1061

Els and G. Lecatsas, 7. gen. Virol. 1972, 17, 129; G.Lecatsas, Lancet, 1974, ii, 524), kindly furnished byDr H. H. Malherbe, each react significantly by thecomplement-fixation technique with hyperimmunesera to the human reovirus-like agent, the N.C.D.V.,and the E.D.i.M. virus. It thus appears that these 5reovirus-like agents are indeed antigenically related.Antisera to the SA-11 and " O " agents are currentlybeing prepared.

HEPATITIS DUE TO AFLATOXICOSIS

An Outbreak in Western India

K. A. V. R. KRISHNAMACHARIV. NAGARAJAN

RAMESH V. BHATT. B. G. TILAK

National Institute of Nutrition, Indian Council ofMedical Research, Jamai-Osmania P.O.,

Hyderabad-500007, India

Summary Parts of Western India have experienced

an outbreak of hepatitis affecting manand dogs and characterised by jaundice, rapidly devel-oping ascites, portal hypertension, and a highmortality-rate. The disease was associated with the

consumption of maize contaminated heavily with

Aspergillus flavus. Analysis of contaminated samplesshowed that affected people could have consumedbetween 2 and 6 mg. of aflatoxin daily over a periodof a month. A specimen of liver obtained at necropsyshowed bileduct proliferation and giant cells. Thedisease appears to be a result of aflatoxicosis.

Introduction

THE immediate and the delayed responses to afla-toxin administration have been studied in various

laboratory animals 1 Though epidemiological studieshave suggested that aflatoxin may be responsible forhuman disease, there have so far been few reportsdirectly incriminating it. A syndrome characterisedby icterus, rapid onset of signs of portal hypertension,and a high rate of mortality, broke out in epidemicform in parts of western India during 1974 and affectedas many as 200 villages. This outbreak has beentraced to consumption of maize heavily contaminatedwith aflatoxin.

Extent and Circumstances

A total of 397 patients in two adjacent states-Gujarat and Rajasthan-were affected, and 106 died.The outbreak started during late October, 1974, andlasted for about two months. It was confined to ruralareas and to tribal population groups whose staplefood was maize. Areas where the disease broke outare chronically drought-stricken, and the general levelof nutrition in the population was poor. In October,1974, there were unseasonal rains which drenched thestanding maize crop, and the episode started a fewweeks after the families had consumed maize badlyaffected as a result of having been stored under adverseconditions. The epidemiology of the outbreak willbe reported in detail elsewhere.

Clinical Features

The disease was noticed simultaneously in all theaffected villages. Jaundice was the presenting feature.It was preceded by a brief febrile episode, vomiting,and anorexia. In some, the course of the disease wasmild and recovery complete. In others, however, ascitesappeared rapidly within a period of two to three weeks,followed by oedema of the lower extremities. Col-lateral vessels on the abdomen were prominent in afew cases. The liver was enlarged and tender in onlya few. The spleen was enlarged, firm, and not tender.There were no cutaneous haemorrhages, though sub-conjunctival ecchymosis was seen in some patients.Death was usually sudden, and in most instances waspreceded by massive gastrointestinal bleeding. Hepaticcoma was rare. The course and progress of thedisease and the mortality pattern are shown in figs 1and 2. Males were affected twice as commonly aswere females. Not a single infant was affected.Contacts were not affected.

During the outbreak and for several weeks after-wards, a large number of dogs developed ascites andicterus, and died within two to three weeks of theonset. It was observed that affected dogs invariablyshared the food of affected households. Other animals,including cattle, which did not share the family dietdid not suffer during the outbreak.

Fig. 2-Age distribution of cases and deaths.

