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Experimental reasoning and clinical efficiency of REKITSEN RD application in cases of children's salmonellosis

версия для печати

Academy "AGREED" N. V. Sadovnikova

Deputy chief of Kirov Territorial Department of Public Health,
Chief of a Department of Protection of Maternity and Childhood

Yu.V. Zolotaryov,
L.V. Zolotaryova, S.M. Krichmar

Kirov — 2000

The necessity of studying different aspects of salmonellosis and improving its therapy is determined by a high level of the infection case rate, its frequent appearance in serious forms, especially for children of early age, and formation of failures, as well as persistent and durable carrying of salmonellas (Учайкин В.Ф., 1999).

Nowadays treatment of salmonellosis with antibacterial drugs rather often follows to unsuccessful results and complications. Firstly, it осcurs because of the growing number of salmonellae strains refractory to widely used antibacterial preparations. Secondly, administering antimicrobic agents is frequently accompanied by allergic and other side effects, including negative influence on intestinal biocenosis.

The search of alternate effective therapeutic agents possessing a minimum of negative influences on a sick child is required. One of such ways is usage of intestinal absorbents, capable to bind pathogenic agents and their toxins, alongside with rendering diverse positive effects, including immunomodulating activity (Беляков Н.Я., 1991; Данилишин Н.И. And соавт., 1991; Андрейчин М.А. And соавт., 1994).

Modern investigators found out enough data indicating expediency of enteric absorbents in complex therapy of intestinal infections (Гайдук И.В., 1991; Воротынцева Н.В. et al., 1992, 1996; Рудакова P.Н. et al., 1992; Прохорчик A.A. et al., 1995; Горелов А.В. et al., 1997, 1999; Харченко Г.А., 1997; Ивахив О.Л., 1998; Павлова Л.A. et al., 2000). However the problem of administering enteric absorbents to the children suffering from salmonellosis is in a stage of accumulation of an actual material (Ивахив О.Л. el al., 1998; Колоколов В.А., 1998). Further search and development of new enteric absorbents, testing them in a complex treatment of the infection in children will result in selection of prepаrations, most effective and not possessing a side effect.

One of such perspective enteric absorbents is REKITSEN RD (registration number). Us efficiency was demonstrated on patients with gastric and duodenal ulcers, chronic colitises, atherosclerosis, but there is no information on REKITSEN RD usage in infantile and children's intestinal infections. Studying of REKITSEN RD effects in a complex treatment of salmonellosis was performed on a faculty of children's infectious diseases (Kirov State Medical Academy) in cooperation with Research Institute of Microbiology (Russian Federation Ministry of Defense).

The check of REKITSEN RD antitoxic and curative properties was carried out in two stages. On the first stage the absorptive capacity and antitoxic activity of the preparation was determined. On the second stage the analysis of REKITSEN RD efficiency in treatment of children with salmonellous infection was carried out using clinic and laboratory tests.

REKITSEN RD was produced by solid phase explantation of specially selected strain of wine yeast on wheaten bran. The content of inactivated cells was 1,0x108 - 1,2x109 per gram of the dry product. Its residual humidity was 5,0+/-1,0%.

Preclinic tests
For determining of REKITSEN RD adsorptive capacity and antitoxic activity we have carried out a series of in vitro bench-tests. The experiments were performed on 18-22 g mass alley white mice and on 75-100 g mass golden hamsters with no division on gender in both cases.

In accordance with the demands of Federal Standard 4453-74 "Coal activated clarifying powdery", REKITSEN RD adsorptive capacity was determined as 4,2 mg of a methylene blue stain per gram of a substance.

The analysis of REKITSEN RD toxin adsorptive activity was carried out in vitro. The experiments were purposefully made using the most active and strong toxins: a botulinic A type toxin, Schigella-derived toxin and Escherichia-derived thermolabile enterotoxine.

Botulinic A type toxin is one of the strongest ones known today. In experiments we used it as I,0xl08 ED/ml glycerine-based solution (1 ED corresponds LDso of a toxin introduced intraperitoneally to white mice).

Schigella-derived toxin (the Shiga toxin or neurotoxin) does not interfere with adenylatcyclase system and causes direct cytotoxic effect. We used glycerine based solutions of the toxin in concentration I,4xl06 ED/ml (1 ED corresponds LDso of a toxin introduced to golden hamster intraperitoneally).

