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Immunotherapy against antibiotic-resistant bacteria: the Russian experience with an antistaphylococcal hyperimmune plasmaand immunoglobulin
Microbes and Infection, 2, 2000, 1383–1392 © 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S1286457900012922/FLA
Review
Immunotherapy against antibiotic-resistant bacteria: the Russian experience with an antistaphylococcal hyperimmune plasma and immunoglobulin Jeanne Kelly Coates & Jarratt, Inc., 4455 Connecticut Avenue, A500, Washington, D.C. 20008, USA
ABSTRACT – The Russian experience with the preparation and clinical application of an antitoxic antistaphylococcal hyperimmune plasma and immunoglobulin is described. The immunotherapies were developed in the late 1960s and put into widespread use in the Soviet Union for the prophylaxis and treatment of sepsis, pneumonia, and other conditions caused by an epidemic of antibiotic-resistant Staphylococcus aureus. © 2000 Éditions scientifiques et médicales Elsevier SAS staphylococcal / antibiotic-resistant / immunotherapy / hyperimmune plasma
1. Introduction In the face of growing bacterial resistance to antibiotics, passive immunotherapy has received renewed attention as an alternative treatment [1–3]. Missing in all such discussions published in the West, however, is any mention of the unique Russian experience with antibacterial immunotherapies from human blood, particularly an antistaphylococcal hyperimmune plasma and immunoglobulin widely used to combat antibiotic-resistant strains of Staphylococcus aureus since the 1970s. In the 1960s and early 1970s multi-drug-resistant strains of S. aureus caused major bouts of nosocomial infections in Europe [4]. In Russia at this time, they reached epidemic proportions. As in Europe, strains 80/81 were identified as those responsible. Faced with alarming rates of mortality from staphylococcal infections throughout the country, Professor Simon Skurkovich, M.D., then chief of the Immunology Laboratory at the Central Institute of Hematology and Blood Transfusion in Moscow, developed what became the first hyperimmune antitoxic–antistaphylococcal plasma and immunoglobulin produced in humans [5]. The preparations are commercialized and widely used in Russia today to treat advanced staphylococcal infections, though demand may far exceed supply. More than 50 articles in the Russian literature describe the production, storage, and application in fresh and dry form of these preparations. They are used for the treatment or prophylaxis of infections in neonates, burn victims, and patients who have undergone cardiac surgery, and for the treatment of pneumonia, endocarditis, meningoencephaMicrobes and Infection 2000, 1383–1392
litis, osteomyelitis, local infections of the ear, nose, throat, and eyes, bronchial asthma of staphylococcal etiology, and other staphylococcal diseases. The use of the preparations in infections due to implanted prosthetic devices, not common in the 1970s and 1980s, is not described. The plasma has been administered intravenously, intraosseously [6], locally [7], topically [8], and even endolymphatically [9] and orally (to neonates) [10]. The immunoglobulin, first developed for intramuscular use, is usually administered in less severe cases or outside the hospital setting. The plasma has also been used to preserve xenoaortic valves before transplant, sprinkled over wounds in powdered form, soaked into bandages, and applied in the form of ointment or drops. More recently (1994), an antistaphylococcal intravenous immunoglobulin (IVIG) was described [11]. Despite their widespread use in Russia and parts of Eastern Europe and the many Russian-language publications on the subject, this author could not find a single reference to the preparations in the English literature. Exceptions are a report in a compendium of papers in English presented at a Soviet–American symposium [12] and an article on the use of the plasma in burns by Russian researchers [13]. So complete is the language barrier, that information on the antistaphylococcal preparations has so far existed in a kind of sealed isolation. Meanwhile, the Russian side appears to have assumed such immune preparations were also available in the West. A reviewer of a 1988 Russian book on the antistaphylococcal plasma chastised the author for omitting discussion of Western 1383
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work on this type of preparation, unaware there was none in use [14]. While some reports can be found in the Western literature on human plasma-derived preparations in experimental staphylococcal infections that have shown promising results, these have apparently not led to testing in humans [15–17]. It should be noted that the language barrier has also to some extent isolated the Russian medical research community and limited discourse with Western research on staphylococcus, little of which is referenced in Russian journals. The present review of the development and application of these preparations was written to break this barrier and make the details of this work available to the Western medical community. Before his emigration, Skurkovich developed other similar antibacterial preparations: one against several types of Escherichia coli [18], another against Pseudomonas aeruginosa exotoxin [19], and an interferon-containing plasma against viral infections [20], all shown to be promising at the phase I or II stage of clinical testing [21, 22], but apparently not put into widespread use after he left the Soviet Union. In 1984 the State Prize, the Soviet Union's highest honor, was awarded for the development and nationwide application of the antistaphylococcal plasma (ASP) and antistaphylococcal immunoglobulin (ASIG) [23]. Professor Skurkovich, best known in the West as the first to discover the role of cytokines in the pathogenesis of autoimmune diseases and a pioneer of anticytokine therapy for these diseases [24, 25], immigrated to the United States in late 1979. Russian physicians and researchers continued to find new uses for and expand production of the plasma and immunoglobulin after his departure. Articles and books on the plasma written in the 1980s emphasize refinements in techniques to maximize production [26] and increase lot-to-lot consistency of the plasma [27]. Experimental work was also conducted at this time to investigate the basis for the immunologic effects of the preparations. This report was prepared based on a careful reading of all available Russian articles and books on the preparations with special attention to controlled clinical trials and experimental work and to information that would allow other researchers to conduct independent investigations of the immunotherapies. All but one or two articles listed in Medline were available at the National Library of Medicine in Bethesda, Maryland.
2. Development and production of ASP and ASIG Choice of immunogen. A widely used Western immunology textbook states that “there are no vaccines against the numerous staphylococcal exotoxins” [28]. In fact ASP was first prepared in Russia by immunizing donors with such an antistaphylococcal vaccine in use since the 1930s, though it is called an anatoxin (toxoid), the word 'vaccine' being reserved for preparations made not from staphylococcal exotoxins but from somatic antigen. The anatoxin is similar in form and preparation to diphtheria and tetanus vaccines, that is, from adsorbed toxoid, as it is called in the West, made by treating staphylococcal exotoxins with 1384
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formalin to destroy toxicity while preserving the original toxins' immunogenicity. Though Skurkovich was the first to produce a hyperimmune antitoxic antistaphylococcal plasma in humans, he followed a long line of research going back to the 1930s and 1940s in the Soviet Union and Europe on staphylococcal toxins and their role in disease. This included work on the induction of high titers of antistaphylococcal antitoxins in human and animal blood by immunization with toxoid and extensive clinical study (in Russia) of toxoid for the prophylaxis and treatment of staphylococcal infections. Development of the antitoxic immune preparations was based on the then current understanding of a close correlation between circulating antitoxic titer and both antitoxic and antimicrobial immunity to staphylococcal infection. This work is summarized in the book Stafilokokkovyye Infektsii (Staphylococcal Infections) (1963) [29] by Vygodchikov, a leading Soviet expert in staphylococcus at the time. Work with rabbits in the 1930s had demonstrated that the higher the titer of antibodies to staphylococcal toxins in the blood, the stronger the immunity to natural or laboratory-induced staphylococcal infection [29]. The Russian Davidovich reported in 1937 – before nonspecific and cell-mediated immunity were understood – that immunization with staphylococcal toxoid produced antitoxins first, with other 'antibodies' raising cellular defenses, namely opsonizing and complement-fixing substances and agglutinins, developing more slowly [29]. Research performed by Delaunay in France (1938) [30] and confirmed by others, demonstrated that in immunized rabbits with antitoxic antibodies in their blood, in contrast to controls without such antibodies, a “strong polymorphonuclear leukocytic reaction and sharp rise in phagocytic activity was observed that localized and overpowered the staphylococci” [29]. Toxins, Vygodchikov concluded, “not only impede phagocytic functions of the leukocytes, but destroy them, exerting a leukocytolytic action.” Thus, by neutralizing the toxicity, antitoxins 'open the staphylococci to phagocytosis' [29]. This view differs from the approach in the West, where, for example, the role in pathology of α-toxin, the major cytolysin in S. aureus, is still under discussion [15], and cell wall antigens have preferably been used in vaccine development [17]. (The U.S. company NABI has reported positive results from the prophylactic use in children of a hyperimmune immunoglobulin prepared from microbial cells of S. aureus.) Capsular antigens as a vaccine candidate were tested in Russia in the 1970s and were found to have a weak protective effect in mice [31]. 2.1. Active vs passive immunization
As mentioned, active immunization with the toxoid was in practice at the time of the plasma development but is considered to produce a weak and not long-lasting immunity in vaccinees. It was not protective for patients who have reached an advanced stage of infection with an already weakened immune system. Passive immunization, on the other hand, immediately delivers high titers of antitoxic antibodies from healthy donors. Microbes and Infection 2000, 1383-1392
Antistaphylococcal hyperimmune plasma
2.2. Vaccine preparation
Skurkovich obtained the toxoid to immunize donors from the Gamaleya Institute of Microbiology, Epidemiology, and Immunology (Moscow). It was produced as described by Vygodchikov from all toxins isolated from the strain of S. aureus known as O-15 (equivalent to Wood-46), considered the most toxigenic [29], inducing antibodies to toxins from a wide range of strains. The Gemaleya Institute routinely supplies the toxoid to blood centers for immunization and ASP production, so details of its preparation are not included in any of the reports on the use of the antistaphylococcal plasma. Vygodchikov's method of preparing the toxoid [29], essentially follows the work of Ramon (1923) [32]. Cultures of S. aureus (taken from pus of infected individuals) were grown at the Gamaleya Institute on a laboratory-prepared 1-L caseine broth for 5 days at 37 °C with 20–30% CO2. On day 5, culture bottles are removed from the thermostat and the microbial mass separated by filtration. The filtrate, containing the toxins, is tested for activity. The hemolytic titer of the toxins for toxoid preparation must be no less than 1 000 Dhm (dosis hamolitica minima) after 1 h of incubation at 37 °C and 1 h at room temperature. To obtain the toxoid, formalin is added to the toxins at a concentration of 0.3–0.5%, and the mixture is incubated at 37 °C from 7–28 days to achieve full detoxification [29]. Today, the same strain of S. aureus is used, though commercially prepared media are undoubtedly available for growing the toxins. In 1965 standard methods for protein purification of the staphylococcal toxoid were described, namely, using precipitation with methanol, fractionation with (NH4)2SO4, ion-exchange chromatography with DEAE-cellulose, and Sephadex gel filtration. From this a toxoid preparation is obtained with an activity of 400 binding units per mg of protein nitrogen and 70% toxoid yield, described as a serologically pure preparation in precipitation reaction in agar with staphylococcal antitoxin [33]. 2.3. Immunization scheme
