The degree of interest with phage therapy showed substantial fall together with the introduction of sulphonamides and then antibiotics. Renewed interest in this problem emerged again 40 years ago and was connected with the appearance of more and more frequent and difficult for curing infections induced by antibiotic and chemotherapeutic-resistant bacteria^2,4. This concerned mainly suppurative postoperative and hospital infections induced not only by antibiotic-resistant Staphylococci but also by Gram-negative bacteria as: Pseudomonas, Klebsiella, Escherichia Proteus and, rarely, by other types of bacteria.

In spite of great interest in phage therapy, majority of reports encountered in the literature are of casuistic type and those based on vast clinical material evaluating the curative value of phage therapy, are rather scarce.

The present work consists of two parts. The first one analyzes the results of application of bacteriophages in 138 cases; the second, presents a detailed analysis of phage therapy in 184 categories of various diseases diagnosed clinically in 138 patients treated with bacteriophages.
 
 

Phage therapy was applied in 138 patients with septic infections resulting from either idiopathic infections or post-operative complications. Age and sex of patients subjected to phage therapy is presented in Table 1. The age 21 - 77 years was predominating (78% patients). The ratio of men to women was almost equal, the number of men being slightly predominating (55.8%:44.2%). The classification into categories was based on the recommendations of the World Health Organization presented in the publication: "Manual of the International Statistical Classification of Diseases, Injuries and Causes of Death", vol. 1, WHO, Genewa 19776. The number of nosologic units in particular categories is depicted in Table 2.

Table 1. Age and sex of patients treated with bacteriophages

Age of	No. of	Sex
patients	cases	M	F
Up to 1 year of age	6 (4.4%)	2	4
1-5	11 (8.0%)	5	6
6-10	5 (3.6%)	1	4
11-20	18 (13.0%)	11	7
21-30	30 (21.7%)	19	11
31 - 40	15 (10.9%)	10	5
41-50	23 (16.7%)	14	9
51 - 60	14 (10.1%)	6	8
61 - 70	15 (10.9%)	9	6
Above 70 years of age	l  (0.7%)	0	1
Together	138	77 (55.8%)	61 (44.2%)

 

Phage therapy was carried out in 10 clinical and hospital departments, mainly on surgical departments (Institute of Surgery, Medical Academy, Wroclaw: Cardiosurgery Clinic, Children Surgery Clinic, Orthopaedic Clinic; Institute of Internal Diseases: Nephrologie Clinic, Clinic of Pulmonary Diseases, K. Dluski Lung Disease Hospital, Wroclaw: Department of Immunotherapy, T. Marciniak Hospital in Wroclaw: Department of Children Surgery; Hospital and Polyclinic of KWMO in Wroclaw: Traumato-Orthopaedic Department; Department of Traumato-Orthopaedic Surgery of ZOZ in Lubin; Department of ZOZ Surgery in Olesnica; Provincial Hospital of Locomotory Organs Diseases in Kamienna Gora).

Table 2. Disease categories according to the International Classification of Diseases (WHO, 1977)

	Disease category	Number of cases
I.	Infectious diseases	2  1.4%
III.	Immunological disorders (humoral, cellular)	2  1.4%
VII.	Diseases of the circulatory system (varicose ulcers of lower extremities)	8  5.8%
VIII.	Disease of the respiratory system (mucopurulent inflammation of the respiratory tract, pneumonia, bronchopneumonia, suppurative upper pericarditis, mediastinitis)	17  12.3%
IX.	Diseases of the digestive system (diseases of the oral cavity, suppurative peritonitis)	17  12.3%
X.	Diseases of the genitourinary system (glomerulonephritis, urinary tract infections)	6  4.4%
XII.	Diseases of the skin and subcutaneous tissue (furunculosis, abscesses cutis, decubitus ulcer)	20  14.5%
XIII.	Diseases of the musculoskeletal system and connective tissue (pyogenic arthritis, infective myositis, postoperative ostitis)	31  22.5%
XVII.	Injuries (contusions, postoperative wounds, burns, open wounds, pyogenic infections after fracture of bones)	35  25.4%
	Total	138

 

Out of 138 cases investigated, in 90 monoinfections and in 48 polyinfections were confirmed. Most frequent cause of monoinfection (Table 3) were pyogenic Staphylococci (67.7%), rarely Pseudomonas (14.4%), Escherichia (10.0%) and Klebsiella (10.0%) bacilli.

In the cases of polyinfection (Table 4), beside Pseudomonas aeruginosa (60.4%)' most commonly isolated were pyogenic Staphylococci (70.8%). Rarely occurring were: Escherichia (33.3%), Klebsiella i31.2%), Proteus (20.2%) bacilli. The remaining microorganisms were isolated sporadically.

Table 3. Species of bacteria in monoinfections

Species of bacteria	No. of cases (n= go)
Salmonella enteriditis	1  1.1%
Shigella sonnei	1 1.1%
Staphylococcus pyogenes	6 67.8%
Klebsiella pneumoniae	5 5.6%
Escherichia coli	9 10.0%
Proteus vulgaris	0
Pseudomonas aeruginosa	13 14.4%

Table 4. Species of bacteria in polyinfections

Species of bacteria	Number of cases
	(n = 48)
Staphylococcus pyogenes	34 70.8%
Klebsiella pneumoniae	15 31.2%
Escherichia coli	16 33.3%
Proteus vulgaris	14 29.2%
Pseudomonas aeruginosa	29 60.4%
Streptococcus fecalis	2 4.2%
Enterobacter sp.	2 4.2%

 

Origin of bacteriophages^* . In the experiments the use was made of 259 virulent bacteriophages including 116 for Staphylococcus of human and animal origin, 42 for Klebsiella, 39 for Escherichia, 30 for Shigella, 20 for Pseudomonas, 11 for Proteus and 1 for Salmonella enteritidis. In the course of therapy 52 Staphylococcus bacteriophages were used, 20 for Escherichia, 17 for Klebsiella, 17 for Pseudomonas, 4 for Proteus and for Shigella and Salmonella—one for each.

