482109178

Journal of Applied Bacteriology 1979,46,247-267

A Review

The Microbial Contamination of Intravenous Infusions During Clinical Use

Department of Pharmacy, The University, Manchester 13, England Received 20 September 1978 and accepted 20 December 1978 Contents 1. Introduction . . . . . . . . . . . . . 2. MicrobialContaminationofIntravenous Infusions . . . . . . . A . Recognition of the problem . . . . . . . . . . B. Contamination of infusions during use . . . . . . . . 3. Sources of In-use Contamination . . . . . . . . . . A. Airborne contamination . . . . . . . . . . . B. Touch contamination . . . . . . . . . . . C. Origins of 'airborne and touch' contamination . . . . . . . D. The potential for contamination of intravenous infusions by airborne skin scales E . Rubber seals . . . . . . . . . . . . . F. Intravenous additives . . . . . . . . . . . G. The site of vein puncture . . . . . . . . . . H. Contaminated disinfectants . . . . . . . . . 4. Other Considerations . . . . . . . . . . . . A . Duration of infusion . . . . . . . . . . . B. In-line membrane filters . . . . . . . . . . C. Pyrogenic reactions . . . . . . . . . . 5. Total Parenteral Nutrition . . . . . . . . . . 6. Survival of Micro-organisms in Intravenous Infusion Solutions . . . . . A . Survival in simple solutions . . . . . . . . . . B. Survival in total parenteral nutrition solutions . . . . . . . C. The survival of 'naturally occurring' micro-organisms . . . . . 7. Clinical Significance of In-use Microbial Contamination of Intravenous Infusions . 8. Conclusions . . . . . . . . . . . . . . 9. References . . . . . . . . . . . . . .

.

. .

. . .

Page 24 7 248 248 249 250 250 252 253 253 254 254 255 255 255 255 256 256 256 257 258 259 260 26 1 262 263

1. Introduction THE TECHNIQUE of intravenous infusion has been established for over 50 years and is used principally in medical therapy for the correction of serious disturbances in electrolyte and fluid balance in the body and the provision of basic nutrition. In recent years, infusions have also been utilized as vehicles for drug administration and to provide a means for total nutritional maintenance when oral feeding is not possible. Freedom from pyrogenic and particulate matter and living micro-organisms is an essential pre-requisite of the safe use of intravenous infusions. This should be assured by adequate manufacturing and quality control procedures. Microbial contamination of

* Present address: Surgical Bacteriology Department, Peter Brent Brigham Hospital, 721 Huntington Ave., Boston, Mass., U.S.A. t Present address: Pharmacy Department, Addenbrookes Hospital, Hills Road, Cambridge. 0021-8847/79/020247

+ 2 1$01.00/0

[2471

0 1979 The Society for Applied Bacteriology

248

C. J. HOLMES AND M. C. ALLWOOD

infusion products during clinical use, which may result in infection, particularly septicaemia, is, however, always possible. This review includes a summary of reported incidents of in-use contamination of intravenous infusions and subsequent investigation into the sources of such contamination. The growth of potential contaminant micro-organisms in various intravenous infusion solutions is also discussed.

2. Microbial Contamination of Intravenous Infusions A . Recognition of the problem First reports of adverse reactions due to contaminated fluids appeared in 1953. O’Hare et al. (1953) reported a fatal case of anaphylactic shock following the intravenous infusion of 10% (w/v) glucose which was ‘inadvertently’ contaminated with Aerobacter aerogenes. In the same year Michaels & Ruebner (1953) reported two cases in which patients receiving intravenous therapy developed severe pyrexia; they attributed both to coliform bacilli originating from the infusion apparatus. Wilmore & Dudrick (1 96 l), in a study assessing the use of in-line membrane filters for intravenous solutions, postulated that the parenteral fluid itself might be the source of infection and suggested that contamination may be introduced to the infusion system by unfiltered air entering the container during the addition of drugs to the infusion, or during intermittent administration of the parenteral medication. Even though reports of several incidents of septicaemia were published, no attempt appears to have been made to discover the source or mechanism of such infection (Appleyard & Lloyd 1969; Robertson 1970; Sack 1970). Between 1970 and 1973, however, four major outbreaks of nosocomial sepsis in the United States of America and Great Britain were attributed to the presence of living micro-organisms in infusion fluids after the process of sterilization (Felts et al. 1972; Phillips el al. 1972: Anon. 1972, 1973). Contamination of fluids was traced either to faulty maintenance of autoclaving equipment or the introduction of contaminated spraycooling water into the fluids via the infusion bottle closures. In three of these outbreaks and in several other reports of sepsis traced to contaminated infusion fluids (Duma et al. 1971; Lapage et al. 1973) the causative organisms were found to be from the tribe Klebsielleae, in particular Enterobacter agglomerans (formcrly classified with Erwinia) and, to a lesser extent, Ent. cloacae and Klebsiella pneumoniae. Enterobacter agglomerans is an unusual human pathogen and is normally regarded as a plantassociated bacterium. The preponderance of such Gram negative bacteria is explained to some extent by the investigations of several workers (Felts et al. 1972; Guyunn et al. 1973; Pierpaoli et aE. 1973; Maki & Martin 1975). They have shown that with most of the bacterial species examined (both Gram positive and negative) growth in virtually all types of intravenous fluid is suppressed for at least 24 h, with the exception of the Klebsiella-Enterobacter group. These particular micro-organisms grow in the fluids and often proliferate to 105-106 viable cells/ml, such numbers not causing any visual turbidity in the solutions. Mackel et al. (1971) have shown that the KlebsiellaEnterobacter group of bacteria may be present in many areas associated with the manufacture of infusion fluids. Such major outbreaks of nosocomial sepsis led to an increased awareness of the problems of sterile fluid production. As a result, however, the

CONTAMINATION OF INTRAVENOUS INFUSIONS

249

hazards associated with the contamination of intravenous infusions during use on the hospital ward were also highlighted.

