A GERMFREE INFANT

168 or monthly intervals, this regimen might prove more convenient for some patients. Live vaccines (polio, vaccinia, and B.C.G.) were given to a third of all patients without apparent ill-effect. The working-party -recommend, however, that immunisation should be undertaken only after careful consideration and after ensuring that normal circulating lymphocytes and normal delayed skin hypersensitivity are present.

fortnightly

The full report of this study is to be published in the Medical Research Council’s Special Report Series. The working-party gratefully acknowledge the help of some two hundred doctors and many other people who supplied information about the patients. Their names will be listed in the forthcoming report of the working-party. Requests for reprints should be addressed to Dr. Lisa Hill, Clinical Research Centre, 164 Tottenham Court Road, London W.l. REFERENCES

Barandun, S. (1959) Das Antikorpermangelsyndrome. Basle. Bruton, O. C. (1952) Pediatrics, 9, 722. Collins, H. D., Dudley, H. R. (1955) New Engl.J. Med. 252, 255. Conn, H. O., Quintiliani, R. (1966) Ann. intern. Med. 65, 528. Fahey, J. L., McKelvey, E. M. (1965) J. Immun. 94, 84. Fudenberg, H., Hirschhorn, K. (1965) Med. Clin. N. Am. 49, 1533. Gitlin, D., Gross, P. A. M., Janeway, C. A. (1959) New Engl.J. Med. 260, 72. Good, R. A., Rotstein, J. (1960) Bull. rheum. Dis. 10, 203. Grant, G. H., Wallace, W. D. (1954) Lancet, ii, 671. Green, I., Litwin, S., Adlersberg, R., Rubin, I. (1966) Archs intern. Med. 118, 592. Hobbs, J. R. (1966) Lect. scient. Basis Med. 106. Hutchison, J. H. (1955) Lancet, ii, 844, 1196. Janeway, C. A., Apt, L., Gitlin, D. (1953) Trans. Ass. Am. Phycns, 66, 200.

Gitlin, D., Craig, J. M., Grice, D. S. (1956) ibid. 69, 93. Kamme, C., Coster, C., Hagelquist, E., Kalen, N., Lindholm, H., Grubb, R. (1966) Acta med. scand. 179, 679. Keidan, S. E., McCarthy, K., Haworth, J. C. (1953) Archs Dis. Childh. 28, —

110.

A., Mackay, M.

R. no. 286.

Kekwick,

E.

(1954) Spec. Rep. Ser. med. Res. Coun.

Vallet, L., Cutbush, M., Mollison, P. L., Thomas, A. R., Gell, H., Soothill, J. F. (1961) J. clin. Path. 14, 470. Kuschner, D. L., Dubin, A., Donlon, W. P., Bronsky, D. (1960) Am. J. Med. 29, 33. Marshall, W. C., Weston, H. J., Bodian, M. (1964) Archs Dis. Childh. 39, 18. Page, A. R., Hansen, A. E., Good, R. A. (1963) Blood, 21, 197. Parkes, R. (1958) Br. med. J. i, 973. Richerson, H. B., Seebohm, P. M. (1966) Archs intern. Med. 117, 568. Rodgers, T. S., Haggie, M. H. K. (1964) Lancet, i, 1042. —

P. G.

A GERMFREE INFANT D. V. I. FAIRWEATHER

R. D. BARNES

JEAN

C. KEANE

HOLLIDAY

ALINA PIESOWICZ

J. F. SOOTHILL

MAUREEN TUFFREY FROM

THE

DEPARTMENTS

HEALTH, AND

OF

HÆMATOLOGY, IMMUNOLOGY,

CHILD

MICROBIOLOGY AT THE INSTITUTE OF CHILD HEALTH AND

CHILDREN, AND THE DEPARTMENT OF OBSTETRICS, UNIVERSITY COLLEGE HOSPITAL, LONDON W.C.1

HOSPITAL FOR SICK

An infant was delivered by cæsarean section and maintained in a " germfree " isolation unit for 6 days during which absence of bacteria was demonstrated by microbiological tests. This procedure was done as part of a programme for the treatment of potential cases of immunity deficiency, but since this infant was not affected with combined immunitydeficiency state, as her brother had been, she was removed from the germfree isolation unit.