During therapy the use was made of the Iysates of virulent bacteriophages which caused a total Iysis of the sensitive bacterial strains isolated from the patients.

Isolation of bacterial strains from patients and determination of their sensitivity to bacteriophages. The bacteria from 18 h broth culture were passaged in broth and afier 4 h incubation at 3 7 ° C (shaker), 2 - 3 ml of the suspension was sneared on a dried plate with WAHL et al. ' medium (for Staphylococci) and on agar plates with phosphate buffer (for Gram-negative bacteria); an excess of the suspension was removed. One drop of bacteriophage (corresponding to the type of strain isolated) was applied to each plate after drying (30 min). For typing, the phages were diluted 1:10. The plates were incubated at 37 ° C for 4 h in the case of Staphylococcus, for 2 - 3 h with Escherichia, Shigella and Klebsiella and for 4 - 5 h with Pseudomonas. Thereafter, the plates were transferred into refrigerator and the result was read on a following day.

Propagation of Staphylococcus bacteriophages. After selection, the phages bringing about a total Iysis on the strain isolated from patient, were propagated in 0.5 ml flasks containing 300 ml of broth, 5 ml of 30% glucose and 2.5 ml of 18 h Staphylococcus broth culture. After I h incubation at 37?C either 5 ml of phage Iysate or agar from the spot showing full Iysis, were added and the mixture was reincubated on a shaker for about 3 h. The strain culture on the same medium but without phage addition constituted the control. After Iysis the flasks were stored overnight in a refrigerator. On the following day, the Iysate was tested for the phage presence. If the Iysate was turbid, it was centrifuged for l h at 3,500 rpm. After confirmation of the phage presence, the Iysate was titrated and thymol crystal was added to kill the remaining bacterial cells. After 3-day storage in a refrigerator, the Iysate from above thymol was collected and poured into ampouls, 10 ml to each and tested for sterility.

Propagation of bacteriophages for Gram-negative bacilli (Pseudomonas, Klebsiella, Escherichia, Proteus, Shigella and Salmonella). Pseudomonas phages were propagated in 10 ml of peptone water to which a young (4 h) bacterial culture in amount I—2 ml and 5 ml of phage Iysate, was added. The control consisted of a flask with the same medium but without the phage. After completion of the Iysis the flasks were transferred into refrigerator. On the following day, the Iysate was tested for the presence of phage. To kill the remaining bacteria, 2 ml of chloroform was added to the Iysates, shaken for 2 min, left for 2 h at room temperature, transferred into refrigerator and on the next day, the Iysate from above chloroform was collected, poured into ampoules and tested for sterility.

Propagation of the remaining Gram-negative bacilli was carried out similarly except that the bacteria were cultured on broth instead of peptone water. Incubation lasted for 2 h and the propagations process of phages— about 3 h.

Therapeutic use of bacteriophages. Bacteriophages were administered orally 3 times a day, in amount of one 10 ml ampoule, 30 min before meal, after neutralization of the gastric juice (gelatum, baking soda or a glass of Vichy water). When applied directly on a wound, moist applications were recommended 3 times per twenty four hours.

When the phage was applied locally, it was made sure that the wound was not washed with any antiseptics to prevent inactivation of phages. If necessary, aseptic broth or physiological salt solution was used.

The bacteriophages were stored at +2 - +4?C. Intravenous administration of phages was not recommended for fear of possible shocks.
 

In the course of treatment regular bacteriological control and evaluation of sensitivity of the isolated bacteria to the bacteriophages aplied were carried out. If necessary, the bacteriophage was changed. In the case of a negative culture, the phages were continued to be applied prophylactically for 14 days.

Final evaluation of phage therapy was based on the notes from case history and on the data from a special inquiry. The inquiry was filled by a therapeutist and sent to the Institute of Immunology and Experimental Therapy, Polish Academy of Sciences in Wroclaw. The results of the treatment were evaluated by a therapeutist.

The results of phage therapy were evaluated according to four-degree scale: + + + + outstanding effect manifesting by a complete recovery, + + + marked effect manifesting by a complete healing of the local lesions and liquidation of the suppurative process, + + marked rmprovement of the local state with a tendency to healing of the lesions and negative result of bacteriological culture, + transitory improvement of the local state.  In the last group the evaluation met with some difficulties.

These cases "difficult for evaluation" are separately discussed.
 

The results obtained were used for the elaboration of a computer program. This program was written in ALGOL 1900 language for Odra 1300 computer series and set to work at the Computation Center at Wroclaw Technical University. The source program together with its numerical and technical characteristics is at disposal at the Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw.

