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1970-79 were available, and all were sensitive to the 10 fig disc. TheMIC of the sensitive strains, including that of control, was below2-44 fig/mi, and that of the resistant strains, including A hydrophila,was 625 fig/mi (except for 1 higher than this).

Pteridine compounds inhibit synthesis of tetrahydrofolic acidfrom p-aminobenzoic acid by blocking dihydrofolate reductase, 6similar to the action of trimethoprim. All the 70 V cholerae 01isolates resistant to 0/ 129 were also resistant to trimethoprim (table).The emergence of resistance to 0/129 among the V cholerae 01 1isolates is most likely to be mediated through a plasmid, as observedin enterobacteria with trimethoprim.7 The high MICs in theseisolates also suggest this possibility.These data indicate that susceptibility to 0/129 cannot be used to

distinguish Vibrio from other closely related genera or for

interspecies differentiation. However, being facultative anaerobes,members of Vibrio can be differentiated from the aerobic

pseudomonas by oxidation-fermentation. Further, they can usuallybe distinguished from aeromonas spp by their ability to grow onthiosulphate citrate bile salt sucrose (TCBS) agar, to decarboxylatelysine and ornithine, and to dihydrolate arginine. Interspeciesdifferentiation within Vibrio can be achieved for Vparahaemolyticusand V alginolyticus by observing their ability to multiply at 8% and10% NaCl, respectively, and by their typical growth on TCBS ;Vfluvialis and V furnissii are distinguishable because theydihydrolate arginine, ferment arabinose, and do not grow withoutNaCl. V hollisae is unable to decarboxylate lysine or ornithine ordihydrolate arginine. Furthermore, in the typical colony, thecharacter of V cholerae Olin different media has always been usefulin identification.The chronological data of this study indicate that resistance

probably emerged first among clinical isolates and then spread tothe environment.

Department of Microbiology,Institute of Medical Sciences,Benares Hindu University,Varanasi 221 005, India

GOPAL NATHSUHAS C. SANYAL

1. Shewan JM, Hodgkiss W, Liston J. A method for rapid differentiation of certainnon-pathogenic, asporogenous bacilli. Nature 1954; 173: 208-09.

2. Furniss AL, Lee JV, Donovan TJ. The Vibrios. Public Health Laboratory ServiceMonograph Series no 11. London: HM Stationery Office, 1978: 21-22.

3. Bauman P, Fumiss AL, Lee JV. Genus I. Vibrio pacini 1854, 411. In: Kreig NR, HoltJG, eds. Bergey’s manual of systematic bacteriology, vol 1. Baltimore: Williams &

Wilkins, 1984: 518-38.4. Lee JV, Hendrie MS, Shewan JM. Identification of Aeromonas, Vibrio and related

organisms. In: Skinner FA, Lovelock DW, eds. Identification methods for

microbiologists. Society for Applied Bacteriology Technical Series no 14. London:Academic Press, 1979: 151-66.

5. West PA, Colwell RR. Identification and classification of Vibrionaceae—an overview.In: Colwell R, ed. Vibrios in the environment. Baltimore: John Wiley, 1984:285-363.

6. McCormack JJ. Structure-activity relationship among pteridine derivatives andrelated quinazolones as inhibitors of dihydrofolate reductase. In. Pfleiderer W, ed.Chemistry and biology of pteridines. Proceedings of 5th International symposium.Konstanz, West Germany, 1975: 125-32.

7. Vrbina R, Prado V, Canelo E. Trimethoprim resistance in enterobactena isolated inChile. J Antimicrob Chemother 1989; 23: 143-49.

Sunlight and choleraSiR,—Cholera in South America was reported in January, 1991,

for the first time since the turn of the century, representing the latestmanifestation of a 30 year pandemic currently involving Asia andAfrica.’ The subsequent Latin American epidemic has beenexplosive, with about 360 000 cases reported in 13 countries in1991.2 Since the major vehicle for local spread of the disease isthought to be contaminated water, several Latin Americancountries have issued national mandates on the exclusive use ofboiled water for drinking. However, fuel for boiling is costly andreportedly in short supply in parts of rural South America.Therefore an alternative method of disinfection would be desirable.

Vibrio cholerae in drinking water is completely inactivated bydirect ultraviolet irradiation. 3 Furthermore, exposure ofcontaminated oral rehydration solutions to sunlight inactivatesseveral pathogenic colifonns.4 It has been proposed that sunlighthas sufficient ultraviolet radiation to inactivate V cholerae incontaminated drinking water. This would provide a practicalalternative to boiling water in regions where fuel is unavailable.

Effect of sunlight on survival of V cholerae in drinking water.

Glass bottle in shade (.) or in sun (0); plastic bottle in shade (.) orsun (D).