1062

Methods of AnalysisFoods.-Randomly selected samples of maize, sorghum,

wheat, and Kodo millet (Paspalum scorbiculatum) col-lected from the households were plated on potato dextroseagar (P.D.A.) and Czapek dox agar (C.D.A.) media aftersurface sterilisation with 0-1% mercuric chloride.2 Theindividual colonies of fungi appearing around the platedseeds were subcultured on P.D.A. slants and were identifiedmorphologically. Aflatoxins were extracted and quanti-tated as described earlier.3 Confirmatory tests were doneaccording to the protocol suggested by Nagarajan et al.4Biological tests were made using day-old ducklings.

Tissue and urine.-One liver, obtained at necropsy, waspreserved in formalin, then washed thoroughly in water,dried, defatted with n-hexane, and extracted withmethanol. The methanol extract was subjected to treat-ment with 20% aqueous basic lead acetate. After filter-ing, the filtrate was extracted with chloroform and usedfor thin-layer chromatography analysis to detect aflatoxinand its metabolites. Similarly, blood-samples obtainedfrom 7 cases were treated with methanol and were sub-sequently extracted with chloroform. Urine samples(50 ml.) from 7 patients were concentrated and evaporatedto dryness; the residue was dissolved in methanol, treatedwith 20% aqueous basic lead acetate, filtered, and extractedwith chloroform for detection of aflatoxin.

Results

Affected maize grains were pale and shrivelled. Allfive maize samples obtained from affected householdsshowed the presence of Aspergillus flavus on P .D.A.

and c.D.A. plates. Samples of wheat and sorghum didnot show signs of fungal contamination. In the maizesamples obtained from affected households, the afla-toxin levels ranged between 6-25 and 15-6 p.p.m.

Thin-layer chromatographic analysis of extracts ofthe liver showed three prominent spots with green,light blue, and bright blue violet fluorescence underultraviolet light. The nature of these spots is beinginvestigated. However, aflatoxin Bl was not detectedin the liver extract. Of the seven serum samplesexamined, two showed detectable levels of aflatoxin.None of the urine samples, however, showed thepresence of aflatoxin Bl.

Liver histopathology.-Microscopic examination ofthe liver obtained at necropsy revealed extensive bile-

Fig. 3-Section of the liver showing bileduct proliferation,periductal fibrosis, cholestasis, and multinuclear giant cells(haematoaylin and eosin, x 100).

Fig. 4-Section of liver exhibiting giant-cell transformation ofhepatocytes (haematoxylin and eosin, x 400).

duct proliferation with periductal fibrosis (fig. 3).These ducts were found growing along the liver cellcords, leaving behind irregular islands of liver cells.Liver cells over wide areas were normal except foroccasional multinucleated giant cells (fig. 4), and at afew other places exhibited foamy cytoplasm. Therewas considerable bile stasis in a few proliferated bile-ducts, dilated biliary canaliculi, and in the cytoplasmof the liver cells and Kupffer cells.

Discussion

The simultaneous outbreak of the disease in over180 widely scattered villages suggested that the diseasewas unlikely to be due to an infection. The observa-tion that infants were completely spared, and thatchildren under 5 years were much less commonlyaffected than were adults, supports this view sinceinfections generally tend to affect these age-groupsto a greater extent. The observation that the outbreak

began with the consumption of spoiled maize andlasted only until the stock was consumed suggestedthat the disease was due to this food item. The

appearance of the disease in dogs of families wherethe disease was seen, and the fact that other householdanimals which did not share the family diet did notsuffer from the disease, strengthened the suspicion thata food toxin was the setiological agent.The clinical course of the disease clearly indicated

hepatic involvement. The maize consumed by theaffected community was heavily contaminated withAspergillus flavus, and several samples of grain hadconsiderable amounts of aflatoxin ranging from 6-25to 15-6 p.p.m. Since an adult consumes about 350 g.and a child about 75-80 g. of maize daily, the

patients would have ingested 2-6 mg. of aflatoxindaily for several weeks. The investigation was under-taken towards the end of the outbreak, and these

figures may underestimate actual intakes, since con-taminated maize consumed earlier in the outbreakcould have contained higher levels of the toxin. Thehistopathological features, which included bileductproliferation, and the presence of aflatoxin Bl in theserum of two patients, further support a causal role foraflatoxin.