Statistical evaluation of results was based on the following tests:
1. Chi-square goodness of fit test⁵:

Number of degrees of freedom r = k - 1.
2. t-test of the differences between means from two independent samples¹, assuming that s ²₁ = s ²₂:

Number of degrees of freedom r= n_l+n₂ - 2.
3. Test of inference on the basis of frequencies¹:

4. Test of comparison of two frequencies of independent samples¹:

Noteworthy is that in 125 cases (90.6%) out of 138 treated, phage therapy was performed on patients resistant to antibiotics and chemotherapeutics as the only way of inhibition and control of the infection. Only in 13 patients (9.4%) not treated previously with antibiotics, the bacteriophages were used.

Of 138 cases treated with bacteriophage, in 129 (93.5%) a good therapeutic result was obtained manifesting by a control of the infection and healing of the local lesions (Table 5).
 
Table 5. General evaluation of phage therapy

	Result of treatment
No. of cases	+	++	+++	++++
138	9	8	99	22
Percentage	6.50%	5.70%	71.70%	16.10%

 
Statistical evaluation of the results obtained during phage therapy was based on the chi-square test of goodness of fit (test 1) and on the test of inference on the basis of frequency (test 3). The results evidence that the effect of phage therapy in 138 subjects was not accidental, and point to great efficacy of the treatment (test 1) x² = 164.3, p < 0.001 with r = 3). As observed, in majority of the patients treated with bacteriophages, a complete recovery or a healing of the local lesions was obtained (test 3, u = 8.77, p < 0.001).

Results of the treatment in relation to patients' age are depicted in Tables 6, 7 and 8. Table 6 presents the results of phage therapy, mean age of the patients and standard deviations. Statistical evaluation of the results obtained was based on the the t test of the differences between two means from independent samples, assuming that s ²₁ = s ²₂ Number of degrees of freedom— n_l + n₂ - 2 (test 2).

As follows from Table 6, the effect of phage therapy is related to the age of patients subjected to it. For instance, the difference of the average age of patients with a transient improvement of the local state (+) and the average age of the completely cured patients (+ + + +) was as high as 13.1. To check whether the relation between the effect of phage therapy and age observed in the study, is significant for the whole population, the use was made of the t test with right-hand critical region to compare the average age of not completely cured patients (group +, ++ with average age 41.6) with that of completely cured (group +++, ++++ with average age 32.2). The obtained value (r = 1.81, p < 0.07 with r = 136) confirms the statistical significance of the dependence between phage therapy and age of the patients.

Table 6. Age of patients and result of phage therapy

		Result of treatment
	+	++	+++	++++
No. of patients	9	8	99	22
(n= 138)	6.5%	5.7%	71.7%	16.1%
Average age (AA)	45.7	36.9	32.1	32.6
Standard deviation of age (SD)	16.2	21.1	20.2	20.1

 
Table 7. Age of patients divided into 4 categories and result of phage therapy

Result of treatment	0-	20-	40-	60-	No. of cases
+	1	2	4	2	9
++	1	4	1	2	8
+++	30	31	27	11	99
++++	6	7	6	3	22
No. of cases	38	44	38	18	138

Calculation of estimated fractions and their errors

Age	0-	20-	40-	60-
Fractions	0.05	0.14	0.13	0.22
	±0.001	±0.004	±0.003	±0.009

Table 8. Result of treatment of suppurative infections in infants and children aged up to 5 years

Disease	No. of		Result of treatment
category	cases	+	++	+ + +	+ + + +
I	1			1
VIII	6		1	4	1
IX	1				1
X	1			1
XII	3			2	1
XIII	3			3
XVIII	2			2
Total	17		1	13	3

 
Table 7 presents a number of patients with various effect of treatment: +, + +, + + +, + + + + depending on age group. Patients from + and + + groups were regarded not completely cured and from + + + and + + + + groups—completely cured. A question arose whether the number of not completely cured patients had a tendency to increase with age. Estimated fractions and their standard errors were calculated from the formula:
 

A marked difference is observable between the extreme age groups i.e. 0 - 19 years and above 60. The former included only 5% of not cured patients and the latter—22%. In the group 20 - 39 years and 40 - 59 years participation of the not cured patients ranged from 13 to 14%.

Of particular importance is a possibility of bacteriophage application in the treatment of intestinal and suppurative infections in infants and in 1 - 5-year-old children. The investigated group included 6 infants aged from 2.5 to 6 months and 11 children aged from 1 to 5 years. In all the cases phage therapy gave very good results. Full characteristics of these groups is depicted in Table 6.

The results obtained in the group of children up to 5 years old confirmed the earlier observed dependence between the age of patient and the result of phage therapy. In this age group as much as 95% of cases were completely cured. As follows from the statistical evaluations (u = 3.39, p < 0.001), such good results may be expected in all children up to 5 years old.