B. Contamination of infusions during use Many studies have shown that even if an infusion is sterile on arrival at the hospital ward it may become contaminated with micro-organisms during administration. After detecting four cases of septicaemia in 1970, which were traced to contaminated intravenous volume-control administration sets, Duma et al. (197 1) also discovered that 35% of such sets in their hospital became contaminated during use, many with more than one type of organism. It was noted that 12% of 40 unused ‘sterile’ administration sets examined as controls were found to be contaminated. Thus, it cannot be assumed that the 35% contamination rate was due entirely to contamination of the sets during use in the hospital. Sixty per cent of the contaminated sets contained Gram negative bacilli. Ordinarily, one might suspect that Gram positive cocci would be more common among organisms originating from personnel but the authors postulated that ‘nearly half of the patients were receiving antibiotics via the infusions, particularly semi-synthetic penicillins’ and consequently selection of Gram negative bacilli may have occurred. Deeb & Natsios (1971) found a 3.8% contamination rate of in-use intravenous bottles; sterility testing of samples of the product before use implied contamination was occurring in the ward rather than at an earlier point in the life of the product. Extrinsic contamination has probably caused sporadic septicaemias ever since the introduction of intravenous infusion therapy, but until 1971, these sources had been unrecognized or attributed to other factors. More recently, many studies have attempted to establish the proportion of intravenous infusion containers that can become contaminated during use. This has often been assessed by examining the small volume of fluid remaining in the container after administration of the infusion for contaminating micro-organisms. Enumeration of micro-organisms is done either by membrane filtration or direct inoculation of a volume of the fluid on to growth supporting media. Such investigations have revealed relatively low contamination rates, ranging from 0-5% (Colvin el al. 1972; Cox 1973; Ernot et al. 1973; Steckel et al. 1973; Woodside et al. 1975). Unfortunately, control experiments to assess the possibility of contamination of the fluids due to the sampling method were not performed in most of these studies. Maki et al. (1974), however, reported a higher contamination rate (1 1%) when filtering ca. 10 ml of fluid remaining in administration sets. Other studies have assessed in-use contamination by filtering the total contents of the infusion container after routine assembly of the intravenous system in hospitals. Rates of contamination for infusions contained in bottles range from zero to as high as 42% (Arnold & Hepler 1971; Hanson & Shelley 1974; Poretz et al. 1974; Newman et al. 1975) and for infusions contained in plastic collapsible bags, between 1 and 25% (Letcher et al. 1972; Kundsin et al. 1973; Poretz et al. 1974). Although Poretz et al. (1974) reported contamination rates of 7.3 and 5 . 5 % respectively for infusions contained in bottles and flexible bags, these figures were not significantly different from control experiments. In all studies contamination was usually with small numbers of non-pathogenic micro-organisms, in particular Gram positive cocci, indicative of their extrinsic origin. Unfortunately, the origin and level of contamination could not be attributed to differences in container types or particular manipulations of the intravenous systems, as often uncontrolled variables existed in the experiments. Often,

250

C. J. HOLMES AND M. C . ALLWOOD

one or more drugs were routinely added to the infusions; this might be an additional cause of contamination and could also influence microbial survival in the infusion fluid. In some reports, the infusion systems were assembled by nursing staff who were not under supervision, and thus variations and errors in technique would not be recognized. Control experiments, as previously mentioned, were regularly omitted.

3. Sources of In-use Contamination Infusion fluids may become contaminated by several routes before being administered to the patient. This may be due either to poorly designed apparatus or poor aseptic technique during manipulation of various components of the intravenous infusion system. A . Airborne contamination Rigid glass containers have to be vented via an air-inlet during infusion of their contents in order to permit fluid flow out of the container. In the U.S.A. a filter may be included in the air-inlet of the administration set (known as a ‘closed system’) or unfiltered air is admitted into the bottle. This unfiltered air usually enters the container through a plastic tube and collects in the air space above the solution (‘open system with tube’) or it may be allowed to bubble through the solution (‘open system’). It has been shown that the introduction of unfiltered air can cause contamination of intravenous infusion fluids (Percival 1966; Hansen 8c Hepler 1973; Arnold 8c Hepler 1971; Holmes 1978). Hansen 8c Hepler (1973) have suggested that the bubbling of air increases the risk of contamination of the infusion fluid. In the United Kingdom, it is usual practice to include a cotton wool filter in the air-inlet set, thus corresponding to the ‘closed system’. In some circumstances, this filter may be wetted accidentally and consequently the cotton wool is removed from the inlet set or replaced by a hypodermic needle to restore the desired flow rate of fluid. Flexible plastic containers avoid the influx of air altogether. A study conducted by Letcher et al. (1972) led to the conclusion that flexible plastic intravenous fluid containers have a low rate of in-use contamination when compared with studies previously reported of other intravenous fluid administration systems. The hazard of touch contamination continues to exist, however, as was shown by the predominance of Staphylococcus epidermidis in the contaminated samples. More recently, Woodside et al. (1975) were able to detect contamination of the contents of flexible plastic bags during use in a hospital although the number examined was relatively small. Conflicting reports, however, have been published by Poretz et al. (1974), who also compared the contamination of plastic and glass containers. They concluded that the potential for contamination of plastic containers may have been five times that of glass bottle units. The source of contamination was unclear but touch appeared to play an important role. Newman et al. (1975) found a relatively high contamination rate of infusions during use, irrespective of the container type employed. As can be appreciated, such investigations did not yield sufficient information to draw definite conclusions about the merits of plastic bags compared with rigid bottles as containers reducing airborne in-use contamination. The authors could not distinguish between contamination from touch (that is from the hands of personnel assembling or manipulating the infusion system) and from an airborne source, but could only speculate as to its origin.