To test this hypothesis, we studied the effect of sunlight onsolutions containing V cholerae Olin two sites in Ecuador underconditions designed to simulate the general application of solardisinfection of drinking water. Fresh stream-water was placed inwidely available 21 plastic bottles and in recyclable 11 glass "Coke"bottles. All bottles were inoculated with the epidemic strain ofV cholerae 01 at 2 x 105 colony-forming units (CFU)/ml, a levelapproximating irrigation water contamination in Peru.6 Half thebottles were placed in midday sunlight in the city of Quito (elevation2850 m, 22 km from equator) and half were placed in the shade.Colony counts (selective TCBS agar) were done in duplicate on allsolutions every 90 min. A similar set of experiments was done inSanto Domingo (elevation 500 m).

In Quito, there was a 1000-fold decrease in CFU/ml in thesun-exposed plastic bottles within 3 h and in the glass bottles within4 h compared with no change in the shaded bottles. Despite theseencouraging results, at a lower elevation with a different watersource, sunlight did not effectively inactivate V cholerae (figure).Thus, solar treatment of drinking water to prevent and control

the spread of cholera cannot be recommended as a widely applicablemethod of disinfection. It is effective only under selected

conditions, possibly related to altitude and intensity of ultravioletradiation.

Department of Medicine,Division of Infectious Disease,University of Colorado Health Sciences Center,Denver, Colorado 80262, USA

THOMAS D. MACKENZIERICHARD T. ELLISON, IIISTEVEN R. MOSTOW

1 Faruque SM, Albert J Genetic relation between Vibrio cholerae O1 strains in Ecuadorand Bangladesh. Lancet 1992; 339: 740-41.

2. Cholera in the Americas. Wkly Epidermiol Rec 1992; 67: 33-39.3. Maurin J, Mazoit P, Dodm A, et al. Application pratique du pouvoir germicide du

rayonnement ultra-violet a l’eau de consommation. Bull Acad Natl Med 1974; 51:35-39.

4. Acra A, Karahagopian Y, Raffoul Z, Dajani R. Disinfection of oral rehydrationsolutions by sunlight. Lancet 1980; ii: 1257-58.

5. Acra A, Jurdi M, Mu’Allem H, Karahagopian Y, Raffoul Z. Sunlight as disinfectant.Lancet 1989; i: 280.

6. Tamplin ML Environmental spread of Vibrio cholerae in Peru. Lancet 1991; 338:1216-17

Comparison of nutrient composition ofrefugee rations and pet foods

SIR,-We join Dr Toole (May 16, p 1214) in expressing ourconcern at the plight of refugees and the nutritional deficiences thatafflict them. A typical refugee ration per day is composed of400-500 g cereal (wheat, rice, corn), 30 g lentils/legumes, 20 g oil,and a small quantity of sugar (5 g). Dried skimmed milk powder isnow rarely given routinely as part of the refugee ration. Whererefugees are unable to supplement their rations by obtaining workor by cultivation, they must live on these rations alone, which lead tooutbreaks of scurvy, pellagra, beri beri, and vitamin A deficiency.The table shows the average vitamin and iron content of a typical

368

COMPARISON OF VITAMIN AND IRON CONTENT IN REFUGEERATIONS* AND PET FOODt

I I I I I I I i

*Based on food consumption tables; ttypical analytical values; tcomparablequantity (on a dry weight basis; §reference nutrient intake per day (values for adult19-50 yr). Dietary reference values for food, energy, and nutrients for the UK (Reporton Health and Social Subjects no 41, London HM Stationery Office, 1991)

refugee ration and compares it with an average pet food. Therefugee ration is not only devoid of vitamins C and A, it is also low inriboflavin, niacin, and iron. The only vitamin in any physiologicallyacceptable level is thiamine, and even that may be inadequate if thecereals are milled inappropriately. By contrast, pet food is not onlybalanced but also has an excellent micronutrient composition.

Classic vitamin depletion studies, conducted in man, may be usedas a guide to assess the potential time needed for the appearance ofvitamin deficiencies within populations living on refugee rations.These are 6-8 weeks for the appearance of niacin deficiency,’ 12-19weeks for riboflavin deficiency, 3-5 weeks for thiamine deficiency,3 3and 10-13 weeks for vitamin C deficiency.4 Deficiency of vitamin Atook 18 months to develop during experimental deprivation studiesof healthy British adults,s but many populations in developingcountries have dangerously low vitamin A stores in the "normal"state. The extra stress of infections such as measles and diarrhoeathat plague refugee camps can precipitate malnutrition-inducedblindness within a few weeks. The depletion times indicate that aidagencies and non-governmental organisations working with

refugees need to be vigilant for potential outbreaks of micronutrientdeficiences among those who have been on standard refugee dietsfor 2-3 months.The pendulum has swung too far in favour of rations acceptable

in energy and protein content alone; the micronutrient content ofsuch rations has been largely neglected. Micronutrient deficienciescan kill. It is essential that refugees are provided with a diet that is atleast as nutritionally adequate as the one we feed to our cats anddogs.Centre for International Child HealthInstitute of Child Health,London WC1N 1EH, UK ANDREW TOMKINS

Centre for the Science of Food and Nutrition,School of Biological and Molecular Sciences,Oxford Polytechnic, Oxford OX3 0PB, UK C. J. K. HENRY

1. Goldsmith GA. Recent advances in nutrition. Arch Intern Med 1951; 88: 93-131.2. Sebrell WH, ed. The vitamins, vol 1. New York: Academic Press, 1954.3. Najjar VA, Holt LE. The biology of thiamin deficiency in man. JAMA 1944; 152:

579-84.4. Barclay W. Vitamin C requirements of the human adult. London: MRC Special

Report no 280, 1953.5. Hume EM, Kreb HA, eds. Vitamin A requirements of human adults. London: MRC

Special Report series no 264, 1949.