Skurkovich and his colleagues tested several schemes of donor immunization using the Gemaleya toxoid and found the most effective to be one requiring that 4 mL of toxoid be given subcutaneously in doses of 1.0, 1.0, and 2.0 mL at 7-day intervals [34]. This scheme permitted the shortest period of immunization and the use of the smallest dose of prepared toxoid vaccine to give the highest antibody titer with no side effects to donors. Donor plasma is collected by plasmapheresis on day 21 after start of immunization, that is, 7 days after the third immunization [34]. Following this scheme, 88% of donors were found to produce an anti-α-toxin (see below) titer ≥ 5 IU/mL with 65% generating a titer > 10 IU/mL, and some generating antibody levels as high as 26 IU/mL or more. The average titer is 12.7 ± 1.28 IU/mL. (Normal serum contains about 0.86 IU/mL.) When in 1974 the Ministry of Health of the Soviet Union mandated that all municipal and regional blood collection centers collect ASP, it was designated that this scheme be used. Occasionally, articles report using a slightly different scheme of 0.5, 1.0, and 2.0 mL. Microbes and Infection 2000, 1383-1392
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Titering of ASP is done by a neutralization assay measuring its ability to protect against the hemolytic action of staphylococcal α-toxin on rabbit erythrocytes, a method developed by Vygodchikov [29]. Because of the use of α-toxin (a sample of which is supplied by the Gemaleya Institute with the toxoid) to determine titer, the ASP is often referred to in the literature as anti-α-staphylotoxin or antiα-toxin. However, it should be noted that all toxins of the O-15 (Wood-46) strain are used for the toxoid. Since a total of only 2 to 3 L of immune plasma can be collected from each donor after a series of immunizations (10 collections of 250 mL, 2–4 times/month followed by a 3-month break), ways were later proposed to refine donor selection, optimize collection, and standardize the product by titer level. For example, a study of donor titer before and after immunization led to an attempt to select out a small percentage of donors found resistant to the action of the vaccine, probably a result of neutralization of the vaccine by pre-existing antibodies in the donors' circulation. Methods were devised to optimize collection by grouping and plasmapheresing donors according to the time their antibody titer reaches its peak, which varies widely among donors [35]. 2.4. Characterization of the toxoid
The Russian literature on the toxoid is extensive and probably deserves separate treatment. (Controlled studies of its use in prophylaxis report postive results, such as a report of the intranasal administration of the toxoid in neonates [36].) Probably because of the importance given toxin neutralization in treating staphylococcal disease with immunotherapy, other immunogenic elements in the toxoid have not been thoroughly investigated. One report, for example, on the antigen-specific and -nonspecific response to the toxoid in prophylaxis, states that in addition to a rise in the level of anti-α-toxin, immunization leads to a rise in titers of antibodies to the staphylococcal cellular wall [37]. Little else was found in the literature on the antibodies to the cell wall, though specific agglutinins to somatic antigen are reported as appearing in the blood of donors immunized for plasma collection (1:640 and above) [34] and are sometimes measured in treated patients. A relatively minor role is attributed to these elements in the overall effect of the plasma. No chromatographic or electrophoretic characterizations of the toxoid preparation were found in the literature or Western blot of patients before and after immunization. A study done on the level of various immunoglobulin classes in the process of immunization and plasmapheresis of plasma donors found that a significant rise in the level of IgM was observed first while class G immunoglobulins rose only at the beginning of plasmapheresis (7 days after third and final immunization), but whether this affected immunoglobulin levels in plasma samples was not investigated [38]. 2.5. Adverse effects
Immunization with the toxoid according to the standard schedule and dosage is reported to produce no local or general reaction in donors, with all clinical, morphological, and biochemical indicators remaining in normal 1385
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bounds during the period of immunization and plasmapheresis [39]. Vygodchikov notes that in practice no case was ever observed in which administration of toxoid was halted because of side effects or complications and that during immunization even with large doses, a rare transient reaction in the form of a rash and slight pain at the site of the injection, accompanied by a small rise in temperature was the worst effect ever observed. This usually disappeared within 24 h [29]. Since this statement, as noted, a more sophisticated method of toxoid purification has been in use. A recently published study of use of the toxoid in prophylaxis also states that no side effects were observed [36]. 2.6. Drying and storage of plasma
Plasma not immediately transfused to patients is kept at –45 °C or lyophylized for intravenous or local administration [40, 41]. The dry antistaphylococcal preparation preserved under various tested conditions (+20, +4, or –30 °C) retains its serological activity at the original level for 3 years (period of observation), though immediately after drying, it shows some degree of reduction in the level of protein. Batches with anti-α-toxin antibodies no less than 6 IU/ mL are used for immediate transfusion to patients. Lower titered batches (plasma with titer from 3–5 IU/mL) are stored and used to prepare ASIG, though ASIG is also obtained from ASP of higher titers (from 6–20 IU/mL). The concentration of anti-α-staphylotoxin antibodies in the prepared ASIG is generally 6–8 times that of standard plasma. In 1994 an antistaphylococcal IVIG, developed by the Institute of Hematology and Blood Transfusion in Kirov, is first mentioned in an article describing its stability in storage. The study reports that the preparations fulfill all requirements for an IVIG and are stable over a period of 12 months when kept at 6 ± 4.0 °C [42].
3. Administration Skurkovich and his colleagues produced the first ASP and ASIG in 1967 [43]. After promising preliminary experience with the plasma in treating advanced cases of infection unresponsive to antibiotic therapy, its use spread as quickly as production permitted because of the urgency of need [5]. The first report describing the clinical application of ASP was published in 1969 [39] and was soon followed by a series of articles reporting results of its use in patients with various diseases of staphylococcal etiology. ASP and ASIG were developed and put into clinical use under conditions of an escalating crisis. Probably because of the constant strain put on the supply of the plasma, which was at first available only sporadically [5], it became standard clinical practice in Russia to use it only after standard antibiotic therapies had failed. In other words, ASP is generally an adjunct therapy used as a reserve defense in critical cases. As described in the literature, it is commonly administered several days, if not weeks or months, after the first signs of infection, its use initiated after unsuccessful treatment with a series of antibiotics, 1386
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normal plasma, γ-globulin or blood transfusions, and supportive care – all referred to as 'complex therapy.' The preparations are reported to produce an immediate evident effect after administration, described in the early literature as 'striking'. especially in advanced cases [44]. These circumstances of the introduction into general use of the preparations may explain why no placebocontrolled, let alone randomized, trials, which would have required withholding them from some patients, were ever conducted. Investigations of the mechanism of action of ASP in experimental models were not performed until years after it was in common use (discussed below). Studies of the clinical use of the immune preparations are numerous, but almost all retrospective. Unfortunately, more often than not, the studies are poorly designed and little primary data is provided except in the form of a few case studies cited as examples. Patients with widely varying sites and severity of infection, complications, and length of disease are often lumped together, though no statistical analysis takes such subgroups into account. Data on the control group are usually not given beyond a statement that the patients in this group were picked on the basis of having comparable disease. Staphylococcal strains isolated from the blood or sites of infection are said to be 'polyresistant' to antibiotics or 'highly resistant' to one or more of a series of antibiotics, but it is not clear how many or which patients were treated with the antibiotics on this list, which include methicillin, oxacillin, chloracid, ristomycin, and others. Vancomycin seems to have been available but is mentioned in only one report [12]. Finally, where P values are provided, the statistical method used is not always given. To provide as much information as concisely as possible on clinical studies, the results of the larger, more detailed ones are summarized in table I. Preliminary studies of fewer than 10 patients were excluded, as were some studies that were so confusing or complex as to defy summary in a table. These included studies on the use of the preparations in burns, eye diseases, and ear, nose and throat diseases. As can be seen from the table, studies of ASP and ASIG generally base evaluation of the preparations either on case-control studies or a kind of within-patient, cross-over study. None of the studies were randomized. Case-control studies compare standard therapy (generally, antibiotics, normal plasma or γ-globulin, vitamins and other supportive care) with and without use of ASP or ASIG in parallel groups of patients. Mortality, recovery, or number of hospital days are the key endpoints, though occasionally laboratory data, in particular on nonspecific immunity, such as phagocytic activity, are statistically compared. The studies are mostly retrospective, with the control group constituted from records of patients not treated with ASP or ASIG because it was not available. One prospective study of the prophylactic use of orally administered ASP in neonates is the most recent of all the published studies (1988) [10]. The second type of study, the within-patient, cross-over studies, were done without statistical analysis. Efficacy is usually considered demonstrated by a rapid normalization of temperature and hematological signs and elimination of Microbes and Infection 2000, 1383-1392
Condition treated
No. of pts.
Treatment/study design
Response
Comments
no control. Given ASIG (58), ASP (12), blood transfusion from immunized donors (18), or combination (16), 5–30 days after CT showed little effect. no control. ASIG (61) and ASP (49) given after failure of CT and surgical intervention.
One death; 103 recovered 72 showed staphlococci in blood after antibiotics, only eight did so after immune preparations added to treatment. five deaths; 45 recovered, 60 improved enough to be released; Drop in CRP significantly correlated with clinical improvement. Attacks stopped in 12 (general improvement after 2–3 transfusions), decreased in eight, little effect in four, but longterm hormone tx reduced in these. 45 complete recovery, 26 significant clinical improvement; 5 chronic osteomyelitis – no effect; tx group: post-tx lysozyme and complement up 2–3x. See table II for comparisons of selected subgroups.
single death attributed to late start of treatment in 2-month-old with meningoencephalitis at birth.
Severe, mostly generalized forms of staphylococcal infection (meningoencephalitis, pneumonia) [45]
104 children: 34 < 3 mos. 22 3–6 mos. 48 1 year
Varied: mostly staphylococcal pneumonia involving lung destruction (50), sepsis (31) [46]
110 children: 45 < 1 year 42 1–3 23 > 3
Allergic form of bronchial asthma of staphylococcal etiology [47]
24:19–71 years
no control. ASP after long-term use of corticosteroids. Antibiotics given in seven cases with chronic pneumonia.
Pyosurgical infections 47 purulent wounds 39 chronic osteomyelitis [48]
118 children: 3–14 years: 86 tx 32 controls
control group of 32 with ‘similar’ diseases given CT. Tx group given CT + ASP. Study retrospective or prospective unknown.
Various diseases of staphylococcal etiology [49]
964: 768 adults and children 196 controls
controlled, retrospective, multiple subgroups by condition. ASIG + CT vs. CT alone, comparisons made by disease.
Neonates with local and generalized staphylococcal infection [50]
80: 50 local, 30 sepsis 20 + 20 controls
Prophylaxis in neonates and premature [36]
125: 75 neonates, incl. 28 premature; 50 controls 58: 31 tx group; 27 controls; ages unknown
controlled, retrospective; ASP + CT vs. CT alone; comparison of days in hospital for one group with local infection and one group with sepsis. controlled, prospective ASP vs. no tx. A single oral dose of 50 IU/kg body weight given after 1st feeding to tx group. controlled; retrospective assumed Tx group: ASP begun after CT proved ineffective; Control group: CT without ASP.
