this fact nay be used in laboratory practice for differential diagnostics of mycobacterium biological properties.

The investigations made have shown the increase in the number of radiation sessions to augment the "Biofon" effect on mycobacteria. The regimen of radiation with the interval of 12 hours and 10-fold increase in the number of sessions have been proved to be optimal.

"Biofon" apparatus application intensifies tuberculostatic action of Isoniazide, Streptomycin and Ethambutol on the MBT sensitive tester strain. Besides, the "Biofon" apparatus affects in a certain way the degree of MBT resistance to antituberculosis drugs. The most essential changes have been marked in the resistance to Streptomycin (resistance increase), and Rifampicin and Kanamycin as well (resistance decrease).

2. Studies of "Biofon" apparatus action on laboratory animals

Simulation of the experimental MBT infection pattern has been conducted on laboratory animals. To determine "Biofon" effect on the development and course of tuberculosis white mice and guinee pigs have been used. Application of white mice allows in comparatively brief terms to get preliminary valid findings. Guinee pigs due to their high susceptibility to tuberculosis are considered to be the most suitable for experiments on TB treatment (Y.N.Pershkin, 1971).

2.1. Determination of the "Biofon" apparatus effect on the course of experimental tuberculosis in white mice

In the experiment,120 mature males weighing 18-22 g have been used. During the experiment the animals were fed and maintained in standard conditions. To induce tuberculosis development mice were infected intravenously with the suspension of laboratory H37 Rv strain in the recommended dose of 0.1 mg. Tuberculosis development was tested according to the general condition of the animals (low-activity, difficult breathing, weight loss). Besides, in 14 days ephthonasia of several mice was made using animals from the control group (untreated animals) and their parenchymatous organs were subjected to microscopic and microbiological analysis. On autopsy multiple foci of TB inflammation in lungs and spleen were revealed. Bacterioscopic examinations showed acid-resistant mycobacteria in the smears. From parenchymatous organs tuberculosis mycobacteria were cultivated. Thus, simulated TB pattern was characterized by the marked and easy-to-determine pathological features corresponding to the standard tuberculosis models.

Work has been carried on to optimize doses and radiation mode. For this purpose radiation of white mice was made by the "Biofon" apparatus according to the standard scheme with 10- 30- 50- and 100-fold increase of the dose. 30-fold mode appeared to be optimal. The next stage of investigations comprised examination of the combined effect of radiation and antituberculosis drugs on the course of experimental tuberculosis.

In the experiment 6 groups of mice were used:

The 1^st group: healthy animals (control No. 1);

The 2^nd group: healthy animals exposed to "Phthisio-Biofon" radiation;

The 3^rd group: infected animals (control No. 1);

The 4^th group: infected animals radiated by the "Phthisio-Biofon";

The 5^th group: infected animals treated with Isoniazide;

The 6^th group: infected animals treated by a combination of Isoniazide and "Phthisio-Biofon" radiation.

Treatment and radiation started in 7 days after infecting and lasted for 30 days. Isoniazide was introduced once a day subcutaneuosly in the dose of 10 mg/kg of the animal weight. Radiation was carried on according to the established scheme. After finishing the experiment ephthanasia with an inhalation anesthesia was done. Animals condition has been followed up during the whole experiment: their behavior, food reception, condition of the fur and mucous membranes, changes in their weight (weekly).

Treatment efficiency was determined by the following parameters:

1. Difference in the mice weight at the beginning and the end of the experiment;
2. An average lung weight;
3. Presence of the specific lesions in the lungs. Index of lung damage was determined using 4 point system (from single vaguely marked foci – 1 point to multiple large nectoric foci – 4 points);
4. A number of mycobacteria in the smears of the lung tissue. This index was also characterized by points: from 1 mycobacterium in 100 visual fields – 1 point to more than 50 mycobacteria in one visual field – 4 points.
5. To count mycobacteria, thin smears from lung tissue 2x1.5 cm in size were prepared, dried in the air, fixed by the fire and stained according to Ziel-Nelson technique.

In order to determine the inoculability of MBT a half of the lung of each mouse was ground in a ceramic cup and poured by 5 ml of 5% sulfuric acid. The obtained mass was quickly transferred into the centrifugal test tube and centrifuged for 10 minutes with 3000 rev./min., then the sulfuric acid was removed, several drops of saline solution were added to the precipitate and then seeded into 2 Test-tubes containing "New" and Levenstein-Yensen media.

