LONGEVITY OF PROTECTION BY ACTIVE IMMUNISATION AGAINST NECROTISING ENTERITIS IN PAPUA NEW GUINEA

389 MUTAGENIC URINE FROM BLADDER CANCER PATIENTS have been found in the urine and of cigarette smokersas well as in chemotherapyl other situations,3but we know of no studies on the urine of patients with bladder cancer. Chemicals have been linked with bladder cancer but the cause(s) of most bladder cancers is unknown.

SIR,-Mutagenic compounds

of people on

Urine samples were collected from patients or controls between noon and 1500 hours. Subjects emptied their bladders before providing the sample used for the mutagenicity assay. Samples were stored at -20°C. Thawed samples of 100 ml urine were filtered through a Whatman no 1 filterpaper and passed through an ’Amberlite XAD-2’ resin column (6 x I 5cm) which had been prewashed with 50 ml double-distilled water. The column was rinsed with 20 ml distilled water and mutagens were eluted with 30 ml methanol. The solvent was removed under reduced pressure and the residue redissolved in 1 ml anhydrous dimethylsulphoxide. 4 a microtitre procedure Mutagenicity was assayed by "fluctuation" thought to be more sensitive than the standard Ames test. The tester strains used were log-phase Salmonella typhimurium TA98 and TA100; assays were done in the presence or absence of a liver preparation from ’Aroclor-1254’ pretreated rats. 0-11 ml urine extract was tested per plate. Positive (4-nitro-ophenylenediamine or 2-acetylaminofluorene) and negative controls were run alongside the test material. 0-01units/ml (3-glucuronidase and 003 units/ml arylsulphatase were added to the stock assay medium. Plates were incubated for 3 days at 37 ° C. The turbid wells were counted and the results analysed by a

assay,

5

chi-square test.5 All samples were tested blind and completed.

decoded

only when the study had been

’

There was no significant difference between the results for males and for females so the data were pooled. Calculation of risk ratios6 and confidence limitsrevealed a relation between urinary mutagens and bladder cancer in both smokers and non-smokers (see table); this’ was significant for non-smokers. This was a pilot study to test whether or not bladder cancer patients have mutagenic urine. We reasoned that, although bladder cancer may have been diagnosed many years after the initiation event(s), the bladder mucosa would still be exposed to the same mutagens as those at the time of tumour initiation. As with so many studies, the influence of cigarette smoking tends to mask the presence of mutagens from non-cigarette sources, so it is worth concentrating more on the data from non-smokers. The results suggest a greater exposure to mutagens in bladder cancer cases. No clear pattern was seen in the mutagenic response when this was analysed with regard to bacterial tester strain or liver activation (data not shown), suggesting that the mutagens were 1 Legator MS, Connor TH, Stoechel M. Detection of mutagenic activity of metronidazole and niridazole in body fluids of humans and mice. Science 1975; 118: 1118-19 2 Yamasaki

E, Ames BN. Concentration of mutagens from urine by absorption with the non-polar resin XAD-2: Cigarette smokers have mutagenic urine. Proc Nat Acad Sci USA 1977; 74: 3555-59. 3. Garner RC. Biological monitoring of aromatic amines and their metabolites using shortterm tests for carcinogenicity. In: Egan H, Fishbein L, Castegnato M, O’Neill IK, and Bartsch H, eds. IARC Scientific Publication no. 40. Environmental carcinogens: selected methods of analysis, vol. 4. Some aromatic amines and azo dyes in the general and industrial environment. Lyon, 1981: 33-50. 4. Gatehouse DG, Delow GF The development of a microtitre fluctuation test for the detection of indirect mutagens and its use in the evaluation of mixed enzyme induction in the liver. Mutation Res 1979; 60: 239-52. 5 Gilbert RI. The analysis of fluctuation tests. Mutation Res 1980; 74: 283-89. 6 Mantel N, Haenszel W. Statistical aspects of the analysis of data from retrospective studies of disease. J Nat Cancer Inst 1959; 22: 719-48. 7. Miettiner OS. Estimability and estimation in case referent studies Am J Epidemiol 1971, 103: 226-35.

chemically diverse from sample to sample. Further research is required to confirm these findings in a larger series. It is not possible from such studies, however, to say whether the excess of mutagens is cause or effect. This could only be answered in a prospective study. This work was supported by the Yorkshire Cancer Research Campaign and a grant from the Yorkshire Regional Health Authority.