CONTAMINATION OF INTRAVENOUS INFUSIONS

25 1

Holmes (1978) attempted to assess the microbial contamination of various intravenous fluid delivery systems by airborne contaminants in a hospital ward after simulated administration of infusions. Three types of delivery system were studied; plastic collapsible bags, glass bottles utilizing a cotton wool plug in each air-inlet set and identical bottles and air-inlet sets from which the cotton wool plugs were removed. Each system was assembled in the hospital ward and the infusions ‘administered’ into empty sterile MRC bottles. Contaminants present in the collected fluid were subsequently recovered by membrane filtration. During the course of each experiment the hospital ward air was sampled intermittently for airborne microbes using a slit sampler. Results are shown in Table 1. TABLE1

Number and type of micro-organisms recovered from infusion j l u ids Total number of viable micro-organisms recovered from four 500 ml containers Type of infusion system* r

Experiment

PVC bagst

Bottle + air filter

Bottle with no air filter

1 2 3

-

4

-

1B lB, 1M

5 6 I 8 9 10 11 12 Total

1s 1s 1s 3

-

2s 3S, 1B 3S, 1B 4S, 1B 2s 3S, 2B 1s

2s

3s

-

1B 1s

*S

-

= Staphylococcus, B = Bacillus, C Ltd., Thetford, Norfolk, U.K.

-

lB, 2s

-

8 = Micrococcus.

2s 1s 31

t Viaflex, Travenol Labs

All possible precautions were taken during the setting up and simulated administration of infusion fluids in the hospital ward to eliminate touch contamination of the fluid, including antiseptic swabbing of all plug surfaces, adherence to strict aseptic technique and the wearing of sterile plastic gloves for all manipulative procedures. Despite this, a small number of micro-organisms was still recovered from the contents of the plastic containers after administration. These contaminants must have gained access to the infusions during the setting up of the infusion systems or during filtration of the collected fluid as no airborne micro-organisms can enter these collapsible containers during administration of the fluid contents. Although more micro-organisms were recovered from the fluid of bottles employing air filters than from the plastic containers, it was shown statistically that this difference was insignificant (t = 1-33;tabulated value at P :0.05 = 1.8 1,for n = 10). It must therefore be assumed that some or all of the micro-organisms recovered from these bottles may have originated from other than air-borne sources. It is clear, however, from the results that the risks of contamination

252

C. J. HOLMES AND M. C. ALLWOOD

are significantly greater if air does not pass through a simple cotton wool filter prior to entering the infusion container. Comparison of the types of micro-organisms recovered from containers and ward air indicated that the species and relative proportions of organisms found in the bottles were typical of that found in the air, confirming that contamination was from the air. It was also noted that no Gram negative bacteria were found contaminating the infusion containers. In 1973, Hansen & Hepler assessed the potential microbial contamination of infusion containers in both plastic bags and bottles employing various air inlet systems in an experimental sampling room in which dust was intentionally disturbed with fans. They concluded that bottles utilizing the ‘closed’ system and plastic bags offered significantly better protection against contamination by airborne micro-organisms than did bottles utilizing the ‘open’ and ‘open with tube’ systems. Holmes & Allwood (1978) have employed an enclosed air space artificially contaminated with viable bacterial spores to assess the airborne contamination of intravenous infusions. As predicted, no spores entered flexible plastic bags from the surrounding air during the administration of the infusion. Infusion fluids contained in bottles allowing the influx of unfiltered air were contaminated with relatively high numbers of spores and these numbers related to those present in the surrounding air. The incorporation of a cotton wool filter, as in commercially prepared air inlet sets, reduced greatly the number of spores gaining access to the infusion fluid. It was observed, however, that a relatively high number of spores was recovered from a small number of such containers. The presence of a loosely packed air filter may cause channelling through the cotton wool, thus, permitting the passage of micro-organisms through the filter (Kluyver & Vissell 1950). Holmes & Allwood (1978) consequently assessed the efficiency of cotton wool plugs as bacterial air filters using the same artificially contaminated environment. Results indicated that both increasing the amount of cotton wool or the force of compression applied when packing it significantly increases filter efficiency; the influence of the degree of compression was most evident with lower weights of cotton wool. In such experiments, large numbers of spores were used to challenge the filters. Previous studies have indicated that the hospital environment contains ca. 1-5 micro-organisms/l of air (Davies & Noble 1962; Greene et a!. 1962). Thus, the likelihood of any micro-organisms gaining access to the contents of intravenous infusion containers is considerably less than was found in the above study. Any inconsistency in the quantity and packing of filters resulting in poor filter efficiency, or complete removal of the filter, could lead, however, to the introduction of microorganisms into the infusion container from the surrounding air during administration.