5. B.E. F 5 years old                     Acute osteitis, persistent postoperative fistula; treatment with antibiotics ineffective; phages administered orally for 16 weeks and applied locally on the
        (041.1)                                    lesion and to the fistula for 12 weeks; complete healing of the local lesions (+++). Simultaneous administration of antibiotics.
6. B.A. M 1.5 years old               Acute osteitis, persistent post-operative fistula; treatment with antibiotics ineffective;
        (041.1, 041.4, 041.6)              phages administered orally (I5 weeks) and locally applied (on the lesion and to the fistula) for 12 weeks; complete healing of the lesions (+++).
13. CH.P. M 3 years old              Urinary tract infection; ineffective treatment with antibiotics; phages administered orally for 2 weeks; complete sterilization of urinary tract (+++).
        (041.7)
36. G.T. F 4 years old                  Acute osteitis, persistent post-operative fistula; ineffective treatment with antibiotics; phages administered orally for 8 weeks; complete healing of the
        (041.7)                                    local lesions (+++).
43. M.M. F 4 years old                Mucopurulent bronchitis; ineffective treatment with antibiotics; phages administered orally for 4 weeks; complete recovery (++++).
        (041.1)
49. K.A. F 1.5 years old               Furunculosis of the face; ineffective treatment with antibiotics; phages administered orally for 5 weeks and applied locally (on the lesion and to the
        (041.1)                                    fistula) for 3weeks; complete healing of the local lesions (+++).
52. K.A. F 4 years old                 Acute pancreatitis (disturbances in absorption), pneumonia; ineffective treatment with antibiotics; phages administered orally for 3 weeks; complete
        (041.7)                                    recovery (+++).
53. K.E. F 1.5 years old               Dysentery; not treated with chemotherapeutics; phages administered orally for 1 week; complete recovery (++++).
        (004.3)
62. K.S. M 2.5 years old              Pneumonia, thoracic empyema with fistula; ineffective treatment with antibiotics; serious state; phages administered orally for 2 weeks; complete recovery
        (041.4)                                    (+++).
82. P.K. F 1.5 years old                Pneumonia, thoracic empyema with fistula, ineffective treatment with antibiotics; phages administered orally for 7 weeks and locally applied on the wound
        (041.3, 041.7)                         and to the fistula for 7 weeks; full recovery (+++).

137. Z. P. M 3 years old               Fracture of fornix of the scull with fistula and purulent subscleral posttraumatic hematoma, contusion of heel; ineffective treatment with antibiotics;
        (041.1, 041.7)                          phages applied orally and locally for 4 weeks; full recovery (+++).

Result of phage therapy in relation to patients' sex is illustrated in Table 9. Statistical evaluation was based on the chi-square test for two-by-two contigence table with regard to frequencies (test 6).

Table 9. Sex of patients and result of bacteriophages treatment

		Result  of		treatment
Sex		+	++	+++	++++	Total
Females	No. of cases	4 6.60%	3 4.90%	43 70.50%	11 18%	61 44.20%
	AA	52.8	26.4	31.1	32.2
	SD	8.9	20.1	22.5	20.8
Males	No. of cases	5 6.50%	5 6.50%	56 72.70%	11 14.30%	77 55.80%
	AA	40	43.2	32.8	32.9
	SD	18.3	19	18.2	19.4

 
To test the above dependence, a comparison was made within the group of completely cured patients (++++) since this group displayed most substantial difference in the effect of treatment between men and women (3.7%). However, no dependence between sex of patients and effect of phage therapy was noted: (x² = 0.36, p > 0.05 with r = 1).

Table 10 presents a general comparison of phage therapy in relation to a type of infection (monoinfections, polyinfections), average age of patients and standard de viation of age. Statistical evaluation of the results obtained was based on the chi-square test for two-by two contingence table with regard to frequencies (test 6).

Table 10. Result of phage therapy in mono- and polyinfections
 

		Result  of		treatment		No. of
Type of infection		+	++	+++	++++	cases
Monoinfection
	No. of cases	5 3.3%	3 74.4%	67 16.8%	15 5.5%	90
	AA	33.3	42	31.8	30.7
	SD	11.5	17.5	19.5	19.9
Polyinfection
	No. of cases	4 8.3%	5 10.4%	32 66.7%	7 14.6%	48
	AA	60.5	33.8	32.7	36.7
	SD	5.5	22.4	21.7	20.0

 
The values of x² test were calculated for all the degrees of recovery (x²₊ = 0.39, x²₊₊ = 2.18, x²₊₊₊ = 0.89, x²₊₊₊₊ = 0.16).

The results obtained did not confirm the possible differences in the effect of phage therapy in mono- and polyinfections (p > 0.05).

Table 11 contains the data concerning results of phage therapy in 90 monoinfection cases in relation to a species of bacteria that induced the monoinfection. Due to a high number of monoinfections induced by Staphylococci a question arose whether the majority of monoinfection is induced by Staphylococci. analysis was based on the test of inference on the basis of frequencies (test 3). The obtained u value (u = 14.65) allows to infere that most monoinfections is caused by Staphylococci (p < 0.001).

Table 11. Monoinfections and result of treatment

Species of bacteria		Result of treatment
	+	++	+++	++++	No. of cases
041.1  Staphylococcus	5	2	43	12	62
					68.9%
041.3  Klebsiella	0	1	4	0	5
					5.6%
041.4  Escherichia	0	0	8	1	9
					10.0%
041.6  Proteus	0	0	0	0	0
041.7  Pseudomonas	0	0	12	0	12
					13.3%
004.3  Shigella	0	0	0	1	1
					2.2%
003.0  Salmonella	0	0	0	1	1
Number of cases	5	3	67	15	90
Average age (AA)	34	42	31.8	30.7
Standard deviations of age (SD)	12	18	19.5	19.9

 
Another problem to solve was whether there is any difference in the effect of the monoinfection. Statistical analysis was based on the chi-square test for two-by-two contingence table with regard to frequencies (test 6). Considering two groups of patients: + and ++, +++ and ++++, no difference was observed in the effect of phage therapy of monoinfections induced by: Staphylococcus and Pseudomonas, Klebsiella and Pseudomonas, and also Escherichia and Pseudomonas. An example of analysis of Staphylococcus and Pseudomonas infections looks as follows: x² for 2 x 2 contingence table with regard to frequencies, r = 1 x²_obs = 1.49 x²_0.05 = 3.84 p > 0.05.