Septic postoperative endocarditis of staphylococcal etiology [44]
Prophylaxis in patients given prosthetic heart valves [44]
29: ages unknown
one local infection in tx group (omphalitis) vs. 12 local, one sepsis in control after 3-month observation. 22/27 died in control (81.5 %) 6/31 died in tx group (19.3 %).
no cases of septic endocarditis developed vs. 21% in those surviving first day after transplant among historic controls.
no information given on control group outcomes other than lack of significant response of lysozyme and complement activity. no complications, 0.6% a little allergic; lysozyme, complement, and phagocytic activity significantly increased compared to controls. P < 0.001 (fewer days for both local and sepsis in tx group).
no serious side effects. Within 24 h, anti-α-toxin in blood up from 0.68 to 2.9 IU/mL, 1.5 IU/ml after 3 months. no details on patients except tx group included more serious heart defects, e.g., Fallot’s tetralogy, Ebstein’s anomaly. Antibiotics included methicillin, oxacillin. no details given on patients. Review
1387
historical controls. ASP was given patients 2–3 days after operations to prevent septic endocarditis.
Tx group vs. controls: 34.8 ± 1.8 days vs. 50.6 ± 3.1 days (sepsis) 17 ± 0.5 days vs. 24 ± 1.4 days (local).
deaths attributed to bacteria of phage type 42E – tx had no effect; 3 children given ASP had quickly passing post-transfusion allergic reaction. one patient on highest dose of ASP had anaphylactic type reaction, two suffered hives; others no reaction.
Antistaphylococcal hyperimmune plasma
Microbes and Infection 2000, 1383-1392
Table I. Summary of studies of use of ASP or ASIG.
ASP, antistaphylococcal plasma; ASIG, antistaphylococcal immunoglobulin; CT, complex therapy, i.e., standard therapy which includes antibiotics, vitamins, blood transfusions and/or normal plasma, prednesolone. Sepsis defined as generalized infection. No distinction between sepsis, severe sepsis, or septic shock. CRP, C reactive protein; tx, treatment; cntrl, control group. treatment;
cntrl,control
group.
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Table II. Use of ASIG + complex therapy (tx) vs complex therapy alone (cntrl) [49].* Condition Diseases of skin and soft tissue Staph. pneumonia -simple staph.pneumonia -complex Acute osteomyelitis Chronic osteomyelitis Staph. enterocolitis
Group
# of Pts.
Full recovery
Satisfactory
No effect
tx cntrl tx cntrl tx cntrl tx cntrl tx cntrl tx cntrl
372 26 64 18 48 48 105 36 52 20 47 12
354 2 44 0 26 3 92 8 6 0 15 0
18 14 20 12 20 9 9 12 46 0 27 2
0 10 0 6 2 36 4 16 0 20 5 10
* ‘Satisfactory’ not defined; tx, treatment; cntrl, control group.
bacteremia within 2–3 days of administration of the immune preparations after antibiotics had been tried and failed – often after a long treatment course – in a group of patients. The time between start of infection and administration of the immune preparations was sometimes one or many more weeks. In all cases, the presence (or absence) of S. aureus was determined by bacteriological investigation. One case is cited in the literature of the plasma having no effect on a particular strain of S. aureus – phage type 42E [46]. In a review article Skurkovich also mentions that in very rare cases the ASP has little effect [51]. Table II provides results of the largest case-controlled study of the use of ASIG. Statistical analyses are not given here, since the results clearly favor the use of ASIG. The study appears to be retrospective. 3.1. Dosage and response
ASP is usually administered intravenously in quantities of 5–10 mL/kg of body weight (activity at least 5 IU/mL) daily or at 1–3-day intervals, depending on the severity of the condition. ASIG is injected intramuscularly at a dose of 5–15 IU/kg of body weight daily or every other day for 5–6 days. No controlled studies comparing clinical or laboratory parameters of patients are reported, but descriptions of clinical responses to ASP administration across all the studies are quite consistent. These can be summarized as follows. When ASP or ASIG is given in early stages, for example, before 7 days to infants with generalized infection, a rapid drop in temperature, a decrease in other signs of intoxication, and improved sense of well-being (e.g., increased appetite, etc.) are apparent by the first or second dose. S. aureus is usually undetectable in the blood after a typical course of transfusions. Given in late stages to infants (after 20 or more days), signs of clinical improvement are observed somewhat later (after 3–5 administrations of the preparation). Reparative processes at the sites of infection and a tendency toward normalization of the peripheral blood is observed in most patients within 1–2 weeks after the end of the treatment in these cases [46]. Normalization of temperature is the key clinical parameter mentioned in most published studies. Blood pressure, 1388
for example, seems not to have been measured. While the published studies of the clinical use of the preparations report uniformly positive results, such as normalization of temperature and hematological signs, few pre- and posttreatment laboratory data are provided on their effects besides antitoxic titers and immunological indices, such as Ig classes, B and T lymphocytes, or indices of nonspecific humoral activity, e.g., lysozyme, complement, and phagocytic activity. 3.2. Antitoxic activity
As reported, when these preparations are given to healthy people, maximum anti-α-toxin activity in the blood is reached within the first day after injection, remains high for 2–3 days, then gradually drops off, staying elevated for 16–19 days. When given to patients with generalized infections – distinctions were not made, as they are today, among sepsis, severe sepsis, and septic shock – no sharp rise in the level of specific antibodies is usually noted in the blood for a number of days, especially in the case of severe intoxication, until a certain neutralizing activity of the preparation on the staphylococcal toxins is reached. In this connection, a discussion in a clinical study notes a direct correlation between a rise in titer of both antitoxins (2–6-fold) and specific agglutinins (2–4-fold) and treatment efficacy. In other words, a high titer several days after infusion signaled greater likelihood of complete recovery. No movement or a drop in titer was associated with a poor prognosis. A close correlation purely between activity and dose of the administered preparation and subsequent rise in titer was not observed. The authors conclude that the protective action of the preparations depends not only on the neutralizing activity of the infused antibodies, but also on 'the activation of antibody-formation in the organism' [46]. This seems to confirm the traditional understanding of the basis for the efficacy of the preparations, namely, that by effectively neutralizing the toxins, the plasma allows the immune system to regain its own defensive processes. A statistically significant rise in percent of lysozyme activity and, as noted below, indices of both ingestion and digestion of microbial cells by neutrophils in patients receiving ASP compared with those not given ASP has also been demonstrated [49, 52]. Microbes and Infection 2000, 1383-1392
Antistaphylococcal hyperimmune plasma
A comparison of the effects of the infusion of ASP vs normal plasma on anti-α-staphylotoxin titer was made in a case-control study examining humoral factors of nonspecific immunity [48]. Titers were measured in children (3–14 years) with suppurative surgical infections before and after treatment with standard antibiotic therapy plus either ASP (47 patients) or normal plasma (32 patients). Pretreatment titers in both patient groups averaged about 1.24 ± 0.19 IU/mL (normal plasma is 0.85 (0.11 IU/mL). On the third day after the ASP transfusion (6–18 IU/mL, 4–8 mL/kg body weight), average titer in the blood reached 1.48 ± 0.16 IU/mL. By the 10th day this had climbed to 3.37 ± 0.33 IU/mL. In contrast, in the patients receiving the normal plasma, a slight but insignificant drop in average titer occurred on day 3, which was still unchanged by day 10. Controlled animal studies conducted in 1984 to investigate the isolated effect of the plasma on the immune system, i.e. without the involvement of antibiotics, also confirm a stimulatory effect of the antitoxic plasma on the immune system [53]. Guinea pigs in whom generalized infections were induced by intramuscular injections of S. aureus cells were inoculated with the ASP (human) and compared with a control group of similarly infected animals not receiving the ASP. The plasma reduced the number of staphylococcal colonies in the spleen and affected mainly the system of neutrophils with a lesser effect on the T and B lymphocytes. For example, by day 3 and day 14 (the moment of resolution of generalization), the percentage of active neutrophils with the Fcγreceptor had increased significantly in the animals receiving the ASP compared with the initial level and with controls. By day 14, the level of lymphocytes with receptors for staphylococci had also increased in comparison to initial level and controls. The authors conclude that the plasma seems to be acting, not only by neutralizing the toxins, but also through its opsonizing and neutrophil- and lymphocyteactivation effects, stimulating the active ingestion and degradation of the microbes [53]. 3.3. Adverse effects of preparations
Most early investigations on the use of ASP and ASIG specifically report no side effects. Very rarely, an occasional brief allergic reaction such as swollen eyelids or hives at the injection site (of ASIG) or a post-transfusion (of ASP) allergic-type reaction in some patients is mentioned. Only one serious side effect, an anaphylactic-type reaction, is reported in one case of a patient with bronchial asthma. The patient was given a very high dose of the plasma in a study testing its use for the first time in 24 patients with bronchial asthma of staphylococcal etiology after failure of hormonal and other treatment [47]. Later reports, after dose and regimes of ASP transfusions became more standardized, usually do not mention side effects. 3.4. Transmission of infection
Only one case of hepatitis (type not given) is mentioned in the ASP literature and that in a case description included in a larger study. This of course could have equally resulted from a normal plasma transfusion as from the ASP, since normal plasma was a part of standard therapy. It is emphaMicrobes and Infection 2000, 1383-1392
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sized elsewhere that donors were screened very strictly for history or signs of viral hepatitis. HIV was unknown when most of the reports discussed were published.
4. Discussion ASP and ASIG have been in wide use in Russia and some countries of Eastern Europe now for nearly 30 years. The massive application of the antistaphylococcal preparations is credited with reducing mortality in the USSR from suppurative septic conditions by 2– 4 times [54]. The available reports on the use of ASP and ASIG of course raise many questions not answered by the published literature. It is hoped that this review will help open dialog with Russian specialists who probably know much more about the preparations than can be found in the written literature. Studies of the plasma have not been done in light of more recent understanding of S. aureus or even of the immune system that exploded after HIV came on the scene, and the primacy given the role of toxins in pathology may have prevented further interrogation of assumptions on the mechanisms of action of the plasma. Staphylococcal superantigens, for example, were not known in this period. (The word 'superantigen' appears only twice in Medline in the Russian language literature.) Cytokines are never mentioned. Given the lack of randomized trials, the scarcity of primary clinical and laboratory data provided, the relatively small numbers of patients in the controlled studies, and the loose structure of the studies, most of which are retrospective, it is not possible to make categorical pronouncements on the efficacy of these preparations. At best the testing is at a phase II level. But when examined together, the results reported are encouraging enough to warrant further study and randomized testing of these preparations in the West, perhaps using the Gemaleya Institute toxoid. Unlike antibiotics, the effect of ASP and immunoglobulin appears to be immediate, its use has not generated drug-resistant bacterial strains, and it apparently produces no more side effects than normal plasma. As an adjunct therapy, it can be administered without the need to isolate and test strains for drug sensitivity, and doses can generally be raised without increased risk. Plasma has been recognized to be more effective than immunoglobulin prepared for intravenous use [55] and has other advantages. No additional costly and time-consuming steps, which may themselves induce modifications of the IgG, are needed to prepare it for use. In combination with antibiotics, it may increase sensitivity of the bacteria to the action of antibiotics through a synergistic effect [56]. In this connection, Fedorovskaya (1969) found in experiments on cultures of staphylococci resistant to several antibiotics, that in vitro contact of the staphylococci with γ-globulin-containing antistaphylococcal agglutinins resulted in the restoration of the sensitivity of some strains of staphylococci to antibiotics, at least temporarily [57]. Plasma also contains many other elements besides antitoxic antibodies, including, as noted, specific antimicrobial agglutinins, other immunoglobulins, albumin, fibrinogens, enzymes, miner1389
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als and other proteins as well as fluid, which are needed by patients in a state of reduced immune defenses and which are lost in isolating the IgG. Lyophylized, the preparation can be easily transported and stored for at least 3 years without loss of activity. An obvious and principal concern raised by use of plasma is the possibility of transmission of viruses, less likely with the use of purified immunoglobulins. This, of course, is not a risk to be ignored, but a risk undoubtedly much lower than that faced by a patient with advanced staphylococcal sepsis refractory to all antibiotics, a situation that may not be far away. In summary, the literature describing the development and use of ASP and ASIG in the Soviet Union during an epidemic of untreatable staphylococcal infections supports serious consideration of these therapies and offers one possible blueprint for an effective response to a threat we could soon be facing. The Russian experience with ASP disproves the contention that human immune preparations against bacterial infections are not feasible and encourages a new look at the further development of such preparations for other bacterial diseases. It still remains a challenge to create therapies that can better the natural defense mechanisms of the human body, which these preparations appear to help restore.