B. Touch contamination Rubber closures of glass bottles may be contaminated with micro-organisms if they are touched accidentally during assembly of the intravenous infusion system. Contamination of the infusion fluid is possible on the subsequent insertion of the administration set connector needle through the closure (Holmes & Allwood 1976). These workers have shown that careful swabbing of bottle plugs with antiseptic (70% propanol + 0.5% chlorhexidine) before insertion of the connector needle is sufficient to kill relatively large numbers of micro-organisms that could be present, confirming the value of this practice. Plastic bags often possess separate entry ports which allow insertion of the connector needle within a closed, sealed compartment. When the

CONTAMINATION OF INTRAVENOUS INFUSIONS

253

connector needle is inserted, the outer part of the entry port is occluded before the internal seal is penetrated. This feature prevents the operator from touching the point of penetration (Petrick et al. 1977). Neither system, however, eliminates the possibility of accidental contamination of the administration set connector needle. It is vulnerable to contamination by touch during the period between removal of its protective sheath and insertion into the infusion container. The higher level of contamination observed with glass bottles (Letcher et al. 1972; Holmes 1978) could be a reflection of the increased amount of handling needed to set them up prior to administration.

C . Origins of ‘airborne and touch’ contamination Viable microbial particles found in the air of a hospital can come from a variety of sources. The frequency and distribution of various bacterial species depends on where air samples are taken in the hospital (Greene et al. 1962). Nevertheless, it has been shown that the predominant types of airborne micro-organisms are Gram positive cocci and diphtheroids (Greene et al. 1962) and that they originate from hospital staff and patients (Speers et al. 1965). This observation was confirmed with respect to patients by Selwyn et al. (1964) who demonstrated that the types of bacteria recovered from the environment in patient areas were related to those directly recoverable from lesions. Ward activity, especially bedmaking, and the number of airborne contaminants have been shown to be closely related (Noble 1962) and it has been known for several years that dust from clothing disseminates airborne micro-organisms (Duguid & Wallace 1948). The majority of airborne bacteria are liberated from the human body surface during the shedding of skin scales (or squames) of the stratum corneum (Davies & Noble 1962). A complete layer of skin scales is shed from the body surface every four days (Halprin 1972; Jansen et al. 1974). In certain disease states, especially eczema and psoriasis, the individual may be a potent source of contaminated skin scales (Noble 1975). Consequently the recovery of micro-organisms such as Staph. epidermidis from an intravenous infusion does not necessarily indicate touch contamination as suggested by previous workers (Letcher et al. 1972; Poretz et al. 1974). This organism is present extensively in a hospital atmosphere but is also a major inhabitant of practically all regions of the skin. D. The potential for contamination of intravenous infusions by airborne skin scales Although the air-inlet is a highly suspect source of contamination of intravenous infusions, some workers have reported the recovery of contaminants from infusions contained in plastic bags after simulated administration of fluid (Hansen & Hepler 1973; Poretz et al. 1974; Holmes 1978). As no air-inlet was employed the only possibility was that contamination occurred during the connecting of the delivery system. Although direct contact between the hands of medical staff and critical areas of the infusion system is always possible, strict measures were taken to avoid such contamination. Meers (1976) has suggested that skin scales shed from the body surface may be attracted on to the connector needles of intravenous infusion administration sets and that viable micro-organisms associated with skin scales may therefore gain access to the infusion by this route. Results of a study by Holmes & Allwood (1977) have indicated that skin scales are attracted on to the connector needles of intravenous infusion administration sets. It may

254

C. J. HOLMES AND M. C. ALLWOOD

be that this attraction is electrostatic in nature as it has been established that skin scales carry a negative surface charge (Lees & Brighton 1972). Although the nature of this attraction is yet to be determined, Holmes & Allwood (1977) observed that skin scales were equally well attracted on to the surfaces of other materials such as borosilicate glass, natural rubber and aluminium. Thus, it appears that an attraction exists between skin scales and solid surfaces. The likelihood of contaminating intravenous infusions in this way will obviously depend on the number of skin scales carrying viable micro-organisms. Davies & Noble (1962) observed concentrations of skin scales ranging from ca. 350-2000/1 of air sampled from hospital ward environments but only a small proportion (0- 1-0.25%) carried viable micro-organisms. Skin scales shed from the body of persons assembling or manipulating an infusion system carry a greater number of viable micro-organisms than are present in the environmental air (Noble & Davies 1965). The longer the connector needle is unavoidably exposed to the environment on removal of the protective sheath and before insertion into the infusion container, the greater will be the chance of it becoming contaminated. The assembly of the infusion system will unavoidably be performed within the natural convective boundary layer of the operator and consequently will expose the system to a higher concentration of skin scales than that found, on average, in hospital ward air. Micro-organisms associated with skin scales trapped in the connection made by the needle and container and not directly introduced to the infusion fluid may gain access to the fluid on subsequent movement of the tubing of the administration set, or when the joint is broken and remade when, for instance, a second intravenous infusion container is attached to the same administration set (Meers 1976). In order to eliminate this source of contamination, the assembly of the infusion system would have to be performed in a laminar flow work station. The practice of administering intravenous fluids from several consecutive containers via a single administration set would also have to be avoided.