There is no significant difference in the effect of phage therapy applied in monoinfection induced by Staphylococcus and Pseudomonas.

Table 12 contains data concerning a frequency of occurrence of particular bacterial species in polyinfections. In statistical analysis of the results obtained, two bacterial species (Staphylococcus and Pseudomonas), most frequently represented on the material investigated, were considered. Of interest was whether on the basis of the results obtained in a group of 138 cases, it may be inferred that among all polyinfections, majority is induced by the above bacteria occurring together or in combination with other microorganisms. Statistical analysis was based on the test of inference on the basis of frequencies (test 3).

Table 12. Polyinfections and result of treatment

		Result of treatment			No of
Species of bacteria	+	++	+++	++++	cases
041.1  Staphylococcus	1	5	22	7	35
					72.90%
041.3  Klebsiella	3	0	13	0	16
					33.30%
041.4  Escherichia	3	1	10	2	16
					33.30%
041.6  Proteus	2	0	10	2	14
					29.20%
041.7  Pseudomonas	2	4	20	3	29
					60.40%
041 Streptococcus	0	0	2	1	3
					10.40%
041.8 Other species	0	0	1	1	2
No. of cases	4	5	32	7	48
Average age (AA)	60.5	33.8	32.7	36.7
Standard deviation of age (SD)	5.5	22.4	21.7	20

 
The obtained results: a) for Staphylococcus u = 11.3, p < 0.001, b) for Pseudomonas u = 8.9, p < 0.001, confirmed that the inference was correct in both cases.

Another worth noting problem was whether the bacteria being a cause of monoinfections and occurring together with other bacteria (polyinfection) may bring a statistically significant difference in the effect of phage therapy. Statistical analysis of the joined groups + and ++, +++ and ++++ was based on the chi-square

test of independence between two variables with r= 1 (test 5). Effect of phage therapy in mono- and polyinfections was compared for four bacteria:
a) Staphylococcus (x² = 0.56, p > 0.05),
b) Klebsiella (x² = 0.02, p > 0.05),
c) Escherichia (x² = 1.06, p > 0.05),
d) Pseudomonas (x² = 1.58, p > 0.05).

For all the above organisms, no dependence between the type of infection and the result of treatment was observed.

Another problem of our concern was whether a given bacterial species occurs with equal frequency in mono- and polyinfections. Statistical analysis was based on the test of comparison of two frequencies from independent samples (test 4).
041.1 u_obs = 049 u_0.001 = 3.29 p > 0.05
Staphylococcus (041.1) occurs equally frequently in mono- and polyinfection.
041.3 u_obs = 4.23 u_0.001 = 3.29 p < 0.001
Klebsiella (041.3) occurs more frequently in polyinfection.
041.4 u_obs = 3.35 u_0.001 = 3.29 p < 0.001
Escherichia (041.4) occurs more frequently in polyinfection.
041.7 u_obs = 5.76 u_0.001 = 3.29 p < 0.001
Pseudomonas (041.7) occurs more frequently in polyinfection.

The influence of age and sex to the appearance of monoinfection and polyinfection was also subjected to statistical analysis (Tables 13 and 14). In the case of age the use was made of the t-test of the differences between two means from independent samples assuming that s ²₁ = s ²₂ and in the case of sex the chi-square test of independence between two variables (test 5) was applied.

Table 13. Age of patients with mono- and polyinfections

Type of infection	Average age	Standard deviation
	(AA)	(SD)
Mono- n = 90	32	19.2
Poli- n = 48	35.7	21.9

 
Table 14. Sex and mono- and polyinfection

Sex	Type of infection
	mono-	poli-	total
Females	41	20	61
	67.20%	32.80%
Males	49	28	77
	63.60%	36.40%

 
In both cases no dependence between the investigated traits and infection type was noted (for age: t = 1.03, p > 0.05, for sex x² = 0.19, p > 0.05, with r = 1).

State of the patients at the beginning of phage therapy is presented in Table 15.. Statistical analysis of the results obtained was based on the chi-square test of independence between two variables (test 5). No significant dependence between the result of treatment and patient's state at its beginning was noted (x² = 1.47, p > 0.05, with r = 6).

Table 15. Severity of the disease and result of the treatment

State of patient at the  beginning of therapy		Result of treatment			No. of cases
	+	+ +	+ + +	+ + + +
Without disturbances in general state	4	3	41	9	57
	70%	53%	71 9%	15.80%	41.30%
AA	46	34.3	32.4	38.4
SD	15.5	20.2	20.4	18.3
Medium state	2	2	37	10	51
	3.90%	3.90%	72.60%	19.60%	37.00%
AA	32	50.4	30.3	24
SD	16	27.1	20.5	18.6
Severe state	3	3	21	3	30
	10%	10%	70%	10%	21.70%
AA	54.3	30.4	34.3	43.7
SD	9.8	27.1	19.4	18.6

 

Phage therapy was performed on 125 patients (90.6%) in whom antibiotic treatment appeared ineffective and the bacteria showed resistance to most of the antibio-tics applied (Table 16). Antibiotic-resistant group was compared with a small, not treated with- antibiotics group of patients. It included 13 subjects (9.4%).