Thermolabile enteric toxin stimulates hypersecretion of an intestinal liquid, containing Na+, K+ and Cl- ions, Sodium hydrocarbonate. In experiments we used glycerine solutions of the toxin in concentration 2,7xl04 ED/ml (1 ED corresponds ЛД5о defined on white mice in the test of edema of a back extremity).

For determination of thermolabile enteric toxin activity we introduced 0,05 ml of its appropriate dilution into one pes of the animal and the same volume of a normal saline solution -into the other pes. In 48 hours we killed animals, extremities were amputated on a level of a knee joint and weighed within 5 mg precision. If the average difference in weight between legs exceeded 65 mg we considered it as an evidence of thermolabile enteric toxin presence.
Samples of 1,0 g REKITSEN RD were brought into containers with 10,0 ml of type Л botulinic toxins, Shigella-derived toxin or thermolabile enteric toxin, diluted down to concentration (1200x1,4) ED in 1,0 ml. The samples were carefully mixed and incubated at 37,0±1,0°C during 45 minutes. After that centrifugation was executed at 4000 rev/min during 20 minutes. Supernatant was used for further studying of toxins content. The control samples were prepared similarly but 1,0 g of bran were added to toxin instead of REKITSEN RD. The results are represented in the table 1.

Table 1. The in vitro toxin absorbing ability of REKITSEN RD

Toxin studied
Concentration of a toxin, ED/ml
Amount of (he absorbed toxin, ED per gram of a sorbent
initial
after interaction with the
REKITSEN RD
bran
Botulinic
1386
562
1298
736,0
Schigella-derived
1275
614
1298
645,0
Thermolabile enteric
1458
539
1382
843


On the final step of stage 1 of the research we performed the analysis of toxin-neutralizing REKITSEN RD properties in vivo. In this series of experiments both botulinic and Schigella-derived toxins as well as REKITSEN RD were administered to white mice and golden hamsters orally. As it is impossible to test thermolabile enteric toxin this way, it was excluded from the series.

Beforehand the experiment laboratory animals were not feed within 12 hours, then they were introduced the substances mentioned above and given forage at once. The experimental group of animals received a mixture of grain and REKITSEN RD in equal proportion, the control group - only grain. The consumed REKITSEN RD dose was determined using residual of a forage within 24-hour intervals and was approximately 1,5-2,0 g per day for every golden hamster. We observed animals within 6 days, including monitoring in 2-hour intervals during first 2 days. The results of animals loss were basic in determination of LDso values for toxins in experimental and control groups. We also obtained for analysis STso index - average time before animals loss.

Where t is time interval;
i - amount of intervals;
n - number of animals lost in time t;
N - total of animals taken in the experiment.
The results are represented in the table 2.

Table 2. Toxin-neutralizing ability of REKITSEN RD in experiments in vivo

Dose of toxins,
Number of animals died after
ST5o (his)2) for animals after administering...
LDso (ED) 3) for animals after administering...
REKITSEN RD
grain
REKITSEN RD
grain
REKITSEN RD
grain
Botulinic toxin
3,15х103
0/6 1)
0/6
>144
>144
1,8х104
8,9х103
6,3х103
0/6
2/6
>144
72; 86
1,25х104
1/6
4/6
90
52+/-14
2,5х104
4/6
6/6
51+/-8
38+/-11
5,0х104
6/6
6/6
38+/-7
29+/-9
1,0х105
6/6
6/6
29+/-5
17+/-4
Schigella-derived toxin
1,2х103
0/5
0/5
>144
>144
5,7х103
2,1х103
2,4х103
0/5
3/5
>144
98+/-19
4,8х103
2/5
5/5
96; 138
74+/-16
9,6х103
4/5
5/5
92+/-18
68+/-11
1,9х104
5/5
5/5
76+/-11
49+/-8
3,8х104
5/5
5/5
59+/-8
36+/-9

The notes.

  1. Numerator - amount of died animals, denominator - number of animals taken in experiment;
  2. the values of mean time of life for animals with 95%-reliability; 3) the LDso was calculated according to the method of Kerber-Ashmarin.

Thus, the data represented in the tables 1 and 2, evidence that REKITSEN RD has adsorptive activity in cases of type A botulinic toxin, Schigella-derived toxin and Escherichia-derived thermolabile enteric toxin. If used immediately after peroral introduction of botulinic type A or Schigella-derived toxins, REKITSEN RD partially binds and neutralizes them. The latter is reflected in augmentation of a lethal dose of toxins and average time before loss of experimental animals.