E. Rubber seals Natural rubber bungs have been shown to contain viable fungi even after autoclaving. These can gain access to the infusion from ‘blisters’ on the bung by ‘coring’ when the bung is pierced by the administration set (Garven & Gunner 1963, 1964). F. Intravenous additives The administration of drugs via the infusion fluid is a widely accepted clinical procedure. Potassium chloride, heparin, oxytocin, antibiotics and lignocaine are common additives either alone or in combination to one container (D’Arcy & Thompson 1974) and it has been estimated that, in the United Kingdom drugs are added to approximately 30% of intravenous infusions (D’Arcy 1975). Several studies have shown little correlation between the addition of drugs and the contamination rate of intravenous infusions (Letcher el al. 1972; Hughes 1973; Hanson & Shelley 1974). In contrast studies conducted by D’Arcy & Woodside (1973) and Woodside et al. (1975) indicate that drug additives may be a causative factor in the in-use contamination of infusion fluids. It Is still as yet unclear whether poor techniques used during this drug addition are a potential source of contamination of intravenous infusions. An increasing number of hospital pharmacies now provide intravenous additive services which permit the

CONTAMINATION OF INTRAVENOUS INFUSIONS

255

addition of prescribed drugs to appropriate infusions under aseptic conditions (Herman 1970).

G. The site of vein puncture Intravenous fluids may become contaminated if micro-organisms on the cannula tip migrate into the administration set tubing. Experiments have revealed that microorganisms can ascend more than five feet against a continuous flow of fluid (Weyrauch & Bassett 1951). No work has yet shown, however, that this mechanism could be a source of contamination of intravenous infusion containers. Indeed, a number of studies utilizing membrane filters situated in the administration set tubing have revealed the presence of micro-organisms in the container side of the filters much more often than on the patient side (Wilmore & Dudrick 1961; Miller & Grogan 1973, 1975), indicating that retrograde contamination is unlikely.

H. Contaminated disinfectants Several epidemics of septicaemia have been documented, in which the origin of the containers has been traced to aqueous solutions of benzalkonium chloride used as skin disinfectants at the site of catheterization (Plotkin & Austrian 1958; Malizia et al. 1960; Frank & Schaffner 1976). In each case, the disinfectant was heavily contaminated with Gram negative bacilli, in particular Pseudomonas spp. Aqueous chlorhexidine solution, contaminated with Pseudomonus cepacia, was also responsible for a case of infusionassociated septicaemia (Speller et al. 1971). Disinfectants may actually be the source of infection, as they can often support the growth of certain micro-organisms. Although few disinfectants are free from risk, benzalkonium chloride solutions are notoriously hazardous in this respect. This disinfectant can be rapidly inactivated by organic material, such as cork stoppers (Anderson & Keynes 1958), absorbent cotton (Lee & Fialkow 1961) and gauze pads (Malizia et al. 1960). Other reported instances of infusion-associated septicaemia have been traced to assorted origins. These include contaminated pressure transducers for arterial pressure monitoring (Phillips et al. 1971; Anon. 1974, 1975a), contaminated ice used to chill syringes for arterial blood specimens (Anon. 19753) and contaminated hand lotions (Morse et al. 1967). Such reports emphasize the numerous potential sources of contamination of intravenous infusion systems that may arise if care is not taken to ensure aseptic manipulation of the infusion system. Aqueous benzalkonium chloride or chlorhexidine solutions should be avoided in intravenous therapy (Maki 1976a).

4. Other Considerations A . Duration of infusion The incidence of in-use contamination of intravenous infusions has been associated repeatedly with increased duration of uninterrupted infusion (Duma et al. 1971; Letcher et al. 1972; Maki et al. 1976). Such observations have prompted the recommendation that the bottle or bag and administration set should be changed at least every 24 h, and preferably at the same time. Adherence to this procedure would eliminate the possibility of the reflux of blood into an infusion fluid via an administration set previously used for blood products. The reflux of blood into the infusion system may provide sufficient nutrients not only to permit rapid multiplication of non-exacting micro-organisms of the

256

C. J. HOLMES AND M. C. ALLWOOD

Klebsiella-Enterobacter type (Felts et al. 1972) but also broaden the spectrum of pathogens capable of proliferation.

B. In-line membranefilters An in-line terminal filter in the administration set of an infusion system is sometimes used to reduce the risk of infusing micro-organisms into the bloodstream of the patient if they are present in the infusion fluid (Wilmore & Dudrick 1961; Myers 1972). Filters with an average pore size of 0.22 pm slow the flow rate of infusing fluid, an effect not observed with the 0.45 pm average pore size filters. The latter, however, may permit the passage of certain microbes, such as Ps. aeruginosa (Rusmin et al. 1975). Other reported problems include obstruction and rupture of the filter possibly causing embolism or the sudden influx of micro-organisms.

C. Pyrogenic reactions Reports of elevated temperature, fever and general malaise in patients following administration of intravenous infusions have been made since the beginning of this century. Jona (19 16) demonstrated that a heat-stable extract of ‘bacillus’ culture, virtually free from protein material, caused fever in rabbits. Early investigators soon discovered that pyrogenic reactions were related only to Gram negative bacteria and the active principle could not necessarily be removed by boiling, autoclaving or filtration. There is now a greater understanding of the origin and chemistry of pyrogenic substances. Such substances are usually constituents of the outer region of the cell walls of Gram negative bacteria and are lipopolysaccharide in nature (Chang & Nowotny 1975). Even though an infusion has been sterilized by autoclaving, endotoxic shock may result shortly after administration of the infusion if the fluid was originally contaminated with Gram negative bacteria. All intravenous infusions, therefore, must be sampled for the presence of pyrogens before use. The rabbit fever response test (Anon. 197 1) and the Limulus amoebocyte lysate test (Jorgensen & Smith 1973) are both employed for this purpose.