Summary

Introduction A SEVERE and fatal form of hypogammaglobulinxmia associated with lymphopenia was first described by Hitzig and Willi (1961). This familial, and possibly inherited,

disease (Hitzig et al. 1968) presents with deficiency of both cellular and humoral immunity-the combined immunitydeficiency syndrome of Soothill (1968a). Foreign-tissue grafts are not rejected. Despite this, attempts at treatment by grafting immunologically competent tissue have been only marginally effective and have not modified the universal mortality in this condition (Hitzig et al. 1965, Harboe et al. 1966, Hong et al. 1968). Failure may have been due to the effects of severe infection at the time the patients were diagnosed and treated, or it may have resulted from the relative maturity of the immunity mechanism and this might; in part, be the effect of the normal antigenic stimulus of infection. In view of this, when the mother of such a child (Thompson 1967) became pregnant again, the child was delivered by caesarean section and maintained in a germfree isolation system while diagnostic tests were done. If the child were affected it was proposed to attempt early grafting of foetal tissue, before the normal bacterial colonisation occurred and whilst the child was, immunologically, relatively " immature ". Methods

Transfer

to

Isolator

The mother, aged 23 was admitted during a normal pregnancy at 38 weeks. Her previous delivery had been normal and clinical examination revealed no abnormality. Elective lower-segment cxsarean section was performed at 39 weeks’ gestation through a sterile, flexible plastic-film surgical isolator described (Barnes, Fairweather, et al. 1968). This isolator was adhered aseptically to the mother’s abdomen which had been previously prepared with an iodine antiseptic solution. The baby, after delivery into the surgical isolator, was then transferred through a connecting tunnel to a mobile plastic transfer isolator for resuscitation. Both isolators, joined (fig. 1) with their enclosed contents (surgical and infant resuscitation requirements), had been sterilised in advance with 4 megarads of gamma irradiation from a cobalt-60 source (U.K. Atomic Energy Authority, Wantage Laboratory). Whilst the infant was being resuscitated in the transfer isolator the connecting tunnel between the two units was sealed and cut between the airtight steel clamps. After resuscitation, the infant was transported in the mobile transfer isolator to the vicinity of the maintenance isolator. The aseptic union, and subsequent incision of the two sterile adhesive membranes incorporated into both the transfer and maintenance isolators formed a common tunnel for the ultimate introduction of the infant into the maintenance isolator. ‘Cornercroft’ type-A human isolator (6g. 2) (Barnes, Tuffrey, and Cook 1968) with minor modification of the sealing of the plastic tent to the consoles was used for the care of the infant. The microbiological integrity of this unit and its sterilisation had been previously confirmed by successful maintenance of germfree mice (Barnes, Holliday, and Cook 1968). Fail-safe devices, similar to those incorporated in the germfree animal isolator (Cook, Tuffrey, and Barnes 1968), were a feature of this isolator and, as a precaution, suction, filtered oxygen i and electricity were also supplied. A metal cot, a sterile plastic film sealed mattress, and a baby incubator were also required I and were introduced into the isolator before sterilisation. ’

Sterilisation The equipment and all supplies likely to be required for the of the infant were sterilised in advance. The method I selected for each item depended upon its ability to withstand : the particular form of sterilisation : care

Gamma irradiation (4 megarads from cobalt-60 source) was used for surgical and connected transfer isolators with enclosed contents and for normal neonatal nursing requirements (instruments &c.) (clothing, bedding, water, sugar, &c.). Gas sterilisation (ethylene oxide) was used to sterilise the air filters, and enclosed incubator in the maintenance isolator; for instruments with glass components, and for the external surfaces of cans of milk.

:

I

169

Fig. 1-Surgical and transfer isolation units. Chemical sterilisation (formalin 10% in 1 %Tego’ followed by 2% peracetic acid) was used to sterilise the assembled maintenance isolator, including the consoles, locks, and plastic tent, and on the external surfaces of drug ampoules. Heat sterilisation was used for evaporated milk.