Statistical analysis was performed on the basis of the chi-square test of independence between two variables (Test 5).

x²_obs = 4.76 x²_0.05 = 7.81 for r = k - 1 = 3 degrees of freedom p > 0.05. The result of treatment is not significantly related to whether antibiotic treatment was. ineffective or not applied prior to phage therapy.

Among 138 bacteriophage treated cases, 125 were antibiotic-resistant (Table 17). An attempt was made to answer a question whether it may be inferred that an increase in the number of patients would bring about an increase of cases resistant to antibiotics.

Table 16. Resistance to antibiotics and result of therapy

Antibiotic treatment before bacteriophage application		Result of treatment			No. of cases
	+	+ +	+ + +	+ + + +
Ineffective	9	6	93	17	125
	7.2%	4.8%	74.4%	13.6%
AA	45.7	32.4	31.8	33	90.6%
SD	16.2	20.7	20.3	20.1
No treatment	0	2	6	6	13
		15.3%	46.2%	38.5%
AA		50.5	36	31.3	9.4%
SD		15.5	17.6	20.3

 
Table 17. Resistance to antibiotics and result of therapy

		Result of treatment			Total
	+	++	+++	++++
Cases treated with bacteriophages	9	8	99	22	138
	6.5%	5.7%	71.7%	16.1%
Cases resistant to antibioticsand treated with bacteriophages	9	6	93	17	125
	7.2%	4.8%	74.4%	13.6%

To this end the test of inference on the basis of frequencies (test 3) was applied.
u_obs = 9.45 u_0.001 = 3.29, p < 0.001

Among all the patients the percentage of those resistant to antibiotics would be about 90%.

Applying the same test it was attempted to check whether among a great number of patients, the number of cured subjects with + + + and + + + + result would be as high as in the investigated group (110 cases cured with +++ and ++++ result and 125 cases resistant to antibiotics). According to the results of our calculations the positive effect of treatment can be also obtained among a great number of patients resistant to antibiotics (u = 8.41, p < 0.001).

Table 18 compares the results of bacteriophage application in the combination with antibiotics and without them. It was decided to test whether phage therapy is more effective with or without antibiotics. Statistical analysis of the results obtained was based on the chi-square test with r = 1 (test 5).

Table 18. Bacteriophage treatment in combination with antibiotics and without antibiotics

Treatment			Result						No. of cases
Without antibiotics	+		++		+++		++++
No of patients	5		8		59		18		90
	5.50%		8.80%		65.50%		20.20%
AA	36.4		36.9		32.6		35.6
SD	14.2		21.1		20.6		21
With antibiotics
No. of patients	4		0		40		4		48
	8.30%				83.40%		8.30%
AA	57.3		0		31.3		19
SD	9.9		0		19.5		45

 
Group cured with ++ result:
x²_obs = 3.08 x²_0.05 = 3.84, p > 0.05
The difference observed is statistically nonsignificant.

Group cured with +++ result:
x²_obs = 4.84 x²_0.05 = 3.84, p > 0.05
The difference observed (to the advantage of the treatment with antibiotics) is statistically significant.

Group cured with ++++ result:
x²_obs = 3.25 x²_0.05 = 3.84, p > 0.05
The difference observed (to the advantage of the treatment without antibiotics) is statistically significant.

Therefore, it cannot be unequivocally stated that phage therapy is more effective in the combination with antibiotics or without them since the differences observed in its effect vary in particular categories of curing.

Of substantial importance in phage therapy is the route of phage administration. In the examined group of 138 patients phages were applied orally and locally (109 cases), orally only (22 cases) and locally only (7 cases). Locally, they were used as moist applications and in some cases as flow drainage:. General result of phage therapy when the three above routes were applied is presented in Table 19.

The Table illustrates the differences related to the mode of phage therapy application. It was of interest to test whether they were significant enough to confirm the concrete dependence between therapy mode and treatment effect. Statistical analysis of the results was based on the chi-square test with 6 degrees of freedom (test 5).

x²_obs = 7.42 x²_0.05 = 12.59, p > 0.05.

The differences resulting from various modes of phage therapy are statistically nonsignificant.

Table 19. Result of phage therapy in relation to bacteriophage administrations

	No. of cases		Result of therapy
Phage therapy		+	++	+++	++++
local only	7	0	0	4	3
				60%	40%
oral only	22	2	2	11	7
		9%	9%	50%	32%
local and oral	109	7	6	84	12
		6.40%	5.60%	77%	11%
No. of cases	138	9	8	99	22

 
In the group of patients treated with ++++ result a great span between the effect of the local and the local-oral phage therapy was observed. However, the scarce number of the subgroups did not allow to draw general conclusion.

Oral administration of bacteriophage. In 22 cases bacteriophages were administered orally only. The result of oral phage therapy is illustrated in Table 20.

Detailed analysis of the cases treated orally is as follows:

Table 20. Oral phage therapy

No. of cases		Result of therapy
	+	++	+++	++++
32*	2	2	11	7
131*	9	8	95	19
	6.9%	6.1%	72.5%	14.5%

* Cases treated orally only
** Cases treated orally and locally

The results obtained confirmed great effectiveness of this mode of phage therapy, particularly in bacterial infections of the digestive system, diseases of the respiratory system (bacterial pneumonia) and even in the skin diseases and suppurative infections of the urinary tract.

Statistical analysis of the results of oral therapy revealed that the markedly positive effect of oral administration of bacteriophages (82% of patients cured with + + + and + + + + result) can be also obtained in a numerous group of patients receiving bacteriophages orally only (test 3, u = 5.93, p < 0.001).