Alongside with definition of REKITSEN RD absorptive activity for toxins, its possible properties as nutritious substratum for microorganisms were investigated. The ingredients for this series of experiments were prepared as follows.

REKITSEN RD. To avoid damaging action of distilled water on a bacterial cell we prepared slip of REKITSEN RD on a normal saline solution. The slip was then pasteurized by withstanding in weakly boiling water medium within 20 minutes.

The cultures of microorganisms were obtained from standard strains of L.A. Tarasevich Institute's Salmonella enteritidis (269), Salmonella typhi murium (47), Escherichia coli (ATCC 25922), supported by regular passage on appropriate optimal bolstering mediums and periodically tested for identity of biochemical and serologic properties.

For the experiment we used fresh (18-20-hrs) culture of microorganisms on a dense nutritious agar. Bacterial mass was taken out by a closed loop and introduced into a normal saline solution to reach an optical density of 1 billion microorganisms per ml according to McFarland turbidity standard.

The experimental technique was as follows. We mixed 100 ml of 2% agar-agar, cooled down to 50°C, with 5 g of REKITSEN RD, stirred and spilled the medium on Petri cups. The cups were then seeded with 0,1 ml of 1-billion suspension of S. typhimurium, S. enteritidis, E.coli and incubated in a thermostat at 37°C. The period of observation varied from 24 up to 96 hrs. 5 series of such experiments were carried out.

Salmonellae didn't show ability to propagate on any experimental crop of these microorganisms. In crops of Escherichia microorganisms the only sporadic colonial growth was registered. Therefore, REKITSEN RD itself is not a nutritious substratum for tested salmonellae strains.

Clinical investigations

The treatment of 147 children with salmonellosis, aged from 3 to 15 years, was complex and based on uniform principles taking into account age, gravity and period of the disease. The causal treatment included application of anti-salmonellous bacteriophage (in mild forms), chemotherapeutic drugs, antibiotics and their combinations (in forms of medium gravity and serious forms). The antibacterial preparations were administered in view of clinical efficiency and sensitivity of salmonellae circulating in the district and allocated from the patients. The average course of treatment ranged 5-7 days. The second course of antibacterial therapy was assigned if necessary.

Control group of sick children (82 persons) were treated according to the traditional schemes of therapy. In the main group (65 children) REKITSEN RD was used alongside with traditional therapy. The patients were included into groups by a random sample method and the groups were corresponding on gravity, age, etiological factor.

REKITSEN RD was applied as 10-20% water suspension both as monotherapy and in combinations with one antibacterial preparation. The daily doses of REKITSEN RD for children aged from 3 to 7 were 5-7 g, from 7 to 10 - 7-10 g, for aged elder 10 - 15 g. Daily dose was divided into 3-5 receptions and was administered in lime intervals different from other medicines to avoid sorption effects. The course of REKITSEN RD treatment did not exceed 7 days, and in cases of earlier normalization of a stools or its delay within a day the drug was cancelled.

Efficiency of treatment was estimated on expressiveness and duration of main clinical signs (termination of a vomiting and diarrhea, restoring of appetite and body mass, normalization of a temperature, disappearance of abdominal pains etc.).

The results show that application of REKITSEN RD in complex therapy of gastrointestinal form of salmonellous infection in children is highly effective just during (he first course of treatment (table 3). The fast retrogress of clinical signs in this group of the patients is apparently caused by binding and elimination of pathologic agents, toxins and products of a pathological metabolism by the examined enteric absorbent.

As it follows from the table data, in REKITSEN RD assignment group, when compared with control, an average duration of an intoxication has reliably decreased, the state of health improved and appetite restored faster as well as febrile period was reduced. On an average in the main group vomiting stopped earlier for 1 day, the expressiveness and duration of abdominal pains was less.