In view of the uncertainty concerning the anticipated length of stay in the isolation unit supplies were initially prepared for a period of 28 days. Before sterilisation, the articles were sealed in two layers of either nylon or polyvinyl-chloride (r.v.c.) film. When ethylene-oxide sterilisation was the method of choice, cottonwool wicks were incorporated into the sealed plastic film to facilitate the entry of the gas. Drugs for parenteral administration were assumed to be sterile and here it was only necessary to treat the external surface of the ampoule with a chemical antiseptic. The evaporated milk was pasteurised by the manufacturer. Numerous bacterial cultures of it were sterile, and the sterility of this product was also confirmed by its successful use in the maintenance of germfree pigs (Trexler 1967). Unlabelled cans, carefully inspected to exclude damaged ones, were sealed in two layers of nylon film, and treated with ethylene oxide to sterilise their outer surfaces. Water (cans) and sugar (preweighted packets) were sealed in nylon and sterilised by gamma irradiation. Access to Isolator All materials required in the maintenance isolator after its

assembly and sterilisation

were introduced through the entry lock. The outer and inner layers of nylon or P.v.c. film were removed below the open outer door of the entry port (fig. 3). Here there was a mechanical barrier against bacterial contamination formed by the door of the lock and also an invisible barrier of a high-velocity stream of sterile filtered air directed out of the lock. Closing the door automatically stopped this flow of sterile air in this chamber and also lit the ultraviolet lights. Both doors of the entry port were locked electrically for a 6-minute ultraviolet sterilisation period, designed to

Fig. 3-Entry port of the maintenance isolator.

eliminate any chance airborne contaminant. Milk-food cans, excreta, linen, &c., were removed through an exit lock. This chamber, with a simple locking device that prevented both doors being opened at the same time, was also provided with an ultraviolet light. The air pressure in the isolator was usually maintained at 0-15 in., but it was increased to 0-40-0-60 in. water-gauge at periods of " high risk "-e.g., for when an object was introduced into or removed from the isolator.

Fig. 2-Cornercroft type

A human isolator.

Clinical Procedures During the 6 days the baby spent in the isolation unit, normal nursing care was achieved without difficulty, as were physical examination with auscultation, ophthalmoscopy, and auroscopy, several heel stabs, a jugular puncture, and an intramuscular injection of vitamin K. Palpation, however, was somewhat limited. Physical examination revealed a normal healthy female infant whose birth-weight was 8 lb. 4 oz. (approximately 4 kg.). The infant reacted completely normally whilst in the isolator and was removed and returned to her mother on the seventh day. In removing her from the isolator special precautions were taken to ensure that her first

170

showed no growth, and all subcultures were sterile except in which Staphylococcus albus were grown, on plates subcultured from the Robertson’s meat medium. These colonies were outside of the line of the inoculum on the plate, and further repeated subculture from the broth

TABLE I-BACTERIOLOGICAL RESULTS

two

showed

bacterial growth. Immunological Competence The patient’s total white-blood-cell and lymphocyte no

and the mean and standard deviation of seven samples of cord blood from other cassarean section delivered infants are shown in table II. The results of incorporation of tritiated thymidine into lymphocyte culture after stimulation by phytohmmagglutinin (P.H.A.) are shown in table III. Immunoglobulins (IgG, IgA, and IgM) were measured by the single-diffusion method, modified from Mancini et al. (1964), using the Medical Research Council provisional-standard serum (fig. 4). counts

exposure to microorganisms was in the environment of the obstetric nursery in close proximity to other infants who had been delivered by normal cassarean section.

Bacteriological Infection

and

Immunological

Tests

Bacterial

Results of examination of gram-stained films of the individual and pooled fxcal specimens taken during and after isolation are shown in table i. One specimen, on day 3, was examined fresh, and the remainder were examined after 6-8 months’ storage at -20°C, as individual specimens (days 2 and 5) or 24-hour pools (days 2, 3, 8, and 9). The specimens on days 8 and 9 showed a normal fxcal flora, providing a control for the storage

technique. Swabs were taken from the infant and from various parts of the isolator (table i). They were plated directly on to two blood-agar plates, and were then broken off into Robertson’s meat culture medium which was incubated at 37 °C for 5 days before subculture on to two more bloodagar plates. All the plates were incubated at 37°C for 48 hours, one of each pair aerobically and one anaerobically. Primary cultures from the infant and the isolator TABLE II-WHITE-BLOOD-CELL COUNT

TABLE III-P.H.A. STIMULATION OF LYMPHOCYTES

(COUNTS

PER

MINUTE)

Fig. 4-Serum-immunoglobulin concentrations.