Results

In mice both healthy and infected, receiving treatment and radiation, weight gain at the end of the experiment has been noted, there being slight differences in this parameter between groups IV, V and VI. An exception was group III (infected untreated mice), where the average weight dropped by 2.5 g compared to the initial one, which was the sign of progressing TB infection.

Findings of microscopic and microscopic studies are presented in Table 7. From the data shown in the table it is seen that an average lung weight in animals received Isoniazide (Group V) and those undergone a combined treatment with radiation (Group VI) did not actually differ from the weight of healthy animals. In mice, received only radiation it was a little bit higher (292 mg), remaining considerably lower (by 1.4 times), than the weight of infected animal lungs in the control group.

Table 7

Efficiency of treatment for experimental TB bacilli with Isoniazide and "Phthisio-Biofon".

Table 8

Inoculability of MBT from white mice organs

Specific changes in the lungs occurred in mice from group III – multiple greyish large foci. In animals from groups V and VI foci were not revealed, in group IV single mycobacteria were found in the visual field in some mice.

Cultural method of investigation showed massive MBT growth in the cultures of lungs and spleen homogenates, obtained from the mice of control group in a month after the moment of seeding (Table 8). Percentage of the positive findings in the lung cultures of the animals, exposed to "Biofon" radiation was twice as low as in the control group, received Isoniazide treatment in combination with radiation and without it showed absence or low viability of mycobacteria.

2.2.Studies of combined effect of radiation and antituberculosis drugs on the development of experimental tuberculosis in guinea pigs.

In the experiment 15 mature animals weighing 270-300 g have been used. During the experiment the animals were fed and maintained in standard conditions. To induce tuberculosis development pigs were infected intravenously by the suspension of laboratory H37 Rv strain in the recommended dose of 1 mg.

Before the experiment Mantoux test was made to all the animals.

Three groups of guinea pigs participated in the experiment:

Group I – infected animals (control);

Group II – healthy animals, radiated by the "Phthisio-Biofon";

Group III – infected by Isoniazide and IR-radiation.

Treatment and radiation started on the next day after infecting the animals and lasted for 30 days. Isoniazide was given per os once a day in the dose of 10 mg per kg of animal weight. Pigs were radiated according to the previously accepted scheme. After the end of the experiment the animals were killed, their parenchymatous organs were subjected to macroscopic and microbiological examination. Phagocytic activity of leukocytes was determined by incubating culture cells with latex particles, and other known tests (V.V.Menshikov, 1987).

Results

Data of macroscopic and microbiological examinations are presented in Tables 9 and 10. In macroscopic examination of parenchymatous organs of untreated pigs (control group) in 30 days after being infected signs of TB infection were revealed. A great number of grey, clearly marked foci, and in some animals, large necrotic foci were found in the lungs. Liver and spleen were enlarged by 2.0-2.5 times and contained multiple TB foci.

Macroscopic picture of the animal internal organs received the treatment differed from the control group. Pigs, radiated by the "Biofon" were found to have some isolated small grey foci in the lungs, and multiple fine TB foci in the liver and spleen. In animals with a combination of drug and radiation TB changes were poorly marked.

Microscopic findings confirmed the data of macroscopic examinations. A number of mycobacteria in the smears obtained from the lungs of pigs from groups II and III was markedly low in comparison with the control group. AT the same time, cultures of parenchymatous organ homogenates obtained from the animals of the control group and animals treated only by radiation in 100% of cases gave positive results, meanwhile in those, treated by Isoniazide MBT, were not revealed.

Studies of phagocytic activity of neutrophils did not reveal any statistically valid deviations of indexes both before and after the beginning of radiation.

Conclusion

Radiation by the "Biofon" had no effect on the condition of the laboratory animals (their behavior, feeding, condition of the fur and mucous membranes and so on), as well as on the characteristics of immunity cell link.

Application of the "Biofon" apparatus for experimental tuberculosis treatment allows to decrease the degree of specific damage manifestations. Combined use of radiation and Isoniazide treatment improves chemotherapeutical potency of the drug.