Acknowledgments I wish to thank Dr Simon Skurkovich for his help in providing a number of published documents on the antistaphylococcal plasma not available in US libraries and for passing on questions to Professor A.K. Akatov of the Gemaleya Institute in Moscow on the staphylococcal strains predominant in the epidemic of the 1960s and 1970s in Russia and on the strains used in the preparation of the anatoxin. Neither had any part in the writing or organization of the manuscript and bear no responsibility for any statements in it.
References [1] Pennington J.E., Newer uses of intravenous immunoglobulins as anti-infective agents, Antimicrob. Agents Chemother. 34 (1990) 1463–1466. [2] Casadevall A., Scharff M.D., Return to the past: the case for antibody-based therapies in infectious diseases, Clin. Infect. Dis. 21 (1995) 150–161. [3] Krause R.M., Dimmock N.J., Morens D.M., Summary of Antibody Workshop: The role of humoral immunity in the treatment and prevention of emerging and extant infectious diseases, J. Infect. Dis. 176 (1997) 549–559. [4] Boyce J.M., Methicillin-resistant Staphylococcus aureus, Infect. Dis. Clin. North Am. 3 (1989) 901–913. [5] Skurkovich S., Facing the Coming Plague, World & I. (1998) 150–158. 1390
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[6] Okhotsky V.P., Skurkovich S.V., Bialik I.F., Demidova N.V., Krokhina M.A., Treatment of post-traumatic osteomyelitis of long tubular bones with antistaphylococcal plasma, Vestn Khirur I. I. Grekova. 114 (1975) 67–71 (in Russian). [7] Malanova N.L., Zakhar'evskaia N.S., Antistaphylococcal gamma-globulin in the treatment of staphylococcal eye infection, Vestn Oftalmol. 6 (1974) 38–40 (in Russian). [8] Kruchinina I.L., Skurkovich S.V., Smolikova V.I., Demidova N.V., Krokhina M.A., Topical use of dry antistaphylococcal plasma in children with ENT-diseases of staphylococcal etiology, Zh Ushn Nos Gorl Bolezn. 5 (1978) 49–52 (in Russian). [9] Eriukhin I.A., Rybkin A.K., Vorob'ev V.V., Kaleko S.P., Use of antegrade endolymphatic administration of antistaphylococcal and antipseudomonal plasma in the treatment of infectious complications in severe multiple trauma, Khirurgiia (Moscow) 3 (1995) 49–51 (in Russian). [10] Manolova E.P., Tret'iakevic Z.N., Khripko S.V., Mechetnaia I.N., Enhancement of antitoxic immunity in newborn infants by peroral administration of donor antistaphylococcal immunoglobulin, Zh Mikrobiol. Epidemiol Immunobiol. 1 (1988) 49–51 (in Russian). [11] Sapozhnikova V.S., Mal'tseva O.V., Stability of antistaphylococcal immunoglobulin for intravenous administration in the storage process, Gematol. Transfuziol. 39 (1994) 39–40 (in Russian). [12] Skurkovich S.V., Krokhina M.A., Demidova N.V., et al., Use of immune preparations possessing a directed action in patients with pyogenic complications after cardiac surgery. Proceedings: First Joint USA-USSR Symposium on Blood Transfusion, Moscow, USSR, October 18–25, 1976, DHEW Publication No. (NIH) 76–1246, 169-181. [13] Kuzin M.I., Sologub V.K., Kol'ker I.I., et al., Actual problems of immunoprophylactic and immunotherapy of burn infection, Burns. Includ. Therm. Inj. 10 (1983) 34–40. [14] Epifanov N.S., Review, in: Konstantinov V.N. (Ed.), Antistaphylococcal plasma (acquisition and clinical application), Alma-Ata, Kazakhstan Publishers, 1989, pp. 63–64 (in Russian). [15] Bhakdi S., Mannhardt U., Muhly M., et al., Human hyperimmune globulin protects against the cytotoxic action of staphylococcal alpha-toxin in vitro and in vivo, Infect. Immun. 57 (1989) 3214–3220. [16] Hungerer K.D., Ronneberger H., In vitro and in vivo efficacy of a toxin-neutralizing human staphylococcal immunoglobulin, Behring Inst. Mitt. 86 (1990) 170–184 (in German). [17] Ramisse F., Szatanik M., Binder P., Alonso J.M., Passive local immunotherapy of experimental staphylococcal pneumonia with human intravenous immunoglobulin, J. Infect. Dis. 168 (1993) 1030–1033. [18] Skurkovich S.V., Likhoded V.G., Krokhina M.A., et al., Investigation of the therapeutic efficacy of plasma of donors immunized with live Escherichia coli vaccines, Material of the 48th Scientific Session of the TNIIGPK on the Basic Results of Scientific Work of the Institute of Moscow, 1975, pp. 100–103 (in Russian). Microbes and Infection 2000, 1383-1392
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[19] Bialik I.F., Krokhina M.A., Davatdarova G.M., Arkhipova N.A., Use of antistaphylococcal, antiescherichia, antipyocyanic plasma in patients with fractures complicated by infection, Sov. Med. 3 (1977) 27–31 (in Russian). [20] Skurkovich S.V., Arkhipova N.A., Olshanskaya N.V., et al., Interferon-containing plasma: the preparation for treatment of viral infections, Biomedicine 29 (1978) 227–228. [21] Krokhina M.A., Davatdarova G.M., Cherkas G.P., Podgornaia L.G., Shinkarenko A.A., Production of a hyperimmune antitoxic donor plasma against Pseudomonas aeruginosa, Zh Mikrobiol. Epidemiol. Immunobiol. 9 (1980) 78–83 (in Russian). [22] Skurkovich S.V., Ol'shanskaia N.V., Eremkina E.I., Arkhipova N.A., Klinova E.G., Obtaining interferoncontaining donor plasma and a study of its therapeutic properties in herpes zoster, Probl. Gematol. Pereliv. Krovi. 22 (1977) 49–51 (in Russian). [23] Izvestia, November 6, 1984 (in Russian). [24] Skurkovich S.V., Klinova E.G., Eremkina E.I., Levina N.V., Immunosuppressive effect of an anti-interferon serum, Nature 417 (1974) 551–552. [25] Skurkovich S.V., Skorikova A.S., Dubrovina N.A., et al., Lymphocytes'cytotoxicity towards cells of human lymphoblastoid lines in patients with rheumatoid arthritis and systemic lupus erythematosus, Ann. Allergy 39 (1977) 344–350. [26] Timofeev G.A., Zhuravlev V.A., Dumkin I.M., Pavlovskaia E.N., Koz'minykh L.F., Means for increasing the preparation of immune antistaphylococcal plasma for the production of immunoglobulins, Gematol Transfuziol. 29 (1984) 55–57 (in Russian). [27] Rusanov V.M., Sapozhnikova V.S., Porokhnenko S.G., Optimal industrial conditions for the production of antistaphylococcal immunoglobulin, Gematol Transfuziol. 35 (1990) 34–36 (in Russian). [28] Roitt I.M., Brostoff J., Male D.K., Immunology. Fifth ed., Mosby, London, 1998, p. 265. [29] Vygodchikov G.V., Staphylococcal Infections (Microbiology, Immunology, and Epidemiology), State Publishers of Medical Literature, Moscow, 1963, pp. 142–166 (in Russian). [30] Delaunay A., L'immunité antistaphylococcique conferée par l'anatoxin specifique et son méchanisme, Rev. Immunol. 4 (1938) 65. [31] Akatov A.K., Prokhorov V.I., Witte W., Kuhn M., Seltmann G., Staphylococcus aureus III. Protective activity of capsular antigen in experiments on mice, Zh Mikrobiol Epidemiol Immunobiol. 11 (1978) 47–52 (in Russian). [32] Ramon G., Sur le pouvoir floculant et les propriétés immunisantes d'une toxine diphtherique rendue anatoxique (anatoxine), C.r. hebd. Séances Acad Sci. 177 (1923) 1338–1340. [33] Schemanova G.F., Akatov A.K., Tolovskaya K.R., Preparation of highly purified preparations of staphylococcus anatoxin, Voprosy Med Khim. 12 (1966) 542–544. [34] Skurkovich S.V., Krokhina M.A., Demidova N.V., Lukovkina E.N., Rodina R.I., Aksenova O.V., Donor immunization schemes for obtaining hyperimmune antistaphylococcal plasma, Probl. Gematol. Pereliv. Krovi. 19 (1974) 58–59 (in Russian). Microbes and Infection 2000, 1383-1392
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[35] Konstantinov V.N., Pozhitkova T.F., Blinetskaia Z.S., Indicators of cellular immunity in donors of antistaphylococcal plasma, Gematol Transfuziol. 30 (1985) 35–39 (in Russian). [36] Manolova E.P., Tretyakevic Z.N., Khripko S.V., Inducing the development of specific immunity to staphylococcal infection in newborn infants by the intranasal administration of adsorbed staphylococcal toxoid, Zh. Mikrobiol. Epidemiol. Immunobiol. 7–9 (1989) 64–67 (in Russia). [37] Katkhanov A.M., Borovikov O.V., Prokhorov V.Y., Ratgauz G.L., Vasilyeva I.V., Noskova I.S., Antigen-specific and antigen-nonspecific reactions of the immunity system after immunization with purified and concentrated staphylococcal toxoid, Zh. Mikrobiol. Epidemiol. Immunobiol. 3 (1987) 34–38 (in Russia). [38] Korepanov A.M., Korepanov A.A., Sokolova I.V., Alypova E.V., Dynamics of the immunoglobulin level and antibody titer in the course of immunization with staphylococcal anatoxin and plasmapheresis of donors, Vrach Delo. 6 (1986) 7–9 (in Russia). [39] Skurkovich S.V., Papko G.F., Rodina R.I., et al., Acquisition and clinical application of hyperimmune antistaphylococcus human plasma and of hyperimmune antistaphylococcal gamma globulin with directed effect, Prob. Gematol. Pereliv. Krovi. 14 (1969) 3–7 (in Russian). [40] Krokhina M.A., Chesnokova V.F., Demidova N.V., Podol'skii M.V., Skurkovich S.V., Production of dry hyperimmune antistaphylococcal plasma and the study of some of its properties during storage, Probl. Gematol. Perliv. Krovi. 17 (1972) 12–13 (in Russian). [41] Krokhina M.A., The acquisition of dry antistaphylococcal plasma and the study of its serological and therapeutic properties, Institute of Hematology and Blood Transfusion, PhD thesis, Moscow, 1974 (in Russian). [42] Sapozhnikova V.S., Mal'tseva O.V., Stability of antistaphylococcal immunoglobulin for intravenous administration in the storage process, Gematol Transfuziol. 39 (1994) 39–40 (in Russian). [43] Skurkovich S.V., Reports of the 43rd plenum of the union of scientists of the Central Institute of Blood Transfusion, Moscow, 22 (1967) 328–330 (in Russian). [44] Nezhlukto A.I., Chekanov V.S., Krokhina M.A., Prophylaxis and treatment of purulent septic complications in cardiac surgery with the use of antistaphylococcal plasma, Grudn Khir. 6 (1981) 39–42 (in Russian). [45] Timofeeva G.A., Krotova T.A., Smirnova A.I., Danilova V.A., Lipatova L.V., Use of antistaphylococcal gamma-globulin, antistaphylococcal plasma, and immune blood of donors in the complex therapy of staphylococcal infections in children of the first year of life, Pediatriia 10 (1978) 18–22 (in Russian). [46] Krotova T.A., Gerasimova M.V., Koloshtivina O.V., Kozlova V.B., Kuzmin V.A., Use of antistaphylococcal polyglobulin and antistaphylococcal plasma in children with various staphylococcal diseases, Probl. Gematol. Pereliv. Krovi. 20 (1975) 28–31 (in Russian). [47] Al'perin P.M., Skurkovich S.V., Dubrovina N.A., Krokhina M.A., Demidova N.V., Use of antistaphylococcal plasma in the treatment of patients with an infectiousallergic form of bronchial asthma, Probl. Gematol. Pereliv. Krov. 20 (1975) 32–37 (in Russian). 1391