The patient had ample blood lymphocytes which transformed with P.H.A., and IgM was present. Discussion of a mother, again pregnant after presentation a with severe familial combined immunitylosing child deficiency syndrome, provided a new opportunity for treatment in this always fatal condition. The report by Hitzig et al. (1968) suggested that there was a 1-in-3 chance that this next infant would also be affected and so we decided to introduce and maintain the newborn infant in a germfree state, in which the infant could be investiand, if affected, treated. gated " Germfree " animal work has been in progress for more than fifty years (Luckey 1963). Relatively cheap flexible plastic film was first used in the construction of animal isolators by Trexler and Reynolds (1957) and shortly afterwards by Landy (1961) and Levenson et al. (1960) for human surgical isolation units. In spite of the demonstration of the suitability of such units for a wide range of human surgery (Levenson et al. 1966) there has been until now no record of an isolator being used for caesarean section. This is surprising since csesarean section is routinely used for introduction of larger animals into the germfree state. The apparatus and technique used for the delivery of this germfree infant is in fact very similar in many respects to apparatus routinely used for germfree animal care (Luckey 1963). The maintenance isolator, although naturally more

The

171 elaborate than most animal isolation units, incorporated similar fail-safe devices to those used and tested in our germfree animal isolator (Cook, Tuffrey, and Barnes 1968). In the human unit the patient was protected from the outside environment by the barrier of plastic film and also by the positive air pressure maintained inside the isolator. The additional feature of the apparatus-the high-velocity laminar air washing through the isolatorwas not used in this case. The microbiological integrity of the Cornercroft maintenance isolator and the techniques for its sterilisation were first demonstrated in the successful maintenance of germfree mice for 7 days (Barnes, Holliday, and Cook 1968). The term " germfree " has been defined as the existence of a single species in the absence of any other detectable viable organisms’(Luckey 1963) and is clearly limited by the methods used to detect such organisms. However, germfree animals are very susceptible to colonisation by bacteria, and bacteria are usually visible on direct films of faeces within 24-36 hours of infection (Luckey 1963). The fact that germfree mice were kept in the maintenance isolator and at the end of 7 days still had no microorganisms detected on gram-stained fxcal smear suggested that the unit was suitable for germfree patientcare.

Microbiological tests were less complete than had been planned. The critical evidence for the germfree state was the absence of microorganisms in gram-stained films of one fresh faecal specimen, two stored fascal specimens, and samples from two stored 24-hour fsecal pools; the stored faeces were kept at -20°C for six months and eight months respectively. Similarly stored pooled fxcal samples, obtained after removal of the child from the isolator, showed a normal faecal flora. Bacterial culture provided further confirmatory evidence of the germfree state of the child. All nineteen primary cultures were sterile; in two subcultures a scanty growth of Staph. albus was obtained, but these were regarded as laboratory contaminants, since the growth was never in the line of the inoculum, and further subculture of the primary media revealed no growth. Such evidence is, of course, confined to bacterial infection; the possibility of virus infection was not investigated. The germfree state of the infant favourably reflects on all the stages of the procedure-including the surgery, the transfer, and the subsequent care in the maintenance isolator. During the 6 days in the isolator unit, no significant practical difficulty was experienced in the nursing care of this infant, who remained well throughout the whole period. The only unusual feature was the absence of smell of fxces and urine, presumably as a result of lack of proteolytic bacteria. We had little information available

which to base a decision to graft immunologically competent tissue. One previous case presented with gross lymphopenia of cord blood (Hitzig 1967), and this together with the striking reduction in in-vitro transformation to P.H.A. (which is a feature of older patients with the combined immunitydeficiency syndrome) were chosen as the critical criteria. But lymphocyte-counts and in-vitro response to P.H.A. were both compatible with control data. Tests of the competence of humoral immunity were more complicated. The serum IgG detected in early infancy is mainly maternal, transmitted across the placenta, but very little IgM or IgA is transmitted across the placenta and the detection of these immunoglobulins on

in the infant’s serum provides evidence of his own immunoglobulin production (Stiehm and Fudenberg 1965). The concentrations of IgM and IgA in this child are shown in fig. 4. IgM was present at birth but the concentration did not rise until after she was removed from the isolator. We considered the possibility that the rise had been delayed until the foreign antigenic stimulus of normal infection occurred, but Allansmith et al. (1968) have shown that this delay may happen in children after normal delivery. Although later IgM levels have been consistently normal, values of IgA (which was not detected in the first 12 days of life) and IgG were rather low for a time. Detailed control data using the M.R.C. standard, for healthy children, are not yet available, but its IgG concentration is about 1000 mg. per 100 ml., and the IgA concentration is less than that of the population mean; the levels shown in fig. 4 are thus compatible with the transient hypogammaglobulinsemia known to present in first-degree relatives of patients with some forms of immunity deficiency, including the combined immunity