R. COLIN GARNER ALLEN J. MOULD VANESSA LINDSAY-SMITH

Cancer Research Unit,

University of York, York YO1 5DD Yorkshire

Regional Cancer Organisation, Cookridge Hospital, Leeds

RAYMOND A. CARTWRIGHT

York District

BRIAN RICHARDS

Hospital

LONGEVITY OF PROTECTION BY ACTIVE IMMUNISATION AGAINST NECROTISING ENTERITIS IN PAPUA NEW GUINEA

SIR,-Necrotising enteritis (pigbel) remains a major cause of illness and death in children in the highlands of Papua New Guinea. The disease was first described nearly 20 years agol2 and its pathogenesis has been elucidated.3-5 This work led to a double-blind controlled trial of the efficacy of active immunisation with adsorbed Clostridium perfringens type C beta-toxoid (’Pigbel Vaccine’; Burroughs Wellcome). The methods of this study and its success in for two years after vaccination have been reported preventing previously. We would like to report the results of continued surveillance of this population. Since the report of Lawrence et al.there have been two further rounds of vaccination in the study area, and the original control population were offered pigbel vaccine. Also there has been considerable population movement into and out of the trial area. For these reasons, the exact size of each population with a given vaccination status is no longer known. However, the number of children who have received two doses of pigbel vaccine is now more than twice as large as the number remaining in the control group (who received tetanus toxoid originally and subsequently declined further vaccination). Surveillance for two years immediately subsequent to the previous report detected only 1 case of pigbel in the group that had received pigbel vaccine, while 4 cases occurred in the smaller control group (p<0-025). Then four years after the vaccinations were original given, pigbel began occurring regularly in the previously protected group. Observation for a further year showed a higher incidence of pigbel in this group although this was not significant (table). So we conclude that two doses of pigbel vaccine confer protection for about four years. As a result of the original study, vaccination against pigbel was introduced into routine maternal child health services throughout the highlands early in 1980. Because a higher antibody response is obtained after three doses of vaccine,present policy is to administer pigbel vaccine at the same time as triple antigen at two, four, and six months of age. Testing has shown that the antibody response to pigbel vaccine was not significantly different when 7 given to babies alone or at the same time as triple antigen. Schoolchildren are also offered a primary course of vaccination. It is clear, though not entirely surprising in view of the acute nature of the disease at the intestinal surface, that a primary course of vaccination does not give life-long immunity. Protection for two

pigbel

TGC, Roth L. Necrotising jejunitis: a newly discovered disease in the highlands ofNew Guinea. Med J Aust 1963; 1: 61-68. Murrell TGC, Roth L, Egerton J, Samels J, Walker PD. Pigbel: enteritis necroticans: a study in diagnosis and management Lancet 1966; i: 217-22. Lawrence G, Walker PD. Pathogenesis of enteritis necroticans in Papua New Guinea.

1. Murrell

URINE MUTAGENICITY RESULTS IN BLADDER CANCER CASES AND

2.

CONTROLS: NON-SMOKERS AND SMOKERS 3.

Lancet 1976; i: 125-26. 4. Lawrence GW. The pathogenesis

of pigbel in Papua New Guinea. Papua New Guinea Med J 1979; 22: 39-49. 5. Lawrence G, Cooke R. Experimental pigbel: the production and pathology of necrotising enteritis due to Clostridium welchii type C in the guinea-pig. Br J Exp Pathol 1980; 61: 261-71. 6. Lawrence G, Shann 7.