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[48] Konstantinov V.N., Sultanbaev T.Z., Blinetskaia Z.S., Humoral factors of nonspecific immunity in children with suppurative surgical infection after antistaphylococcal plasma transfusions, Vestn Khir. 129 (1982) 134–138 (in Russian). [49] Golosova T.V., Anikina T.P., Zakhar'evskaia N.S., Antistaphylococcal γ-globulin in complex therapy of suppurativeinflammatory staphylococcal diseases, Klin. Med. (Mosk). 52 (1974) 115–120 (in Russian). [50] Ebels I.G., Dumesh V.I., Vasina E.F., et al., Clinical aspects of antistaphylococcal plasma use in infants with various manifestations of staphylococcal infection, Pediatriia (1981) 49–50 (in Russian). [51] Skurkovich S.V., Immunoglobulins (hyperimmune plasma) of directed action, Sov. Med. 7 (1977) 103–108 (in Russian). [52] Kuzin M.I., Sologub V.K., Kolker I.I., Papko G.F., Panova I.U.M., Clinico-immunological substantiation for application of hyperimmune antistaphylococcal plasma in the complex treatment of patients with severe burns, Vestn. Akad. Med. Nauk. SSSR (1978) 19–25 (in Russian).
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[53] Snastina T.I., Belotskii S.M., Filiukova O.B., Therapy of experimental staphylococcal infection with a hyperimmune antistaphylococcal plasma, Zh. Mikrobiol. Epidemiol. Immunobiol. 8 (1984) 34–37 (in Russian). [54] Konstantinov V.N., Antistaphylococcal plasma (acquisition and clinical application) (1988) 38 (in Russian). [55] Dwyer J.M., Thirty years of supplying the missing link. History of gamma globulin therapy for immunodeficient states, Am. J. Med. 76 (1984) 46–52. [56] Sonea S., Borduas A., Frappier A., Combined protective action of human gamma globulin and antibiotics when administered simultaneously in experimental staphylococcal infections, Rev. Can. Biol. 17 (1958) 110–115. [57] Fedorovskaia E.A., Effect of antistaphylococcal gamma globulin on staphylococcus resistant to antibiotics, Vrach. Delo. 4 (1969) 117–120 (in Russian).
Microbes and Infection 2000, 1383-1392
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Immunotherapy against antibiotic-resistant bacteria: the Russian experience with an antistaphylococcal hyperimmune plasma and immunoglobulin Jeanne Kelly Coates & Jarratt, Inc., 4455 Connecticut Avenue, A500, Washington, D.C. 20008, USA
ABSTRACT – The Russian experience with the preparation and clinical application of an antitoxic antistaphylococcal hyperimmune plasma and immunoglobulin is described. The immunotherapies were developed in the late 1960s and put into widespread use in the Soviet Union for the prophylaxis and treatment of sepsis, pneumonia, and other conditions caused by an epidemic of antibiotic-resistant Staphylococcus aureus. © 2000 Éditions scientifiques et médicales Elsevier SAS staphylococcal / antibiotic-resistant / immunotherapy / hyperimmune plasma
1. Introduction In the face of growing bacterial resistance to antibiotics, passive immunotherapy has received renewed attention as an alternative treatment [1–3]. Missing in all such discussions published in the West, however, is any mention of the unique Russian experience with antibacterial immunotherapies from human blood, particularly an antistaphylococcal hyperimmune plasma and immunoglobulin widely used to combat antibiotic-resistant strains of Staphylococcus aureus since the 1970s. In the 1960s and early 1970s multi-drug-resistant strains of S. aureus caused major bouts of nosocomial infections in Europe [4]. In Russia at this time, they reached epidemic proportions. As in Europe, strains 80/81 were identified as those responsible. Faced with alarming rates of mortality from staphylococcal infections throughout the country, Professor Simon Skurkovich, M.D., then chief of the Immunology Laboratory at the Central Institute of Hematology and Blood Transfusion in Moscow, developed what became the first hyperimmune antitoxic–antistaphylococcal plasma and immunoglobulin produced in humans [5]. The preparations are commercialized and widely used in Russia today to treat advanced staphylococcal infections, though demand may far exceed supply. More than 50 articles in the Russian literature describe the production, storage, and application in fresh and dry form of these preparations. They are used for the treatment or prophylaxis of infections in neonates, burn victims, and patients who have undergone cardiac surgery, and for the treatment of pneumonia, endocarditis, meningoencephaMicrobes and Infection 2000, 1383–1392
litis, osteomyelitis, local infections of the ear, nose, throat, and eyes, bronchial asthma of staphylococcal etiology, and other staphylococcal diseases. The use of the preparations in infections due to implanted prosthetic devices, not common in the 1970s and 1980s, is not described. The plasma has been administered intravenously, intraosseously [6], locally [7], topically [8], and even endolymphatically [9] and orally (to neonates) [10]. The immunoglobulin, first developed for intramuscular use, is usually administered in less severe cases or outside the hospital setting. The plasma has also been used to preserve xenoaortic valves before transplant, sprinkled over wounds in powdered form, soaked into bandages, and applied in the form of ointment or drops. More recently (1994), an antistaphylococcal intravenous immunoglobulin (IVIG) was described [11]. Despite their widespread use in Russia and parts of Eastern Europe and the many Russian-language publications on the subject, this author could not find a single reference to the preparations in the English literature. Exceptions are a report in a compendium of papers in English presented at a Soviet–American symposium [12] and an article on the use of the plasma in burns by Russian researchers [13]. So complete is the language barrier, that information on the antistaphylococcal preparations has so far existed in a kind of sealed isolation. Meanwhile, the Russian side appears to have assumed such immune preparations were also available in the West. A reviewer of a 1988 Russian book on the antistaphylococcal plasma chastised the author for omitting discussion of Western 1383
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work on this type of preparation, unaware there was none in use [14]. While some reports can be found in the Western literature on human plasma-derived preparations in experimental staphylococcal infections that have shown promising results, these have apparently not led to testing in humans [15–17]. It should be noted that the language barrier has also to some extent isolated the Russian medical research community and limited discourse with Western research on staphylococcus, little of which is referenced in Russian journals. The present review of the development and application of these preparations was written to break this barrier and make the details of this work available to the Western medical community. Before his emigration, Skurkovich developed other similar antibacterial preparations: one against several types of Escherichia coli [18], another against Pseudomonas aeruginosa exotoxin [19], and an interferon-containing plasma against viral infections [20], all shown to be promising at the phase I or II stage of clinical testing [21, 22], but apparently not put into widespread use after he left the Soviet Union. In 1984 the State Prize, the Soviet Union's highest honor, was awarded for the development and nationwide application of the antistaphylococcal plasma (ASP) and antistaphylococcal immunoglobulin (ASIG) [23]. Professor Skurkovich, best known in the West as the first to discover the role of cytokines in the pathogenesis of autoimmune diseases and a pioneer of anticytokine therapy for these diseases [24, 25], immigrated to the United States in late 1979. Russian physicians and researchers continued to find new uses for and expand production of the plasma and immunoglobulin after his departure. Articles and books on the plasma written in the 1980s emphasize refinements in techniques to maximize production [26] and increase lot-to-lot consistency of the plasma [27]. Experimental work was also conducted at this time to investigate the basis for the immunologic effects of the preparations. This report was prepared based on a careful reading of all available Russian articles and books on the preparations with special attention to controlled clinical trials and experimental work and to information that would allow other researchers to conduct independent investigations of the immunotherapies. All but one or two articles listed in Medline were available at the National Library of Medicine in Bethesda, Maryland.