deficiency syndrome (Soothill 1968b). Although the therapeutic stage of the procedure was needed, the microbiological success and lack of problems in care for the child were such that we would have confidence in repeating the whole procedure. Furthermore, we regard this success as a substantiation of the systems used for germfree surgery and patient-care, and that they will have far wider applications. We thank Mr. W. A. Cope, Mr. Z. Kumarassamy, Mr. D. Mackay, Dr. W. C. Marshall, Mr. D. Pope, Miss Janet Anderson, and Miss Kate Mackay for their enthusiastic help; the nursing staffs at the Great Ormond Street Hospital for Sick Children and the University College Hospital obstetric unit, the many commercial organisations, and the U.K. Atomic Energy Authority, and the M.R.E. Division of the Ministry of Defence for their help; the joint research board of the Institute of Child Health and Great Ormond Street Hospital, the Camilla Samuel Trust, and the Wellcome Trust for financial support; the Medical Research Council for their support to the research group in immunology; and Mr. R. Cook, Dr. J. A. Dudgeon, Dr. R. M. Hardisty, Dr. P. Trexler, and Prof. O. H. Wolff for their expert advice. Requests for reprints should be addressed to R. D. B., Institute of Child Health, 30 Guilford Street, London W.C.I. REFERENCES

Allansmith, M., McClellan, B. H., Butterworth, M., Maloney, J. R. (1968). Pediatrics, Springfield, 72, 276. Barnes, R. D., Fairweather, D. V. I., Reynolds, E. O. R., Tuffrey, M., Holliday, J. (1968) J. Obstet. Gynœc. Br. Commonw. 75, 689. Holliday, J., Cook, R. (1968) J. appl. Bact. 31, 349. Tuffrey, M., Cook, R. (1968) Lancet, i, 622. Cook, R., Tuffrey, M., Barnes, R. D. (1968) Lab. Anim. 2, 51. Harboe, M., Pande, H., Brandtzaeg, P., Tveter, K. J., Hjort, P. F. (1966) Scand. J. Hœmat. 3, 351. Hitzig, W. H. (1967) Personal communication. Barandum, S., Cottier, H. (1968) in Ergebnisse der inneren Medizin und Kinderheilkunde (edited by L. Heilmeyer, A. Muller, A. Prader, and R. Schoen); p. 79. Berlin. — Kay, H. E. M., Cottier, H. (1965) Lancet, ii, 151. Willi, H. (1961) Schweiz. med. Wschr. 91, 1625. Hong, R., Kay, H. E. M., Cooper, M. D., Meuwissen, H., Allan, M. J. G., Good, R. A. (1968) Lancet, i, 503. Landy, J. J. (1961) J. Ark. med. Soc. 57, 503. Levenson, S. M., Del Guercio, L., La Duke, M., Kranz, P., Johnson, M., Alpert, S., Saltzman, T. (1966) in Research in Burns (edited by A. B. Wallace and A. W. Wilkinson); p. 563. Edinburgh. Trexler, P. C., Malm., O. J., Horowitz, R. E., Moncrieff, W. H. (1960) Surg. Forum, 11, 306. Luckey, T. D. (1963) Germfree Life and Gnotobiology. New York. Mancini, G., Vaerman, J. P., Carbonara, A. O., Heremans, J. F. (1964) XI Colloquim on Protides of the Biological Fluids (edited by H. Peeters); p. 370. Amsterdam. Soothill, J. F. (1968a) in Clinical Aspects of Immunology (edited by P. G. H. Gell and R. R. A. Coombs); p. 540. Oxford. (1968b) Lancet, i, 1001. Stiehm, E. R., Fudenberg, H. H. (1965) Pediatrics, Springfield, 35, 229. Thompson, E. M. (1967) Proc. R. Soc. Med. 60, 895. Trexler, P. C. (1967) Personal communication. Reynolds, L. I. (1957) Appl. Microbiol. 5, 406. -

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