F, Freestone DS, Walker PD. Prevention of necrotising enteritis in

Papua New Guinea by active immunisation. Lancet 1979; i: 227-30. Davis MW. Pigbel: Where now? Proceedings of 17th annual symposium ofthe Medical Society of Papua New Guinea (Rabaul, 1981)

390 CASES OF PIGBEL IN VACCINATED AND CONTROL POPULATIONS

lamps (Sylvania F20F12G) to see if longer wavelength light can lower the serum bilirubin in jaundiced babies. The spectral output of the green lamp lies almost completely in the narrow range

spectrum from

500-570 nm with a maximum at 525 nm; the similar lamp has been reported by Parshad et al.3

I

I

I

powermeter,

four years accords with the results obtained in protection against enterotoxaemias in adult animals where annual revaccination is practised. Further work is investigating the need for a booster dose and the possibility of vaccine modification. to

Papua New Guinea Institute of Medical Research, Goroka, Eastern Highlands Province,

MICHAEL DAVIS

Papua New Guinea Queensland Institute of Medical Brisbane, Australia

Research,

Goroka Base Hospital, Papua New Guinea

GREGOR LAWRENCE

FRANK SHANN

Bacteriology Research and Development, Wellcome Research Laboratories, Beckenham, Kent BR3 3BS

P. D. WALKER

SUBARACHNOID ADMINISTRATION OF CALCITONIN: A WARNING et al. describe in the European Journal of their interesting findings on the amelioration of Pharmacology severe and intractable pain in man following subarachnoid administration of calcitonin at a dose of 300 IU per 70 kg. Since the material Fraioli used was manufactured by us for intramuscular use, we deemed it prudent to do standard animal toxicology studies of the subarachnoid route of administration, despite the lack of significant adverse effects in Fraioli’s clinical experience. To our surprise, dogs given salmon calcitonin by the intrathecal route at doses equivalent to those used by Fraioli et al. in their human study showed significant respiratory distress. Doses three times greater than those used by Fraioli’s group caused several of the dogs to become moribund, and they had to be killed. Paradoxically, calcitonin given to anaesthetised dogs by the intrathecal route produced no similar toxicity. Further studies in the dog and rodent by the intravenous or intramuscular routes at doses twelve times greater than those used by Fraioli et al. were not associated with similar findings. In the baboon, however, intrathecal calcitonin at the equivalent dose used by Fraioli did

SIR,-Fraioli

produce toxicity. In view of these findings, Armour Pharmaceutical Co. Ltd deem it to withhold further human experimentation with subarachnoid calcitonin until additional animal studies are completed and all the data have been carefully evaluated, even though we know of no definitive association between the animal findings and possible human experience.

prudent

Armour Pharmaceutical Co. Ltd., Eastbourne BN21 3YG

Low-birthweight infants with mean gestational age of 35 weeks (25-41) and mean birthweight of 2110 g (800-2550) were assigned at random to green lamps (50 babies) or to daylight fluorescent lamps (Sylkvania F20F12DA) (50 babies). The power density at the

body surface,

Total population in each group shown in parentheses. Incidence rates were analysed by the method of Katz et al.I

measured with a 210 Coherent 3 - 14 mW/cm2for both groups.