5. Total Parenteral Nutrition In debilitated patients where self-feeding or feeding via a naso-gastric tube is excluded, the technique of Total Parenteral Nutrition (TPN) must be employed, whereby all essential nutrients required for the restoration of an anabolic state are supplied intravenously. At present the most commonly employed TPN solutions consist of solutions of either protein hydrolysates or crystalline amino acid solutions and carbohydrates. In order to meet the calorific demands of patients, hypertonic carbohydrate solutions must be employed. Such solutions rapidly cause thrombosis of peripheral veins and cannulation of the central circulation is normally required. Catheters sited to deliver fluid in a region of high blood flow (e.g. subclavian or internal jugular veins) must remain in situ for prolonged periods. It is not surprising therefore, that sepsis associated with TPN might be anticipated. Since the introduction of plastic catheters in 1945, there have been numerous reports of complications, especially septicaemia and thrombophlebitis (Bentley & Lepper 1968; Collins et al. 1968; Glover et al. 197 1). Documented work about catheter sepsis has resulted in the widely accepted recommendation that plastic catheters be replaced every 48 h if at all possible. A strong correlation has been shown to exist between skin flora at the catheter site and micro-

CONTAMINATION OF INTRAVENOUS INFUSIONS

25 7

organisms recovered from catheter tips (Darrell & Garrod 1969; Banks et al. 1970; Bernard el al. 197 1). Evidence from this work suggests that skin micro-organisms gain access to the cannula tip both at the moment of insertion, and, subsequently, by migration along the interface between catheter and tissue. Microbes may also gain access to the catheter if the intravenous fluid flowing through it is contaminated. Since 1969, incidences of sepsis associated with the employment of TPN have been widespread (Maki 1976b). In-use contamination rates of 1 and 2% (Sanderson & Deital 1973; Copeland et al. 1974) and up to 22 and 27% (Ashcraft & Leape 1970; Curry & Quie 1971) have been reported. The majority of reported incidents of septicaemia associated with TPN therapy have been attributed directly or indirectly to in-dwelling catheters (Boekman & Krill 1970; Gelbart et al. 1973; Maki et al. 1973). These reports are strengthened by the fact that only a relatively small number of incidents of contaminated TPN solutions have been reported (Wilmore & Dudrick 1961; Deeb & Natsios 1971; Sanderson & Deital 1973; Miller & Grogan 1973, 1975). In four of these incidents little contamination was found; only Deeb & Natsios (197 1) found a high rate of contamination (38%). Although culprit organisms have been typical of infection associated with conventional intravenous therapy, the incidence of fungal, and especially candida septicaemia, has been disproportionately high. In fact, ca. 50% of reported septicaemias associated with TPN have been caused by Candida albicans and many of them were fatal. In particular, Curry & Quie (1 97 1) reported the development of fungal septicaemia in 22 patients, 18 of whom died, with septicaemia as the primary cause of death. Patients requiring TPN are often in an extremely poor state of health. They may also be treated concurrently with broad spectrum antibiotics, steroids and immunosuppressants, all of which predispose the patient to fungaemia. Further, the use of certain topical antibiotic ointments often applied to the catheter site may predispose colonization with candida, allied with the use of occlusive dressings which alter the microbial flora of the skin, providing a moist environment for the proliferation of the fungi (Maki et al. 1973). Antibiotic therapy does not seem to promote infection with candida, but only causes an increase in its rate of growth (Cormane & Goslings 1963) attributed to the inhibition of the bacterial flora (Paine 1952; Rosebury et al. 1954). Indeed, it has been reported that C. albicans may utilize certain antibiotics such as chlortetracycline and oxytetracycline as nitrogen sources (Gram 1956; Cormane & Goslings 1963).

6. The Survival of Micro-organisms in Intravenous Infusion Solutions Over the past decade, it has been realized that infusion solutions of simple chemical composition can support the growth and sometimes permit the rapid multiplication of certain micro-organisms. Michaels & Ruebner (1953) proposed that ‘the characteristics of the intravenous infusion fluid’ itself influences the type of micro-organisms found to contaminate it. It would also influence rates of multiplication. Subsequent in uitro studies of this phenomenon have examined the ability of various micro-organisms to remain viable and multiply in solutions employed in intravenous therapy. The results of such experiments yield useful information on the probable fate of a variety of microorganisms in various commercially available intravenous infusion fluids, once contamination of the fluid has occurred.