2. Development and production of ASP and ASIG Choice of immunogen. A widely used Western immunology textbook states that “there are no vaccines against the numerous staphylococcal exotoxins” [28]. In fact ASP was first prepared in Russia by immunizing donors with such an antistaphylococcal vaccine in use since the 1930s, though it is called an anatoxin (toxoid), the word 'vaccine' being reserved for preparations made not from staphylococcal exotoxins but from somatic antigen. The anatoxin is similar in form and preparation to diphtheria and tetanus vaccines, that is, from adsorbed toxoid, as it is called in the West, made by treating staphylococcal exotoxins with 1384
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formalin to destroy toxicity while preserving the original toxins' immunogenicity. Though Skurkovich was the first to produce a hyperimmune antitoxic antistaphylococcal plasma in humans, he followed a long line of research going back to the 1930s and 1940s in the Soviet Union and Europe on staphylococcal toxins and their role in disease. This included work on the induction of high titers of antistaphylococcal antitoxins in human and animal blood by immunization with toxoid and extensive clinical study (in Russia) of toxoid for the prophylaxis and treatment of staphylococcal infections. Development of the antitoxic immune preparations was based on the then current understanding of a close correlation between circulating antitoxic titer and both antitoxic and antimicrobial immunity to staphylococcal infection. This work is summarized in the book Stafilokokkovyye Infektsii (Staphylococcal Infections) (1963) [29] by Vygodchikov, a leading Soviet expert in staphylococcus at the time. Work with rabbits in the 1930s had demonstrated that the higher the titer of antibodies to staphylococcal toxins in the blood, the stronger the immunity to natural or laboratory-induced staphylococcal infection [29]. The Russian Davidovich reported in 1937 – before nonspecific and cell-mediated immunity were understood – that immunization with staphylococcal toxoid produced antitoxins first, with other 'antibodies' raising cellular defenses, namely opsonizing and complement-fixing substances and agglutinins, developing more slowly [29]. Research performed by Delaunay in France (1938) [30] and confirmed by others, demonstrated that in immunized rabbits with antitoxic antibodies in their blood, in contrast to controls without such antibodies, a “strong polymorphonuclear leukocytic reaction and sharp rise in phagocytic activity was observed that localized and overpowered the staphylococci” [29]. Toxins, Vygodchikov concluded, “not only impede phagocytic functions of the leukocytes, but destroy them, exerting a leukocytolytic action.” Thus, by neutralizing the toxicity, antitoxins 'open the staphylococci to phagocytosis' [29]. This view differs from the approach in the West, where, for example, the role in pathology of α-toxin, the major cytolysin in S. aureus, is still under discussion [15], and cell wall antigens have preferably been used in vaccine development [17]. (The U.S. company NABI has reported positive results from the prophylactic use in children of a hyperimmune immunoglobulin prepared from microbial cells of S. aureus.) Capsular antigens as a vaccine candidate were tested in Russia in the 1970s and were found to have a weak protective effect in mice [31]. 2.1. Active vs passive immunization
As mentioned, active immunization with the toxoid was in practice at the time of the plasma development but is considered to produce a weak and not long-lasting immunity in vaccinees. It was not protective for patients who have reached an advanced stage of infection with an already weakened immune system. Passive immunization, on the other hand, immediately delivers high titers of antitoxic antibodies from healthy donors. Microbes and Infection 2000, 1383-1392
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2.2. Vaccine preparation
Skurkovich obtained the toxoid to immunize donors from the Gamaleya Institute of Microbiology, Epidemiology, and Immunology (Moscow). It was produced as described by Vygodchikov from all toxins isolated from the strain of S. aureus known as O-15 (equivalent to Wood-46), considered the most toxigenic [29], inducing antibodies to toxins from a wide range of strains. The Gemaleya Institute routinely supplies the toxoid to blood centers for immunization and ASP production, so details of its preparation are not included in any of the reports on the use of the antistaphylococcal plasma. Vygodchikov's method of preparing the toxoid [29], essentially follows the work of Ramon (1923) [32]. Cultures of S. aureus (taken from pus of infected individuals) were grown at the Gamaleya Institute on a laboratory-prepared 1-L caseine broth for 5 days at 37 °C with 20–30% CO2. On day 5, culture bottles are removed from the thermostat and the microbial mass separated by filtration. The filtrate, containing the toxins, is tested for activity. The hemolytic titer of the toxins for toxoid preparation must be no less than 1 000 Dhm (dosis hamolitica minima) after 1 h of incubation at 37 °C and 1 h at room temperature. To obtain the toxoid, formalin is added to the toxins at a concentration of 0.3–0.5%, and the mixture is incubated at 37 °C from 7–28 days to achieve full detoxification [29]. Today, the same strain of S. aureus is used, though commercially prepared media are undoubtedly available for growing the toxins. In 1965 standard methods for protein purification of the staphylococcal toxoid were described, namely, using precipitation with methanol, fractionation with (NH4)2SO4, ion-exchange chromatography with DEAE-cellulose, and Sephadex gel filtration. From this a toxoid preparation is obtained with an activity of 400 binding units per mg of protein nitrogen and 70% toxoid yield, described as a serologically pure preparation in precipitation reaction in agar with staphylococcal antitoxin [33]. 2.3. Immunization scheme
Skurkovich and his colleagues tested several schemes of donor immunization using the Gemaleya toxoid and found the most effective to be one requiring that 4 mL of toxoid be given subcutaneously in doses of 1.0, 1.0, and 2.0 mL at 7-day intervals [34]. This scheme permitted the shortest period of immunization and the use of the smallest dose of prepared toxoid vaccine to give the highest antibody titer with no side effects to donors. Donor plasma is collected by plasmapheresis on day 21 after start of immunization, that is, 7 days after the third immunization [34]. Following this scheme, 88% of donors were found to produce an anti-α-toxin (see below) titer ≥ 5 IU/mL with 65% generating a titer > 10 IU/mL, and some generating antibody levels as high as 26 IU/mL or more. The average titer is 12.7 ± 1.28 IU/mL. (Normal serum contains about 0.86 IU/mL.) When in 1974 the Ministry of Health of the Soviet Union mandated that all municipal and regional blood collection centers collect ASP, it was designated that this scheme be used. Occasionally, articles report using a slightly different scheme of 0.5, 1.0, and 2.0 mL. Microbes and Infection 2000, 1383-1392
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Titering of ASP is done by a neutralization assay measuring its ability to protect against the hemolytic action of staphylococcal α-toxin on rabbit erythrocytes, a method developed by Vygodchikov [29]. Because of the use of α-toxin (a sample of which is supplied by the Gemaleya Institute with the toxoid) to determine titer, the ASP is often referred to in the literature as anti-α-staphylotoxin or antiα-toxin. However, it should be noted that all toxins of the O-15 (Wood-46) strain are used for the toxoid. Since a total of only 2 to 3 L of immune plasma can be collected from each donor after a series of immunizations (10 collections of 250 mL, 2–4 times/month followed by a 3-month break), ways were later proposed to refine donor selection, optimize collection, and standardize the product by titer level. For example, a study of donor titer before and after immunization led to an attempt to select out a small percentage of donors found resistant to the action of the vaccine, probably a result of neutralization of the vaccine by pre-existing antibodies in the donors' circulation. Methods were devised to optimize collection by grouping and plasmapheresing donors according to the time their antibody titer reaches its peak, which varies widely among donors [35]. 2.4. Characterization of the toxoid
The Russian literature on the toxoid is extensive and probably deserves separate treatment. (Controlled studies of its use in prophylaxis report postive results, such as a report of the intranasal administration of the toxoid in neonates [36].) Probably because of the importance given toxin neutralization in treating staphylococcal disease with immunotherapy, other immunogenic elements in the toxoid have not been thoroughly investigated. One report, for example, on the antigen-specific and -nonspecific response to the toxoid in prophylaxis, states that in addition to a rise in the level of anti-α-toxin, immunization leads to a rise in titers of antibodies to the staphylococcal cellular wall [37]. Little else was found in the literature on the antibodies to the cell wall, though specific agglutinins to somatic antigen are reported as appearing in the blood of donors immunized for plasma collection (1:640 and above) [34] and are sometimes measured in treated patients. A relatively minor role is attributed to these elements in the overall effect of the plasma. No chromatographic or electrophoretic characterizations of the toxoid preparation were found in the literature or Western blot of patients before and after immunization. A study done on the level of various immunoglobulin classes in the process of immunization and plasmapheresis of plasma donors found that a significant rise in the level of IgM was observed first while class G immunoglobulins rose only at the beginning of plasmapheresis (7 days after third and final immunization), but whether this affected immunoglobulin levels in plasma samples was not investigated [38]. 2.5. Adverse effects
Immunization with the toxoid according to the standard schedule and dosage is reported to produce no local or general reaction in donors, with all clinical, morphological, and biochemical indicators remaining in normal 1385
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bounds during the period of immunization and plasmapheresis [39]. Vygodchikov notes that in practice no case was ever observed in which administration of toxoid was halted because of side effects or complications and that during immunization even with large doses, a rare transient reaction in the form of a rash and slight pain at the site of the injection, accompanied by a small rise in temperature was the worst effect ever observed. This usually disappeared within 24 h [29]. Since this statement, as noted, a more sophisticated method of toxoid purification has been in use. A recently published study of use of the toxoid in prophylaxis also states that no side effects were observed [36]. 2.6. Drying and storage of plasma
Plasma not immediately transfused to patients is kept at –45 °C or lyophylized for intravenous or local administration [40, 41]. The dry antistaphylococcal preparation preserved under various tested conditions (+20, +4, or –30 °C) retains its serological activity at the original level for 3 years (period of observation), though immediately after drying, it shows some degree of reduction in the level of protein. Batches with anti-α-toxin antibodies no less than 6 IU/ mL are used for immediate transfusion to patients. Lower titered batches (plasma with titer from 3–5 IU/mL) are stored and used to prepare ASIG, though ASIG is also obtained from ASP of higher titers (from 6–20 IU/mL). The concentration of anti-α-staphylotoxin antibodies in the prepared ASIG is generally 6–8 times that of standard plasma. In 1994 an antistaphylococcal IVIG, developed by the Institute of Hematology and Blood Transfusion in Kirov, is first mentioned in an article describing its stability in storage. The study reports that the preparations fulfill all requirements for an IVIG and are stable over a period of 12 months when kept at 6 ± 4.0 °C [42].