Local application of bacteriophage. In 7 cases bacteriophage was applied locally only. The results are depicted in Table 21. Though the number of cases was rather small, the effect is very encouraging. This mode of treatment may find its application in the healing of burns complicated by bacterial infections, suppurative diseases of the upper respiratory tract, particularly of pansinusitis and maxillar sinusitis as well as in the diseases of conjunctival sac, middle ear and skin furunculosis.

Table 21. Local phage therapy

No. of cases		Result of		treatment
	+	++	+++		++++
7*			4		3
1164*	7	6	88		15
	6.0%	5.2%	75.9%		12.9%

* Cases treated locally only
** Cases treated locally and orally

1. A.A. F 10 years old             Pyogenic arthritis, sepsis, chronic osteomyelitis; ineffective treatment with antibiotics; phages administered orally (26 weeks) and applied locally on the
        (041.3, 041.4, 041.6,          wound and to the fistula, flow drainage; recovery (+++).
        041.7)
26. D.F. M 43 years old           Chronic maxillar sinusitis, antibiotics — ineffective, phages administered orally for 2 weeks and applied locally (flow drainage); recovery (+++).
        (041.1)
45. J.T. M 49 years old            Gastric ulcer with perforation, suppurative peritonitis (postoperative); antibiotics were not given; phages given orally for 3 weeks and locally on the lesion,
        (041.4)                                to the fistula and as flow drainage to the peritoneal cavity (3 weeks); recovery (+++).
59. K.A. F 2.5 months             Thoracic empyema with a fistula, pneumonia; antibiotics ineffective phages given orally for 4 weeks and locally applied on the wound and to the fistula as
        old (041.1, 041.7)               flow drainage. Marked improvement (++).
67. M.Z. F 12 years old           Chronic osteitis, suppurative myositis, fistula, sepsis, antibiotics— ineffective phage administered orally for 13 weeks and locally on the lesion, to the fistula
        (041.1,041.0,041.7)            and as flow drainage; recovery (+++).
73. M.W. F 47 years old          Mucopurulent bronchitis, thoracic empyema with a fistula. Opening of the wound after operation of esophagus cancer antibiotics—ineffective; phages
        (041.1, 041.3)                     administered orally (7 weeks) and applied locally (4 weeks) on the wound and to the fistula and as flow drainage; recovery (+ + +).
92. R.W. M 52 years old         Acute inflammation of the gall-bladder, acute pancreatitis, suppurative-peritonitis bronchopneumonia. Antibiotic treatment ineffective; phages administered
        (041.3, 041.4)                     orally (5 weeks) and locally (1 week) applied on the wound, to the fistula and as a drainage; recovery (+++).
95. S.M. F 53 years old            Urinary tract infection, antibiotics—ineffective, phages administered orally (4 weeks)
        (041.7)                                phage-lavage of the urinary bladder (1 week); recovery (+++).
 

Effect of treatment in relation to the oral or local application of bacteriophages is illustrated in Table 23. From the Table it follows that at oral administration of bacteriophage, the effect of treatment is conditioned rather by other factors than the duration period. It was well evidenced by the cases with the transient improvement of the local state (+) with the longest period of treatment.

Table 22. Period of phage administration with regard to the route of administration and number of cases treated

Period of phage Administration	Route of phage administration
	orally +	Locally	only
			orally	locally
1 week	0	7	3
1.5 weeks	5	4	1
2.0 weeks	14	16	3	3
—5 weeks	41	41	12	3
36 - 10 weeks	33	29	2
11 - 15 weeks	8	4	1	1
16 - 20 weeks	5	5
20 weeks	3	3
No. of cases		109	22	7
		79.0%	15.9%	5.1%

Table 23. Significance of the period of bacteriophage administration (the mean does not include the patients being treated by other than local or oral route)

Period of bacteriophage administration in weeks		Result of treatment
	+	++	+++	++++
Orally
Mean	7.3	4.6	6.4	5.8
	±4.9	±2.3	±4.8	±5.1
AA	45.7	36.9	31.4	29.3
SD	16.2	21.1
Locally
Mean	4.6	3.5	5.6	5.7
	± 1.9	± 1.4	±4.7	±4.1
AA	44.9	33.2	34.2	36.5
SD

 

At local application, however, the tendency toward better therapy effect was growing with the duration of phage administration. The cases difficult for analysis were the exception.
The observed tendency has not yet found a statistical confirmation as manifested by the results of Wilcoxon test (test 7) (e.g. for ++ and +++ classes, W = 62, p > 0.05).
Detailed analysis of the values obtained in the above study is presented in Part II.

Table 24 illustrates the results of bacteriophage treatment in particular disease categories. The aim of the statistical analysis was to test whether there exists a dependence between the result of the treatment and category of the disease. To answer this question, patients cured with + and + + result were joined together to form one group; the same concerned patients cured with + + + and + + + + result. Patients with diseases of VIII category appeared to have poor chance of recovery (+++, ++++) (58.8%), the best chance (excluding categories of diseases of a number ?7) had patients with the following disease categories:
XII 95%, IX 94%, XVII 88.5%, XIII 90.3%
Category VIII was compared with-all the above categories and the values of x² test with r = 1 were calcula> 

Transfer interrupted!