Radiation

After the first 24 h exposure the mean decrements in bilirubin concentration were 20’3% for green lamps and 15 - 8% for control

daylight lamps (p<0 . 00 1). Thus standard fluorescent green lamps can replace fluorescent daylight lamps (still largely used in Italy and other countries)4 and provide more effective therapy for neonatal jaundice. Estimates of absorption and emission curves suggest that bilirubin will absorb more photons from daylight than from green fluorescent lamps. Perhaps the green light penetrates deeper into the skin. The best spectral range for efficient photodegradation of bilirubin may be the result of a balance between absorption and skin penetration. Blue and special blue lamps may not be the most effective: blue radiation better matches the absorption band of bilirubin but penetrates less far into the skin because of high light scattering and absorption by competing skin pigments. Recent investigations show that the near-visible light (400-500 nm) emitted by standard fluorescent tubes may be toxic and mutagenic in mammalian cell cultures5while light of wavelengths above 500 nm is not.6,7 Although in vitro findings should be extrapolated to man with caution the potential side effects of bluelight phototherapy are well known and increased chromosome alterations of peripheral blood lymphocytes during phototherapy have been observed.Green-light phototherapy offers the following advantages: greater efficiency than daylight lamp therapy; no shortterm side-effects in infants; greater safety; complete tolerance by nurses; and reduced risk of retinal damage to doctors and nurses (the threshold for non-thermal lesions is ten times smaller for green light 9 than for blue

light).9

One ofus (R. P.) acknowledges discussion of this problem with: W. T. Ham, Jr, R. Landry, J. A. Parrish, M. Wolbarsht, and, in particular, B. Zuzolo, R. Zuzolo, and I. A. Magnus. Institute

of Puericulture,

University of Florence, Ospedale Meyer, 50132 Florence, Italy

C. VECCHI G. P. DONZELLI

Institute of Physical Chemistry, University of Florence

M. G. MIGLIORINI

CNR Centre for Study of the Chemistry and Structure of Heterocyclic Compounds, Florence

G. SBRANA

Higher Institute of Physics, University of Florence, and CNR Institute of Quantum Electronics

R. PRATESI

3. Parshad

SIR,-We have shown2that the green line at 514’ 5 nm of an argon laser can photodegrade bilirubin in vitro, the effect being

comparable with that achieved by blue lines at 488 -0and 457-99 nm. We have been experimenting with commercial fluorescent green 8. Katz O, Baptista J, Azen SP, Pike MC. Obtaining confidence intervals for the risk ratio in cohort studies. Biometrics 1978; 34: 469-74. 1. Fraioli F, Fabbri A, Genessi L, Moretti C, Santoro C, Felici M. Subarachnoid injection of salmon calcitonin induces analgesia in man. Eur J Pharmacol 1982; 78: 381-82. 2. Sbrana G, Migliorini MG, Vecchi C, Donzelli GP. Laser photolysis of bilirubin.

Pediatr Res 1981; 15: 1517-19.

was

H. LAURENCE SHAW

NEW LIGHT IN PHOTOTHERAPY

a

4.

5. 6.

7.

8. 9.

R, Sanford KK, Jones GM, Tarone RE. Fluorescent light-induced chromosome damage and its prevention in mouse cells in culture. Proc Natl Acad Sci USA 1978; 75: 1830-33. Parshad R, Gantt R, Sanford KK, Jones GM, Camalier RF. Light-induced chromatid damage in human skin fibroblasts in culture in relation to their neoplastic potential. Int J Cancer 1981; 28: 335-40. Burki HJ, Lam CK. Comparison of the lethal and mutagenic effects of gold and white fluorescent lights on cultured mammalian cells. Mut Res 1978; 54: 373-77. Parshad R, Sanford KK, Taylor WG, Tarone RE, Jones GM, Baek AE. Effect of the intensity and wavelength of fluorescent light on chromosome damage in cultured mouse cells. Photochem Photobiol 1979; 29: 971-75. Cohen AN, Ostrow JD. New concepts in phototherapy: photoisomerization of bilirubin IX&agr; and potential toxic effects of light. Pediatrics 1980; 65: 740-50. Schwartz AL, Cole FS, Fiedorek F, et al. Effect of phototherapy on sister chromatid exchange in premature infants. Lancet 1978; ii: 157. Ham WT Jr, Mueller HA, Sliney DA. Retinal sensitivity to damage from short wavelength light. Nature 1976; 260: 153-55.