3. Administration Skurkovich and his colleagues produced the first ASP and ASIG in 1967 [43]. After promising preliminary experience with the plasma in treating advanced cases of infection unresponsive to antibiotic therapy, its use spread as quickly as production permitted because of the urgency of need [5]. The first report describing the clinical application of ASP was published in 1969 [39] and was soon followed by a series of articles reporting results of its use in patients with various diseases of staphylococcal etiology. ASP and ASIG were developed and put into clinical use under conditions of an escalating crisis. Probably because of the constant strain put on the supply of the plasma, which was at first available only sporadically [5], it became standard clinical practice in Russia to use it only after standard antibiotic therapies had failed. In other words, ASP is generally an adjunct therapy used as a reserve defense in critical cases. As described in the literature, it is commonly administered several days, if not weeks or months, after the first signs of infection, its use initiated after unsuccessful treatment with a series of antibiotics, 1386
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normal plasma, γ-globulin or blood transfusions, and supportive care – all referred to as 'complex therapy.' The preparations are reported to produce an immediate evident effect after administration, described in the early literature as 'striking'. especially in advanced cases [44]. These circumstances of the introduction into general use of the preparations may explain why no placebocontrolled, let alone randomized, trials, which would have required withholding them from some patients, were ever conducted. Investigations of the mechanism of action of ASP in experimental models were not performed until years after it was in common use (discussed below). Studies of the clinical use of the immune preparations are numerous, but almost all retrospective. Unfortunately, more often than not, the studies are poorly designed and little primary data is provided except in the form of a few case studies cited as examples. Patients with widely varying sites and severity of infection, complications, and length of disease are often lumped together, though no statistical analysis takes such subgroups into account. Data on the control group are usually not given beyond a statement that the patients in this group were picked on the basis of having comparable disease. Staphylococcal strains isolated from the blood or sites of infection are said to be 'polyresistant' to antibiotics or 'highly resistant' to one or more of a series of antibiotics, but it is not clear how many or which patients were treated with the antibiotics on this list, which include methicillin, oxacillin, chloracid, ristomycin, and others. Vancomycin seems to have been available but is mentioned in only one report [12]. Finally, where P values are provided, the statistical method used is not always given. To provide as much information as concisely as possible on clinical studies, the results of the larger, more detailed ones are summarized in table I. Preliminary studies of fewer than 10 patients were excluded, as were some studies that were so confusing or complex as to defy summary in a table. These included studies on the use of the preparations in burns, eye diseases, and ear, nose and throat diseases. As can be seen from the table, studies of ASP and ASIG generally base evaluation of the preparations either on case-control studies or a kind of within-patient, cross-over study. None of the studies were randomized. Case-control studies compare standard therapy (generally, antibiotics, normal plasma or γ-globulin, vitamins and other supportive care) with and without use of ASP or ASIG in parallel groups of patients. Mortality, recovery, or number of hospital days are the key endpoints, though occasionally laboratory data, in particular on nonspecific immunity, such as phagocytic activity, are statistically compared. The studies are mostly retrospective, with the control group constituted from records of patients not treated with ASP or ASIG because it was not available. One prospective study of the prophylactic use of orally administered ASP in neonates is the most recent of all the published studies (1988) [10]. The second type of study, the within-patient, cross-over studies, were done without statistical analysis. Efficacy is usually considered demonstrated by a rapid normalization of temperature and hematological signs and elimination of Microbes and Infection 2000, 1383-1392
Condition treated
No. of pts.
Treatment/study design
Response
Comments
no control. Given ASIG (58), ASP (12), blood transfusion from immunized donors (18), or combination (16), 5–30 days after CT showed little effect. no control. ASIG (61) and ASP (49) given after failure of CT and surgical intervention.
One death; 103 recovered 72 showed staphlococci in blood after antibiotics, only eight did so after immune preparations added to treatment. five deaths; 45 recovered, 60 improved enough to be released; Drop in CRP significantly correlated with clinical improvement. Attacks stopped in 12 (general improvement after 2–3 transfusions), decreased in eight, little effect in four, but longterm hormone tx reduced in these. 45 complete recovery, 26 significant clinical improvement; 5 chronic osteomyelitis – no effect; tx group: post-tx lysozyme and complement up 2–3x. See table II for comparisons of selected subgroups.
single death attributed to late start of treatment in 2-month-old with meningoencephalitis at birth.
Severe, mostly generalized forms of staphylococcal infection (meningoencephalitis, pneumonia) [45]
104 children: 34 < 3 mos. 22 3–6 mos. 48 1 year
Varied: mostly staphylococcal pneumonia involving lung destruction (50), sepsis (31) [46]
110 children: 45 < 1 year 42 1–3 23 > 3
Allergic form of bronchial asthma of staphylococcal etiology [47]
24:19–71 years
no control. ASP after long-term use of corticosteroids. Antibiotics given in seven cases with chronic pneumonia.
Pyosurgical infections 47 purulent wounds 39 chronic osteomyelitis [48]
118 children: 3–14 years: 86 tx 32 controls
control group of 32 with ‘similar’ diseases given CT. Tx group given CT + ASP. Study retrospective or prospective unknown.
Various diseases of staphylococcal etiology [49]
964: 768 adults and children 196 controls
controlled, retrospective, multiple subgroups by condition. ASIG + CT vs. CT alone, comparisons made by disease.
Neonates with local and generalized staphylococcal infection [50]
80: 50 local, 30 sepsis 20 + 20 controls
Prophylaxis in neonates and premature [36]
125: 75 neonates, incl. 28 premature; 50 controls 58: 31 tx group; 27 controls; ages unknown
controlled, retrospective; ASP + CT vs. CT alone; comparison of days in hospital for one group with local infection and one group with sepsis. controlled, prospective ASP vs. no tx. A single oral dose of 50 IU/kg body weight given after 1st feeding to tx group. controlled; retrospective assumed Tx group: ASP begun after CT proved ineffective; Control group: CT without ASP.
Septic postoperative endocarditis of staphylococcal etiology [44]
Prophylaxis in patients given prosthetic heart valves [44]
29: ages unknown
one local infection in tx group (omphalitis) vs. 12 local, one sepsis in control after 3-month observation. 22/27 died in control (81.5 %) 6/31 died in tx group (19.3 %).
no cases of septic endocarditis developed vs. 21% in those surviving first day after transplant among historic controls.
no information given on control group outcomes other than lack of significant response of lysozyme and complement activity. no complications, 0.6% a little allergic; lysozyme, complement, and phagocytic activity significantly increased compared to controls. P < 0.001 (fewer days for both local and sepsis in tx group).
no serious side effects. Within 24 h, anti-α-toxin in blood up from 0.68 to 2.9 IU/mL, 1.5 IU/ml after 3 months. no details on patients except tx group included more serious heart defects, e.g., Fallot’s tetralogy, Ebstein’s anomaly. Antibiotics included methicillin, oxacillin. no details given on patients. Review
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historical controls. ASP was given patients 2–3 days after operations to prevent septic endocarditis.
Tx group vs. controls: 34.8 ± 1.8 days vs. 50.6 ± 3.1 days (sepsis) 17 ± 0.5 days vs. 24 ± 1.4 days (local).
deaths attributed to bacteria of phage type 42E – tx had no effect; 3 children given ASP had quickly passing post-transfusion allergic reaction. one patient on highest dose of ASP had anaphylactic type reaction, two suffered hives; others no reaction.
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Table I. Summary of studies of use of ASP or ASIG.
ASP, antistaphylococcal plasma; ASIG, antistaphylococcal immunoglobulin; CT, complex therapy, i.e., standard therapy which includes antibiotics, vitamins, blood transfusions and/or normal plasma, prednesolone. Sepsis defined as generalized infection. No distinction between sepsis, severe sepsis, or septic shock. CRP, C reactive protein; tx, treatment; cntrl, control group. treatment;
cntrl,control
group.
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Table II. Use of ASIG + complex therapy (tx) vs complex therapy alone (cntrl) [49].* Condition Diseases of skin and soft tissue Staph. pneumonia -simple staph.pneumonia -complex Acute osteomyelitis Chronic osteomyelitis Staph. enterocolitis
Group
# of Pts.
Full recovery
Satisfactory
No effect
tx cntrl tx cntrl tx cntrl tx cntrl tx cntrl tx cntrl
372 26 64 18 48 48 105 36 52 20 47 12
354 2 44 0 26 3 92 8 6 0 15 0
18 14 20 12 20 9 9 12 46 0 27 2
0 10 0 6 2 36 4 16 0 20 5 10
* ‘Satisfactory’ not defined; tx, treatment; cntrl, control group.
bacteremia within 2–3 days of administration of the immune preparations after antibiotics had been tried and failed – often after a long treatment course – in a group of patients. The time between start of infection and administration of the immune preparations was sometimes one or many more weeks. In all cases, the presence (or absence) of S. aureus was determined by bacteriological investigation. One case is cited in the literature of the plasma having no effect on a particular strain of S. aureus – phage type 42E [46]. In a review article Skurkovich also mentions that in very rare cases the ASP has little effect [51]. Table II provides results of the largest case-controlled study of the use of ASIG. Statistical analyses are not given here, since the results clearly favor the use of ASIG. The study appears to be retrospective. 3.1. Dosage and response
ASP is usually administered intravenously in quantities of 5–10 mL/kg of body weight (activity at least 5 IU/mL) daily or at 1–3-day intervals, depending on the severity of the condition. ASIG is injected intramuscularly at a dose of 5–15 IU/kg of body weight daily or every other day for 5–6 days. No controlled studies comparing clinical or laboratory parameters of patients are reported, but descriptions of clinical responses to ASP administration across all the studies are quite consistent. These can be summarized as follows. When ASP or ASIG is given in early stages, for example, before 7 days to infants with generalized infection, a rapid drop in temperature, a decrease in other signs of intoxication, and improved sense of well-being (e.g., increased appetite, etc.) are apparent by the first or second dose. S. aureus is usually undetectable in the blood after a typical course of transfusions. Given in late stages to infants (after 20 or more days), signs of clinical improvement are observed somewhat later (after 3–5 administrations of the preparation). Reparative processes at the sites of infection and a tendency toward normalization of the peripheral blood is observed in most patients within 1–2 weeks after the end of the treatment in these cases [46]. Normalization of temperature is the key clinical parameter mentioned in most published studies. Blood pressure, 1388
for example, seems not to have been measured. While the published studies of the clinical use of the preparations report uniformly positive results, such as normalization of temperature and hematological signs, few pre- and posttreatment laboratory data are provided on their effects besides antitoxic titers and immunological indices, such as Ig classes, B and T lymphocytes, or indices of nonspecific humoral activity, e.g., lysozyme, complement, and phagocytic activity. 3.2. Antitoxic activity
As reported, when these preparations are given to healthy people, maximum anti-α-toxin activity in the blood is reached within the first day after injection, remains high for 2–3 days, then gradually drops off, staying elevated for 16–19 days. When given to patients with generalized infections – distinctions were not made, as they are today, among sepsis, severe sepsis, and septic shock – no sharp rise in the level of specific antibodies is usually noted in the blood for a number of days, especially in the case of severe intoxication, until a certain neutralizing activity of the preparation on the staphylococcal toxins is reached. In this connection, a discussion in a clinical study notes a direct correlation between a rise in titer of both antitoxins (2–6-fold) and specific agglutinins (2–4-fold) and treatment efficacy. In other words, a high titer several days after infusion signaled greater likelihood of complete recovery. No movement or a drop in titer was associated with a poor prognosis. A close correlation purely between activity and dose of the administered preparation and subsequent rise in titer was not observed. The authors conclude that the protective action of the preparations depends not only on the neutralizing activity of the infused antibodies, but also on 'the activation of antibody-formation in the organism' [46]. This seems to confirm the traditional understanding of the basis for the efficacy of the preparations, namely, that by effectively neutralizing the toxins, the plasma allows the immune system to regain its own defensive processes. A statistically significant rise in percent of lysozyme activity and, as noted below, indices of both ingestion and digestion of microbial cells by neutrophils in patients receiving ASP compared with those not given ASP has also been demonstrated [49, 52]. Microbes and Infection 2000, 1383-1392
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A comparison of the effects of the infusion of ASP vs normal plasma on anti-α-staphylotoxin titer was made in a case-control study examining humoral factors of nonspecific immunity [48]. Titers were measured in children (3–14 years) with suppurative surgical infections before and after treatment with standard antibiotic therapy plus either ASP (47 patients) or normal plasma (32 patients). Pretreatment titers in both patient groups averaged about 1.24 ± 0.19 IU/mL (normal plasma is 0.85 (0.11 IU/mL). On the third day after the ASP transfusion (6–18 IU/mL, 4–8 mL/kg body weight), average titer in the blood reached 1.48 ± 0.16 IU/mL. By the 10th day this had climbed to 3.37 ± 0.33 IU/mL. In contrast, in the patients receiving the normal plasma, a slight but insignificant drop in average titer occurred on day 3, which was still unchanged by day 10. Controlled animal studies conducted in 1984 to investigate the isolated effect of the plasma on the immune system, i.e. without the involvement of antibiotics, also confirm a stimulatory effect of the antitoxic plasma on the immune system [53]. Guinea pigs in whom generalized infections were induced by intramuscular injections of S. aureus cells were inoculated with the ASP (human) and compared with a control group of similarly infected animals not receiving the ASP. The plasma reduced the number of staphylococcal colonies in the spleen and affected mainly the system of neutrophils with a lesser effect on the T and B lymphocytes. For example, by day 3 and day 14 (the moment of resolution of generalization), the percentage of active neutrophils with the Fcγreceptor had increased significantly in the animals receiving the ASP compared with the initial level and with controls. By day 14, the level of lymphocytes with receptors for staphylococci had also increased in comparison to initial level and controls. The authors conclude that the plasma seems to be acting, not only by neutralizing the toxins, but also through its opsonizing and neutrophil- and lymphocyteactivation effects, stimulating the active ingestion and degradation of the microbes [53]. 3.3. Adverse effects of preparations
Most early investigations on the use of ASP and ASIG specifically report no side effects. Very rarely, an occasional brief allergic reaction such as swollen eyelids or hives at the injection site (of ASIG) or a post-transfusion (of ASP) allergic-type reaction in some patients is mentioned. Only one serious side effect, an anaphylactic-type reaction, is reported in one case of a patient with bronchial asthma. The patient was given a very high dose of the plasma in a study testing its use for the first time in 24 patients with bronchial asthma of staphylococcal etiology after failure of hormonal and other treatment [47]. Later reports, after dose and regimes of ASP transfusions became more standardized, usually do not mention side effects. 3.4. Transmission of infection
Only one case of hepatitis (type not given) is mentioned in the ASP literature and that in a case description included in a larger study. This of course could have equally resulted from a normal plasma transfusion as from the ASP, since normal plasma was a part of standard therapy. It is emphaMicrobes and Infection 2000, 1383-1392
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sized elsewhere that donors were screened very strictly for history or signs of viral hepatitis. HIV was unknown when most of the reports discussed were published.