258

C. J. HOLMES AND M. C. ALLWOOD

A . Survival in simple solutions Gram negative organisms of the tribe Klebsielleae have been observed to multiply by up to 3 or 4 log cycles within 48 h in both 5% (w/v) glucose and 0.9% (w/v) sodium chloride solutions in the majority of studies (Chrichton 1973; Felts et al. 1972; Guyunn et al. 1973; Wilkinson et al. 1973; Pierpaoli et al. 1973; Gelbart et al. 1973; Maki & Martin 1975; Holmes & Allwood 1978). Many members of the tribe Klebsielleae have nitrogen fixing capabilities and can utilize trace amounts of inorganic elements present in commercial fluids (Maki 1976~).Holmes & Allwood (in press) have found that proliferation of Ent. aerogenes also occurred in 15% (w/v) glucose solutions but not in higher concentrations. The growth of Ent. aerogenes was characterized in both 5 and 15% (w/v) glucose solutions by a reduction in the concentration of viable cells during the initial 24 h, followed by rapid multiplication. An explanation for this phenomenon has been proposed by Pierpaoli et al. (1973) who have suggested an initial period during the first 24 h in which many cells die and the remaining cells, which have adapted their nutritional requirements, reproduce at a rapid rate. There is little if any consistency between reports of the growth patterns of other Gram negative rods, such as Esch. coli and several Pseudomonas spp., in either 5% (w/v) glucose or 0.9% (w/v) sodium chloride solution, Staphylococcus spp. seem unable to survive in either of the latter solutions and death usually occurs after 48 h IGelbart et al. 1973; Guyunn et al. 1973; Wilkinson et al. 1973; Holmes & Allwood (in press)]. The low pH characteristic of glucose solutions possibly contributes to loss of viability of Staphylococcus spp., Esch. coli and, in particular, Pseudomorzas spp. In addition, lack of essential nutrients and growth factors may have discouraged the survival of the more nutritionally demanding Staphylococcus spp. Studies using fungi as test organisms have primarily employed candidas. Pierpaoli et al. (1973) and Wilkinson et al. (1973) found Candida albicans unable to survive in 5% (w/v) glucose solution, unlike Geibart el al. (1973) and Maki & Martin (1975) who noted that the number of viable candida cells increased slowly during 72 h incubation. Pierpaoli et al. (1973) also observed that 0.9% (w/v) sodium chloride solution lacked the ability to support fungal growth. Holmes & Allwood (in press) have found that the number of viable C. albicans cells remained approximately constant over a 72 h period in 0.9% (w/v) sodium chloride and in concentrations of glucose up to 50% (w/v). The fungus appeared not to be influenced significantly by either low pH or high osmotic pressure, unlike bacterial species examined. Low pH and the presence of glucose have been implicated as factors associated with inhibition of C. albicans in studies by Zacherle & Charache (1970) who examined the survival of micro-organisms in peritoneal dialysis solutions. Thus it seems likely that such factors, in combination with the deficiency of specific nutrients in glucose solutions, may prevent the proliferation of Candida spp. Although some species of Pseudomonas, Serratia and also Esch. coli have been shown to multiply in distilled water after 72 h (Bigger & Nelson 1943; Favero et al. 1971; Carson et al. 1973; Chrichton 1973), little information is available on the ability of distilled water to sustain microbial growth. Holmes & Allwood (in press) observed that freshly distilled water permitted the multiplication of Esch. coli, Ps. aeruginosa and Ent. aerogenes. It was also noted that, with the exception of Ent. aerogenes, the presence of glucose or sodium chloride inhibited the growth and multiplication of various micro-organisms examined (including Gram positive and negative bacteria).

CONTAMINATION OF INTRAVENOUS INFUSIONS

259

B. Survival in total paren feral nutrition solutions The introduction of TPN solutions to intravenous therapy has prompted investigations into the ability of these solutions of complex composition to support microbial growth and multiplication. It has been found that TPN solutions comprising a mixture of 5% (w/v) protein hydrolysate (usually casein or fibrin) and 25% (w/v) glucose always permits the proliferation of the fungus Candida albicans (Brennan et al. 1971; Wilkinson et al. 1973; Pierpaoli et al. 1973; Gelbart et al. 1973). Little consistency exists, however, between studies of the survival of bacteria in TPN solutions. Goldman et al. (1 973) have observed the proliferation of Klebsiella pneumoniae, Ent. cloacae, Esch. coli, Proteus mirabilis, Serratia marcescens and Staph. aureus in TPN solutions containing protein hydrolysates. Pierpaoli et al. (1973) also noted the multiplication of Ent. cloacae and Staph. aureus but not S . marcescens. They did discover, however, that Erwinia spp. could multiply in these solutions. Wilkinson et al. (1973) have reported the multiplication of Staph. aureus only, the population of Ent. cloacae, Ps. aeruginosa and K. pneurnoniae remaining approximately constant over a 72 h period. Gelbart et al. (1 973) found that none of the bacteria mentioned could multiply in protein hydrolysate and glucose mixtures. Conflicting reports have been made concerning the survival of bacteria in TPN solutions consisting of crystalline amino acids and glucose. Several workers have found that these solutions are unable to support the growth of the various bacteria examined (Gelbart et al. 1973; Goldman et al. 1973; Wilkinson et al. 1973). It has been suggested that this might be due to lack of certain growth factors in crystalline amino acid solutions that are present in protein hydrolysates (Goldman et al. 1973; Wilkinson et al. 1973). These factors might be provided by the peptide portion of hydrolysates, or possibly due to the relatively small amounts of preformed ammonia present in protein hydrolysates which might serve as an available nitrogen source for micro-organisms as well as the fact that they are relatively impure solutions. Holmes & Allwood (in press), however, did observe multiplication of Ps. aeruginosa and Ent. aerogenes in a solution of 4.25% (w/v) crystalline amino acids and 25% (w/v) glucose. Multiplication of Ps. aeruginosa was likewise noted in a solution of 4.25% (w/v) crystalline amino acid and 15% (w/v) glucose. These results suggested that such solutions provide adequate nutrients in their optimal proportions for growth of Ps. aeruginosa and Ent. aerogenes without subjecting the organisms to excessive adverse conditions of pH and osmolarity. Candida albicans, in contrast, has been observed to multiply fairly rapidly in these solutions, increasing their viable numbers by 3-4 log cycles in 72 h [Gelbart et al. 1973; Goldman ef al. 1973; Wilkinson et al. 1973; Holmes & Allwood (in press)]. Although it has been shown that amino acids are not essential for the growth of C. albicans (Forni et a/. 1957), Brennan et al. (1971) have demonstrated that varying the proportions of amino acids in an amino acid solution alters its ability to support the growth of C. albicans. They discovered subsequently that dilution of an amino acid solution increased the rate of multiplication of C. albicans and suggested therefore that there may be some inhibitory factor present in the solution. In contrast, Holmes & Allwood (in press) observed that crystalline amino acid solutions permitted the proliferation of C. albicans, unlike infusion solutions of simple composition, indicating that such solutions provide nutrients essential for the multiplication of the fungus. Goldman et al. (1973) likewise reported an increased rate of multiplication of fungi on dilution of TPN solutions, proposing that the high osmotic pressure of the TPN solutions could have been