Table 24. Result of treatment in particular disease categories

Category	No. of cases		Result of treatment
		+	++	+++		++++
I	2					2
III	2					2
VIII	8		1	5	85.7%	2
VIII	17	3	4	8	58.8%	2
IX	17	1		15	94.1%	1
X	6			6
XII	20	1		14	95%	5
XIII	31	3	0	24	90.3%	4
XVII	35	1	3	27	88.5%	4
No. of cases	138	9	8	99		22

 
The results obtained revealed that the differences in phage therapy effect between categories VIII and categories XII, XIII, XVII are statistically significant (p < 0.05). From the above it follows that patients with VIII category of disease have poorer chances of recovery than those with XII, XVII and XIII categories. Can it be, thus, inferred that among all the patients with XII, XIII and XVII categories a percentage of the cured would be as high?

To this end the values of "u" variable were calculated (test 3)

XII: u_obs = 3.79 u_0.001 = 3.29 p < 0.001
XIII: u_obs = 4.31 u_0.001 = 3.29 p < 0.001
XVII: u_obs = 4.71 u_0.001 = 3.29 p < 0.001

The findings of the calculations allow the possibility that among all patients with XII, XIII and XVII categories of disease, the percentage of cases cured with +++ and ++++ result can be as high as in Table 24 (XII — 95%, XIII — 90.3%, XVII — 88.5%).

Side effects in the course of phage therapy are very rare (Table 25). Out of 138 treated subjects, only 3 cases with side effects were recorded of which 2 displayed drug intolerance at oral administration and 1 allergic symptoms at local application on the wound. Statistical analysis was based on the test of inference on the basis of frequencies (test 3).

u_obs = 4.71 u_0.001 = 3.29 p < 0.001

Table 25. Side effects after phage therapy

	Occurred	Did not occur
No. of cases	3	135
	2.2%	97.8%
AA (average age)	24.3	33.2
SD (standard deviation)	4.9	20.1

With the increased number of patients, the number of subjects without side symptoms would be as high as in the cases observed.

On the basis of case history the following observations were made: mostly on day 3 - 5 of phage therapy, hepatalgia occurred which lasted several hours. This can be accounted for mass liberation of endotoxins resulting from phage effect on bacteria. ID severe cases with sepsis, an increase of temperature occurred on day 7 - 8 of phage administration which lasted 24 h.

Therefore, up to the 8th day of treatment with bacteriophages, the patients should be closely observed. Generally, on the 7th day marked improvement in the general feeling of patients was observed, the pains subsided, and the temperature and ESR fell down.

Local application of phage directly OD a wound or to a fistuls caused a purification of a wound on day 3 - 4 and also exceptionally quick granulation.
 

In the available literature we have not encountered any reports concerned conditions of therapeutic effect of bacteriophages. The present report is an attempt of analysis of the dependence between phage therapy effect and various factors such as: age, sex, infection type (monoinfections, polyinfections), bacterial species inducing the suppurative process, severity of disease course, susceptibility and resistance to antibiotic and chemotherapeutic treatment, combined treatment with antibiotics, route and period of phage administration disease category and side effects.

Since phages are applied mostly when all ways of treatment appeared ineffective, thus, the criteria used for evaluation of the results, in majority of cases, cannot be preserved. Besides, the state of patients treated with bacteriophages as the only effective remedy is often serious, even agonal. Thus, in spite of a markedly favorable effect of phage therapy (healing of the local wounds), due to the advanced anatomopathological changes of the internal organs, a death follows.

The results obtained so far showed that bacteriophages are a valuable and often the only effective factor in the bacterial infections.

• Bacteriophages are safe, side effects are rather rare and present no danger for a patient, they are transient and easy for restraint.
• Bacteriophages effectively control the infectious process, irrespectively of its localization, rebuilding the immunity forces of an organism. They are effective in the treatment of infections complicating immunological disorders.
• Bacteriophages can be administered orally or applied locally, they easily penetrate from the gastrointestinal tract to the blood, body fluids and urine exerting substantial therapeutic effect. It is of particular importance in the cases when they cannot be used directly.
• Bacteriophages can be used irrespectively of age, results of the treatment of suppurative infection in infants and small children are very promising.
• Bacteriophages can be prepared at appropriate laboratories, individually for each case which ensures reliability of their effect.
• Worth noting is also a low cost of their preparation.

Acknowledgment. The authors wish to express their gratitude to the heads and medical staff of the clinical and hospital departments for their fruitful cooperation.

1. ARMITAGE P.: Metody statystyczne w badaniach medycznych. PZWL, Warszawa, 1978.

2. BARBER B.: Hospital infection yesterday and today. J. Clin. Path., 1961, 14, 2 - 10.

3. BRUYNOGHE R. and MAISIN J.: Essais de thérapeutique au moyen du bacteriophage. C. R. SOC.. Biol.,., 1921, 85, 1120 - 1121.

4. KLASTERSKY J.: Prevalence des différentes espéces bactériennes oportunistes. Ann. Microbiol.. (Institut Pasteur), 1979, 130A, 1 - 2.

5. SAWICKI F.: Elementy statystyki dla lekarzy. PZWL, Warszawa, 1974. World Health Organization: International Classification of Diseases, Manual of the International Statistical Classification of Diseases, Injures and Causes of Death, Geneva, 1977, Vol. 1.

6. WAHL R. and LAPEYRE-MENSIGNAC P.: L identification des Staphylocoques par les bacteriophages Annales de l'Institut Pasteur, 1950, 78, 353 - 364.

7. ZIELINSKI R.: Tablice statystyczne, PWN, Warszawa, 1972.
 

Received in September 1982