4. Discussion ASP and ASIG have been in wide use in Russia and some countries of Eastern Europe now for nearly 30 years. The massive application of the antistaphylococcal preparations is credited with reducing mortality in the USSR from suppurative septic conditions by 2– 4 times [54]. The available reports on the use of ASP and ASIG of course raise many questions not answered by the published literature. It is hoped that this review will help open dialog with Russian specialists who probably know much more about the preparations than can be found in the written literature. Studies of the plasma have not been done in light of more recent understanding of S. aureus or even of the immune system that exploded after HIV came on the scene, and the primacy given the role of toxins in pathology may have prevented further interrogation of assumptions on the mechanisms of action of the plasma. Staphylococcal superantigens, for example, were not known in this period. (The word 'superantigen' appears only twice in Medline in the Russian language literature.) Cytokines are never mentioned. Given the lack of randomized trials, the scarcity of primary clinical and laboratory data provided, the relatively small numbers of patients in the controlled studies, and the loose structure of the studies, most of which are retrospective, it is not possible to make categorical pronouncements on the efficacy of these preparations. At best the testing is at a phase II level. But when examined together, the results reported are encouraging enough to warrant further study and randomized testing of these preparations in the West, perhaps using the Gemaleya Institute toxoid. Unlike antibiotics, the effect of ASP and immunoglobulin appears to be immediate, its use has not generated drug-resistant bacterial strains, and it apparently produces no more side effects than normal plasma. As an adjunct therapy, it can be administered without the need to isolate and test strains for drug sensitivity, and doses can generally be raised without increased risk. Plasma has been recognized to be more effective than immunoglobulin prepared for intravenous use [55] and has other advantages. No additional costly and time-consuming steps, which may themselves induce modifications of the IgG, are needed to prepare it for use. In combination with antibiotics, it may increase sensitivity of the bacteria to the action of antibiotics through a synergistic effect [56]. In this connection, Fedorovskaya (1969) found in experiments on cultures of staphylococci resistant to several antibiotics, that in vitro contact of the staphylococci with γ-globulin-containing antistaphylococcal agglutinins resulted in the restoration of the sensitivity of some strains of staphylococci to antibiotics, at least temporarily [57]. Plasma also contains many other elements besides antitoxic antibodies, including, as noted, specific antimicrobial agglutinins, other immunoglobulins, albumin, fibrinogens, enzymes, miner1389
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als and other proteins as well as fluid, which are needed by patients in a state of reduced immune defenses and which are lost in isolating the IgG. Lyophylized, the preparation can be easily transported and stored for at least 3 years without loss of activity. An obvious and principal concern raised by use of plasma is the possibility of transmission of viruses, less likely with the use of purified immunoglobulins. This, of course, is not a risk to be ignored, but a risk undoubtedly much lower than that faced by a patient with advanced staphylococcal sepsis refractory to all antibiotics, a situation that may not be far away. In summary, the literature describing the development and use of ASP and ASIG in the Soviet Union during an epidemic of untreatable staphylococcal infections supports serious consideration of these therapies and offers one possible blueprint for an effective response to a threat we could soon be facing. The Russian experience with ASP disproves the contention that human immune preparations against bacterial infections are not feasible and encourages a new look at the further development of such preparations for other bacterial diseases. It still remains a challenge to create therapies that can better the natural defense mechanisms of the human body, which these preparations appear to help restore.
Acknowledgments I wish to thank Dr Simon Skurkovich for his help in providing a number of published documents on the antistaphylococcal plasma not available in US libraries and for passing on questions to Professor A.K. Akatov of the Gemaleya Institute in Moscow on the staphylococcal strains predominant in the epidemic of the 1960s and 1970s in Russia and on the strains used in the preparation of the anatoxin. Neither had any part in the writing or organization of the manuscript and bear no responsibility for any statements in it.
References [1] Pennington J.E., Newer uses of intravenous immunoglobulins as anti-infective agents, Antimicrob. Agents Chemother. 34 (1990) 1463–1466. [2] Casadevall A., Scharff M.D., Return to the past: the case for antibody-based therapies in infectious diseases, Clin. Infect. Dis. 21 (1995) 150–161. [3] Krause R.M., Dimmock N.J., Morens D.M., Summary of Antibody Workshop: The role of humoral immunity in the treatment and prevention of emerging and extant infectious diseases, J. Infect. Dis. 176 (1997) 549–559. [4] Boyce J.M., Methicillin-resistant Staphylococcus aureus, Infect. Dis. Clin. North Am. 3 (1989) 901–913. [5] Skurkovich S., Facing the Coming Plague, World & I. (1998) 150–158. 1390
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[6] Okhotsky V.P., Skurkovich S.V., Bialik I.F., Demidova N.V., Krokhina M.A., Treatment of post-traumatic osteomyelitis of long tubular bones with antistaphylococcal plasma, Vestn Khirur I. I. Grekova. 114 (1975) 67–71 (in Russian). [7] Malanova N.L., Zakhar'evskaia N.S., Antistaphylococcal gamma-globulin in the treatment of staphylococcal eye infection, Vestn Oftalmol. 6 (1974) 38–40 (in Russian). [8] Kruchinina I.L., Skurkovich S.V., Smolikova V.I., Demidova N.V., Krokhina M.A., Topical use of dry antistaphylococcal plasma in children with ENT-diseases of staphylococcal etiology, Zh Ushn Nos Gorl Bolezn. 5 (1978) 49–52 (in Russian). [9] Eriukhin I.A., Rybkin A.K., Vorob'ev V.V., Kaleko S.P., Use of antegrade endolymphatic administration of antistaphylococcal and antipseudomonal plasma in the treatment of infectious complications in severe multiple trauma, Khirurgiia (Moscow) 3 (1995) 49–51 (in Russian). [10] Manolova E.P., Tret'iakevic Z.N., Khripko S.V., Mechetnaia I.N., Enhancement of antitoxic immunity in newborn infants by peroral administration of donor antistaphylococcal immunoglobulin, Zh Mikrobiol. Epidemiol Immunobiol. 1 (1988) 49–51 (in Russian). [11] Sapozhnikova V.S., Mal'tseva O.V., Stability of antistaphylococcal immunoglobulin for intravenous administration in the storage process, Gematol. Transfuziol. 39 (1994) 39–40 (in Russian). [12] Skurkovich S.V., Krokhina M.A., Demidova N.V., et al., Use of immune preparations possessing a directed action in patients with pyogenic complications after cardiac surgery. Proceedings: First Joint USA-USSR Symposium on Blood Transfusion, Moscow, USSR, October 18–25, 1976, DHEW Publication No. (NIH) 76–1246, 169-181. [13] Kuzin M.I., Sologub V.K., Kol'ker I.I., et al., Actual problems of immunoprophylactic and immunotherapy of burn infection, Burns. Includ. Therm. Inj. 10 (1983) 34–40. [14] Epifanov N.S., Review, in: Konstantinov V.N. (Ed.), Antistaphylococcal plasma (acquisition and clinical application), Alma-Ata, Kazakhstan Publishers, 1989, pp. 63–64 (in Russian). [15] Bhakdi S., Mannhardt U., Muhly M., et al., Human hyperimmune globulin protects against the cytotoxic action of staphylococcal alpha-toxin in vitro and in vivo, Infect. Immun. 57 (1989) 3214–3220. [16] Hungerer K.D., Ronneberger H., In vitro and in vivo efficacy of a toxin-neutralizing human staphylococcal immunoglobulin, Behring Inst. Mitt. 86 (1990) 170–184 (in German). [17] Ramisse F., Szatanik M., Binder P., Alonso J.M., Passive local immunotherapy of experimental staphylococcal pneumonia with human intravenous immunoglobulin, J. Infect. Dis. 168 (1993) 1030–1033. [18] Skurkovich S.V., Likhoded V.G., Krokhina M.A., et al., Investigation of the therapeutic efficacy of plasma of donors immunized with live Escherichia coli vaccines, Material of the 48th Scientific Session of the TNIIGPK on the Basic Results of Scientific Work of the Institute of Moscow, 1975, pp. 100–103 (in Russian). Microbes and Infection 2000, 1383-1392
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