260

C. J. HOLMES A N D M. C. ALLWOOD

responsible for inhibition. Again, in contrast, Holmes & Allwood (in press) noted that C. albicans did not appear to be inhibited by high osmotic pressure and grew well in all of the T P N solutions examined. Factors known to influence survival and multiplication of Candida spp. include potassium and glucose. McClary (1952) has shown that potassium is essential for the growth of Candida spp.; T P N solutions d o supply adequate amounts of potassium in their formulation. Zacherle & Charache (1970) have shown that acetate and dextrose in peritoneal dialysis solutions are primary factors associated with the inhibition of C. albicans. Peritoneal dialysis solutions often contain acetate in concentrations comparable to that in T P N solutions but employ much smaller amounts of glucose ( ( 5 % w/v).

C. The sumival of ‘naturallyoccurring’ micro-organisms Previous investigations of the growth of micro-organisms in intravenous infusion solutions have always employed subcultural strains. There is some evidence that microorganisms found occurring naturally appear to be able to withstand physical and chemical stresses to a greater extent than cells of the same micro-organism which have been subcultured on nutrient medium. Favero & Drake (1966) found that naturally occurring Ps. alcaligenes growing in iodinated water from swimming pools were significantly more resistant to free iodine than subcultured cells of the same organism. Similarly, Bassett et al. (1970) found that Ps. multivorans, isolated from infected surgical patients and traced to antiseptic solutions used for pre-operative skin preparation, were far more resistant to this disinfectant before rather than after culture in broth. Although there are obvious differences between these situations and the growth of micro-organisms in T P N solution, the principle of differing physiological capabilities of naturally occurring cells may still apply. Another recent example of this principle is the observations of Favero et al. (1971) who compared survival iates of normally occurring Ps. aeruginosa in buffered distilled water with cell suspensions of the same organism after a single subculture on tryptone soya agar. Naturally occurring cells were isolated from distilled water of ‘mist therapy unit’ (MTU) reservoirs in a hospital. The population of these cells proliferated in the buffered distilled water after an initial decrease, unlike the subcultured cells which lost complete viability within 48 h. Earlier experiments by Favero et al. (1971) had established that MTU water permitted rapid multiplication of the pseudomonad probably because of the presence of organic compounds dissolved in the water during storage. Naturally occurring cells inoculated into the buffered distilled water were, however, suspended in MTU water, which may therefore have influenced the results of their study. It has been argued by Maki (1976a) that in vitro studies of the growth of laboratory subcultured micro-organisms are, nevertheless, clinically relevant. During the 1970-7 1 outbreak of nosocomial sepsis traced to intravenous infusions, 22 microbial species, of which few belonged to the tribe Klebsielleae, were recovered from the interior of screw cap closures of infusion bottles and yet nearly all associated infections were caused by tribe Klebsielleae bacteria. Likewise, in other surveys during this period, many different species of ‘naturally occurring’ micro-organisms were isolated from infusions sampled during use in hospital environments; yet, again, members of the tribe Klebsielleae were the only bacteria that multiplied. Results of in uitro experiments have subsequently indicated that only tribe Klebsielleae organisms are likely to proliferate in infusion

CONTAMINATION O F INTRAVENOUS INFUSIONS

26 1

solutions of simple composition, thus implying that the behaviour of naturally occurring micro-organisms is similar to that of laboratory subcultured strains. In vitro tests involving TPN solutions have shown without exception that fungi, and in particular Candida spp., exhibit preferential growth over all examined bacteria. Once again, such results are comparable to the clinical situation as a high proportion of infections associated with TPN are fungal. Likewise, an investigation by Holmes (1978) indicated that naturally occurring bacteria, acquired from the air of a hospital environment, do not survive adverse environmental conditions that might be imposed on them by TPN solutions better than subcultured cells. The proliferation of a naturally occurring fungus was observed in the TPN solution employed for this study; such behaviour was, however, previously reported with several types of subcultured fungi.

7. Clinical Significance of In-use Microbial Contamination of Intravenous Infusions Previous reports have indicated that usually only small numbers of micro-organisms (