Table 9

Efficiency of Treatment for Experimental TB Infection by Isoniazide and "Phthisio-Biofon" Apparatus

Table 10

Inoculability of MBT from guinea pig organs

3. Investigations of infra-red radiation effectof the "Phthisio-Biofon" apparatus on the patients with urogenital tuberculosis

Urogenital tuberculosis is characterized by a prolonged chronic course, special feature of which depends on the complexity of relations between tuberculosis, mycobacteria and patient's microorganism. Depending on the reactivity of the organism and agent virulency the course of the disease may be various. Besides, tuberculosis development and specific therapy almost always result in the changes in microorganism reactivity, which manifests itself in different disturbances of the patients immune status. Antibacterial therapy for this category of patients lasts for more than 12 months. It is often difficult to reach stable remission of the disease. Urogenital tuberculosis is usually revealed at the ages of 25-59, and is always accompanied by some unfavourable conditions, including chronic unspecified diseases of the urogenital system.

The aim of this section of the research work was to study the opportunity of "Phthisio-Biofon" application for treating patients with urogenital tuberculosis.

Fifteen patients (volunteers only) were selected for our work. Selection was made by clinicists after complete examination and on the basis of clinical and laboratory findings. All of them underwent a course of antibacterial therapy, and 5 of them were additionally radiated with IR according to the standard scheme, proved by experiments in vitro and in the course of treatment for experimental tuberculosis in laboratory animals. The basic scheme used was suggested by Chief of the Department of Phthisiatrics of the Medical Academy in Izhevsk, M.D. prof. E.E.Polushkina. We increased the number of simultaneous sessions of radiation to 10, and took into consideration time parameters of daily radiation multiplicity. Thus, the recommended scheme of the IR-radiation for patients with urogenital tuberculosis is as follows: the 1^st day – 4 times with 6hour-intervals; the 2^nd day – 3 times with 8-hour intervals; form the 3^rd day up to the 7^th day – twice a day with 12-hour interval; beginning with the 8^th day and till the 30^th day – once a day. The number of sessions in each radiation equals to 10.

In the process of our work we also studied the effect of Infra-red radiation on:

1. general clinical and immunological indexes;
2. biochemical parameters;
3. findings of bacteriological examintaion.

The results obtained are being analysed in the following section of this chapter.

3.1. Investigations of the IR-effect on the general clinical and immunological indexes of blood in the patients with urogenital tuberculosis.

Two groups of patients were investigated (volunteers). In all of them urine was tested on MTB before starting the treatment. All of them underwent a course of common antibacterial therapy. In the first group 10 patients with TB received additionally IR-radiation by the "Phthisio-Biofon" apparatus according to the above mentioned scheme. The rest of the patients composed group No. 2

Blood samples for tests were taken before radiation and every other 10 days. hematological parameters were determined: hemoglobin – by unified hemoglobincyanidic method of counting in Goryaev chamber; erythrocyte sedimentation rate by the unified Panchenkov's method, leukogram with staining according to Romanovsky. Immunogram was estimated according to the main indexes: relative and absolute number of T-lymphocytes and their subpopulations (theophylline-sensitive and resistant) by the method of spontaneous rosette-forming with ram erythrocyte; - relative and absolute number of B-lumphocytes by the method of rosette-fotming with mouse erythrocytes; - phagocytic activity of neutrophils in NST – test and by incubation with latexl – circulating immune complexes in blood serum by precipitaion with 3.5% solution of polyethylenglycol with the following colorimetry on the spectrophotometer. The obtained findings are shown in Tables 11 and 12.

Table 11

Hematologic indexes in patients with urogenital tuberculosis

Table 12

Immunological Indexes in Patients with Urogenital TB

Analysing obtained data one can make the conclusion that radiation by "Phthisio-Biofon" apparatus does not affect much hematologic and immunologic parameters of blood in patients with urogenital TB.

3.2 Investigations of biochemical shifts in the organisms of the patients with urogenital tuberculosis, undergone IR-radiation by the "Phthisio-Biofon" apparatus.

Biochemical parameters characterize the main physiological functions of the organism systems and are important characteristics of the safe radiation action on the organisms of the patients with urogenital tuberculosis.

Fifteen patients were examined. All of them receive standard antibacterial therapy. Ten of them underwent radiation according to the standard scheme and composed group No. 1. The rest were included into group No. 2. The following biochemical parameters, characterizing functional condition of the kidneys and liver are listed below:

1. determination of the general protein content in the blood serum using a unified method of biuret reaction (a set of chemicals of Rajchem Co.).
2. determination of globulin-timol-lipid complex by the unified timotic test in the veronal buffer;
3. determination of protein-fractions in blood serum by the unified method of AlAt and AsAt enzyme activity with the chemicals produced by Rajchem Co.
4. determination of bilirubin by the unified method according to Jendrassjk L., Cleghorn R. diazoreaction.
5. determination of the urea and kreatine by the unified methods

Findings are presented in Table 13.