Diagnosis of Leber’s hereditary opticneuropathy without neurological

abnormalities

SIR,-Leber’s hereditary optic neuropathy (LHON), the mostcommon inherited disease of the optic nerve, is a maternallyinherited defect with mitochondrial transmission, and ischaracterised by acute or subacute bilateral loss of central vision inyoung adults with phenotypic heterogeneity among familymembers. Wallace et all discovered the first mutation associated

with LHON, a G-to-A transition in mitochondrial DNA (mtDNA)at position 11778 of the NADH dehydrogenase subunit 4 gene(ND4). This mutation, which converts a highly conserved arginineat position 340 of the ND4 protein into histidine (ND4/R340H),eliminates an Sfa NI restriction site. Two mitochondrial mutationsof the NAD dehydrogenase subunit 1 gene (ND 1) have beenassociated with different clinical forms of LHON. Huoponen et allfound a G-to-A mutation at position 3460, which converts analanine into a threonine at position 52 (ND 1 /A52T) and eliminatesan Aha II restriction site. In a large Queensland family, Howell etall found a mutation at nucleotide 4160 of the NDl gene, whichcorresponds to a severe form of optic neuropathy with severalneurological abnormalities and infantile encephalopathy.We looked for the mutations at positions 3460 and 11778 in

leucocyte mtDNA from affected or non-affected members of 28French families with LHON but without neurological disorders,with the polymerase chain reaction.2,4 These families represent 170subjects with LHON or risk for LHON. The ND4/R340Hmutation was found in 23 multigeneration families. In the 5 familieswithout the ND4 mutation, the mtDNA had a ND1/A52Tmutation. Moreover, heteroplasmy was observed in 2 families, 1with an ND 1 mutation and another with the ND4 mutation. Noneof 40 controls without LHON history had the ND1 or ND4mutations.

Therefore the presence of one of these two mutations of mtDNA

provides an easy and rapid diagnosis in a young patient when themost common form of LHON is suspected, whereas their absencemay represent evidence against the diagnosis of LHON withoutneurological abnormalities. The method has already proven to beefficient for the diagnosis of LHON in 2 patients without apparentfamily history.

Ophthalmologyand Department of Biochemistry.

Centre Hospitalier Régional,F-59000 Lille, France

PIERRE LABALETTE

JEAN-CLAUDE HACHEBERNARD HEMERYBERNARD PUECHPIERRE FRANCOISVIVIANE DUMURGUY LALAUPIERRE-OLIVIER HEMERYPASCAL BOONEPHILIPPE ROUSSEL

1. Wallace DC, Singh G, Lott MT, et al. Mitochondrial DNA mutation associated withLeber’s hereditary optic neuropathy. Science 1988; 242: 1427-30.

2. Huoponen K, Vilkki J, Aula P, Nikoskelainen EK, Savantaus ML. A new mt DNAmutation associated with Leber hereditary optic neuroretinopathy. Am J HumGenet 1991; 48: 1147-53.

3. Howell N, Kubacka I, Xu M, McCullough DA. Leber hereditary optic neuropathy.involvement of the mitochondrial ND1 gene and evidence for an intragenicsuppressor mutation. Am J Hum Genet 1991; 48: 935-42.

4. Dumur V, Lalau G, Boone P, et al. Rapid diagnosis of mitochondrial mutation atposition 11778 associated with Leber’s hereditary optic neuropathy. Clin Chem (inpress).

Remission of Leber’s hereditary opticneuropathy with idebenone

SiR,—There is an association between Leber’s hereditary opticneuropathy (LHON) and a G-to-A substitution of mitochondrialDNA at position 11778 of NADH dehydrogenase subunit 4 in theelectron transport chain. In most patients with LHON, the severeloss of visual acuity is permanent and there is no effective treatment.Patients with the 11778 mutation have an especially poor prognosisfor recovery of vision.2 We have treated a patient with subacuteLHON carrying the homoplasmic 11778 mutation with oralidebenone (Takeda Chemical Industries, Japan) for a year.Idebenone is a quinol that stimulates net ATP formation in cerebralmetabolism and inhibits lipid peroxidation in the mitochondrialmembrane.3 In addition, idebenone readily enters rat brain,localising in mitochondria.4A 10-year-old boy was seen in Keio University in August, 1990,

after complaining of poor vision in both eyes. He had developednormally until this experience although the actual time of onset ofvisual loss is uncertain. Corrected visual acuity was 6/90 in botheyes. The optic discs were hyperaemic and slightly raised, and small