Table 3. The comparative characteristic of main clinical parameters in cases of a salmonellosis in children depending on therapy (Х+/-м)

Parameter, days Group 1 (REKITSEN RD) n=65 Group 2 (control) n=82
Р
Intoxication
2,7 +/- 0,4
3,9 +/- 0,3
<0,05
Fever
2,5 +/- 0,3
3,4 +/- 0,2
<0,001
Vomiting
1,8 +/- 0,1
2,4 +/- 0,1
<0,001
Abdominal pains
2,0 +/- 0,1
2,9 +/- 0,4
<0,05
Exicose I-III grades
2,2 +/- 0,2
2,7 +/- 0,1
<0,05
Complete normalization of stools
4,7 +/- 0,8
6,5 +/- 0,4
<0,05
Clinical efficiency of 1-st course of treatment
80,9 +/- 4,9%
65,6 +/- 5,9%
<0,05
Repeated carrying of the agent
17,6 +/- 4,7%
31,9 +/- 5,1%
<0,05
Average staying in bed
7,9 +/- 0,5
13,2 +/- 1,1
<0,001

In majority cases by the end of the second day from a beginning of REKITSEN RD course frequency and volume of stools decreased, that in turn, followed to improvement of water and electrolyte homeostasis and decreased exicose duration (P<0,05).

The complete normalization of stools in the group of patients adhibiting REKITSEN RD, occurred 1,4 times faster, than on the basis of traditional treatment (P<0,05). The normalization of stools within the first 3 days from a beginning of REKITSEN RD therapy took place in 68% of the patients and only for 51% of patients of control group. The further augmentation of stools frequency within first 3 days of treatment was marked in 31% of patients adhibiting only antibacterial preparations (and in 8,5% cases for 4-5 days of treatment alongside with it even impurity of a blood was registered), and only in 16% of patients in main group. On a background of treatment in control group the raise of fever was observed more frequently (in 17% cases against 6% in main group) as well as vomiting increase (in 14 and 10% cases accordingly).

The most considerable therapeutic effect of REKITSEN RD was observed for the patients with an injury of upper parts of a gastrointestinal tract. For example the average period of a diarrhea in cases of salmonellous gastroenteritis and enteritis was 4,1±0,4 days, in gastroenterocolitis and coloenteritis it achieved 5,2±0,2 days (P<0,05). The difference in the effect of REKITSEN RD is probably explained by fast saturation of these part of a gastrointestinal tract by particles of a preparation with the subsequent inaclivation of toxins, responsible for development of a watery diarrhea. Besides the dependence of clinical convalescence velocity was delected on time of the beginning of REKITSEN RD administration. In cases, when enteric absorbent was prescribed on the first day of disease, the parameter was 2 times higher in comparison with the cases, when the preparation was administered for the first time on 3-4 day from the moment of disease.

The medical effect of REKITSEN RD was insufficient only for the patients with serious forms of salmonellous infection. In such cases we combined REKITSEN RD with antibiotics selected according to sensitivity of microbal agent. REKITSEN RD decreased expressiveness and duration of toxicosis and exicosis and reduced duration of infusion therapy, decreased its volume and frequency of assignment. The average duration of infusion therapy for children with serious forms of salmonellosis on the background REKITSEN RD decreased from 2,8±0,3 days to 2,1 ±0,2 days (P<0,05).

In mild and medium grave forms of salmonellosis single course of oral rehydratation in combination with REKITSEN RD provided 83,6% clinical efficiency, as well as in control group (anti-salmonellous bacteriophage and classic antibacterial therapy) this parameter was only 68,6%.

Similar results were obtained for the patients with serious forms of salmonellosis. Single course of complex antibacterial therapy combined with REKITSEN RD has shown 78% clinical efficiency, while in control group it was 60,5%.

Courses of therapy including REKITSEN RD exceeded those in a control group in sanifying efficiency. The parameter of repeated carrying of pathogenic agent in main group was 1,8 times less, than in control (P<0,05) (table 3). Microbal carrying and insufficient efficiency of treatment for the observed patients, are apparently caused by intracellular pathogen location.

Thus, the analysis of clinical efficiency of REKITSEN RD applying in treatment of salmonellosis in children has shown its advantages when compared to traditional methods of a causal treatment. Undoubtedly important features of REKITSEN RD in new economic conditions are its low cost and therefore possibility to diminish expenditures on expensive antibacterial preparations and solutions for oral and parenteral application. REKITSEN RD applying can also considerably decrease staying in bed economic parameter (P<0,001). We did not observe any side effect during treatment of our patients with REKITSEN RD, that indicates its good acceptability.

According to literature data, in cases of intestinal infections one of the most simple objective and convenient in practical usage indicators of intoxication level is leukocytic index of intoxication (LII), calculated on the basis of the common blood analysis using Kalf-Kaliffs formula:

LII is also useful in estimation of therapy effectiveness in cases of intestinal infections. For healthy children LII is 0,58±0,09.