Table 13

Biochemical parameters in patients with urogenital tuberculosis

As can be seen from the Table, biochemical studies made on the patients with urogenital tuberculosis after IR-radiation by the "Phthisio-Biofon" application did not reveal any statistically significant deviations from physiologic standard parameters, characterizing liver functions. Indexes of kidney function do not have statistically significant difference between the groups.

3.3. Bacteriological examination of patients with urogenital tuberculosis ( a limited group, volunteers) radiated by the "Phthisio-Biofon" apparatus under standard therapy conditions.

In the present conditions of urogenital tuberculosis course it is necessary to take into consideration the difficulty in diagnosing the disease, it's late recognition and as a consequence, unefficient therapy. One of the main links in making diagnosis is bacteriologic examination of urine for tuberculosis mycobacteria.

The main material for the bacteriological examinataion of the patients with urogenital tuberculosis was the whole portion of the morning urine, collected (after careful washing of the external genital organs) into sterile dishes. Jars with the urine were treated with 70% ethylene alcohol, settled for 3 hours, after which liquid above the precipitate was poured, equal quantity of 10% trisodium phosphate was added to the precipitate and jars were settled for 2 hours. After that the liquid above the deposit was poured out, the precipitate was put into a centrifugal test-tube and centrifuged (3000 rev/min) for 10-15 min., after which the liquid was poured out again, and 10% solution of trisodium phosphate was added to the precipitate in 1:1 proportion, it then was termostated at 37ºC for 18-24 hours. On the next day the test-tubes were removed from the thermostat and centrifuged in the same mode. When the above-precipitate liquid was removed once again, the precipitate was neutralized with 1% solution of citric acid in 1:1 proportion, and the remaining part put on the three media, the rest two drops were put on the slides for straining and microscopy with the luminescent "Luman-IZ" microscope. Urine inoculation was carried out before the beginning and at the end of radiation. Before radiation 7 of 10 patients discharged bacteria in the 1^st group, and 3 of 5 patients – in the second group. Since patients were radiated by "Phthisio-Biofon" concamitantly with the standard antibacterial therapy, while material collection for bacteriologic cultures is reasonable to make only on the 3^rd day after cancellation of drug intake, checking up the end of bacteriodischarging process was done only after finishing the whole course of therapy. Final results of bacteriological tests for MTB are not ready yet. It should be noted that during the experiment two patients from group No. 1 were complaining of insomnia condition, one was of still one more patient became worse due to chronic renal insufficiency. The remaining patients did not complain of anything not connected with their main disease.

Conclusion

As a result of the investigations made to examine "Biofon" apparatus effect on mycobacteria, as well as on experimental animal bodies and patients with urogenital tuberculosis, it was estimated that:

1. radiation by the "Biofon" apparatus causes a suppressive, inhibiting action on the growth activity of the virulent MBT strains, stimulating avirulent mycobacteruim growth in the experiments in vitro. Direct relation of the inhibiting bacterium growth effect to the virulence degree has been revealed.

Radiation by the "Biofon" apparatus increases tuberculostatic action of Isoniazide, Streptomycin and Ethambutol on the sensitive MBT tester strains. Besides, the apparatus influences the degree of MBT resistance to antituberculosis drugs. The most significant changes has been noted in the resistance to Streptomycin (increase in the resistance), and to Rifampicin and Kanamycin (decrease in the resistance).

2. The "Biofon" apparatus does not cause any harm or death to the laboratory animals (white mice and guinea pigs), or any changes in their behavior, condition of hair coverage and mucous membranes, refusal to eat and drink, weight loss or indexes of the immunity cell link.

"Biofon" application for experimental TB treatment allows to decrease the activity of specific damage manifestations. Combined use of radiation and Isoniazide therapy improves chemotherapeutic potency of the drug.

3. Biochemical studies have not revealed statistically significant deviations from physiological standards, characterizing liver functions in patients with urogenital tuberculosis after IR-radiation by the "Phthisio-Biofon" apparatus.

Thus, radiation of MBT by the "Biofon" apparatus results in the decrease of their growth activity. The apparatus is harmless for experimental animals and inhibits the progressing TB process, augmenting the action of antituberculosis preparations. Radiation does not cause any marked harmful effect on the patients with urogenital TB, though full and objective evaluation of the apparatus efficiency and safety requires additional investigations with a wider group of patients with this pathology.

Literature used

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