At entering in a hospital the compared groups did not differ from each other in LII index. LII values depended only on gravity of the disease. In the main group LII was 1,8±0,4 in mild forms, 2,8±0,5 in medium grave forms, and 6,5±0,7 in serious forms. In control group LII values were 1,6±0,2; 2,8±0,3 and 6,7±0,5 accordingly.

Usage of intestinal absorbent REKITSEN RD in treatment of salmonellosis in children promotes faster and appreciable downstroke of LII. This is the objective evidence of a faster decrease of intoxication alongside with microbal invasion and toxicosis (table 4).

Table 4. LII in the course of treatment of salmonellosis in children

Days of definition
Treatment
Р
main group
control
n=65
n=82
Entering hospital
4,2+/-0,6
4,4+/-0,4
>0,05
5-7 days of treatment
1,2+/-0,2
2,2+/-0,4
>0,05

Serum levels of immunoglobulins of main classes in our patients tightly depend on gravity of the disease and on a variant of therapy used (drawings 1, 2, 3).

In children of a control group sick with mild forms of salmonellosis during height of disease there took place rising of M and A immunoglobulins with continuing growing during fading clinical signs period. Thus the concentrations of immunoglobulins exceeded those for healthy on 27,5% for class M and on 20% - for class A. During a convalescence period IgM and IgA levels decreased and for the majority of the patients were on a high bound of normal values, while the level of class G immunoglobulins a little bit increased.

Similar dynamics was observed for the patients with medium grave forms of salmonellous infection, but all shifts in this group of children were expressed more clearly. The IgM concentration during height of disease exceeded normal values on 50% (P<0,05), JgA - on 24% (P<0,05). Subsequently JgM synthesis according to legitimacies of the immune response in infectious diseases switched to IgG synthesis.

I - mild form
II - medium grave form
III - serious form
a -period of height
b - period of fading of clinical signs
с - period of convalescence
1 - therapy using REKITSEN RD
2 - therapy without REKITSEN RD

Drawing 1. IgM serum levels in salmonellosis patients depending on kind of therapy. A level of IgM for healthy is accepted for 100%

I - mild form
II - medium grave form
III - serious form
a - period of height
b - period of fading of clinical signs
с - period of convalescence
1 - therapy using REKITSEN RD
2 - therapy without REKITSEN RD

Drawing 2. IgA serum levels in salmonellosis patients depending on kind of therapy. A level of IgA for healthy is accepted for 100%

I - mild form
II - medium grave form
III - serious form
a - period of height
b - period of fading of clinical signs
с - period of convalescence
1 - therapy using REKITSEN RD
2 - therapy without REKITSEN RD

Drawing 3. IgG serum levels in salmonellosis patients depending on kind of therapy. A level of IgG for healthy is accepted for 100%

For children with serious forms of salmonellosis, during height of disease uncertain decrease of concentration in all classes of immunoglobulins was observed, especially of class G. We consider that as a depressive influence of appreciable toxic and antigenic loadings on antibodies production. In fading of clinical signs and convalescence periods the sequential augmentation of concentration of immunoglobulins of all classes was observed, but their maximum values only a little bit exceeded ones for healthy.

On the background of treatment with REKITSEN RD in the mild forms of salmonellosis the contents of immunoglobulins of all classes in periods of fading of clinical signs and convalescence corresponded to that for children of control group. In medium grave forms after REKITSEN RD treatment the content of classes M and A immunoglobulins was reliably less, than in control group. Similar but less expressed legitimacy was observed in the period of early convalescence.

Administering of REKITSEN RD by patients with serious forms of a salmonellosis promoted reliable augmentation in all classes of immunoglobulins by the period of fading of clinical manifestations. The levels in question exceeded the ones for children of control group. Apparently, including REKITSEN RD in complex therapy of the serious forms of a salmonellosis promoted a cupping of secondary immunological failure (Колоколов В. А., 1998). During a convalescence period the moderate decrease in classes M and A immunoglobulins average levels was observed, that evidence for the earlier reduction of an antigenic boring.

Thus, serum immunoglobulin levels in cases of salmonellosis submit to general legitimacies of the immune response at infectious diseases. Course of REKITSEN RD in mild and medium grave forms of salmonellous infection in children promotes earlier change of IgM synthesis to IgG synthesis. In serious forms of the disease the substance promotes diminishing of secondary immunological failure appearances.

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