1063

These observations show that, besides groundnut,which is highly susceptible to aflatoxin contamination,other staple foodgrains can become sufficiently infectedto result in disease. These findings also point to theneed for devising means of averting fungal contamina-tion of foodgrains.We thank Dr S. G. Srikantia, Director, and Dr P. G.

Tulpule, Deputy Director, National Institute of Nutrition, fortheir guidance and keen interest in this study; and the Direc-torates of Medical and Health Services of Gujarat and

Rajasthan for their cooperation.

REFERENCES

1. Detroy, R. W., Lillehoj, E. B., Ciegler, C. in Microbial Toxins:vol. VI, Fungal Toxins (edited by A. Ciegler, S. Kadis, and S. J.Ajl); p. 178. London, 1971.

2. Booth, C. Methods in Microbiology: vol. IV (edited by C. Booth);p. 49. London, 1971.

3. Nagarajan, V., Bhat, R. V. J. agric. Fd Chem. 1972, 20, 911.4. Nagarajan, V., Bhat, R. V., Tulpule, P. G. Environ. Physiol. Biochem.

1973, 3, 13.

SUNLIGHT AND HYPERCALCIURIA

E. S. PARRY

Leishman Laboratory, Cambridge Military Hospital,Aldershot

I. S. LISTER

Queen Alexandra Military Hospital, Millbank,London SW1

Summary Urinary calcium and magnesiumexcretion was measured in two groups

of soldiers leaving the temperate climate of the UnitedKingdom for service in the Persian Gulf. In one

group urinary calcium levels and magnesium/calciumratios were similar, ten days after arrival in the Gulfduring the " cold season ", to those found in the U.K.The other group went to the Gulf in the "hot season",and calcium excretion rose immediately to levels com-parable with those found in the first group after eightmonths. Mg/Ca ratios fell to levels seen in stone-

formers, and 2 of 91 soldiers followed up for three yearshave had urinary calculi. Increased exposure to sun-

light seems to be the most likely cause of the hyper-calciuria.

Introduction

EPIDEMIOLOGICAL studies have revealed seasonalvariations in the incidence of idiopathic urinarylithiasis and an increased incidence in the tropics 1-3Calculi are very common in Service personnel servingin a tropical climate,4,5 and most cases present within afew months of arrival.l,6 Soldiers moving from atemperate to a tropical climate provide a convenient

means of investigating the effect of climate on calciumhomoeostasis.

We have investigated changes in urine calcium andmagnesium excretion in soldiers moving from theUnited Kingdom to Bahrein in the Persian Gulf.The climate in the Gulf, though hot by U.K. stan-dards, has,a striking seasonal variation, and it was,therefore, possible to study soldiers arriving duringthe " cool " and " hot " seasons.

Method

134 soldiers took part as two separate groups. Group Aarrived in the Gulf during the November " cool season ",and group B during the " hot season " in July.Group A initially numbered 74 men who collected theirurine in the U.K. and at ten days and eight months afterarrival in the Gulf. The 63 men of group B collectedurine in the U.K. and again ten days after arrival in theGulf.On each occasion three consecutive twenty-four-hour

urine samples were collected, and acidified and refrigeratedat the end of each of the collection periods. Calcium and

magnesium content were measured by atomic absorptionin the same laboratory in the U.K.

Environmental temperature, humidity, and average dailysunlight were measured on location or data were subse-quently obtained from the Meteorological Office.

Results

Table i shows the groups, times, places, and meteoro-logical data during the five urine collection periods.51 men from group A (mean age twenty-four years)and 40 from group B (mean age twenty-eight years)completed all collections. The remaining men failedto complete one or more collections mainly for militaryor medical reasons. The results from group A werefrom 51 men who collected urine in the U.K. duringOctober (group Al), in the Gulf during November(group A2), and again in the Gulf during late Julyof the following year (group A3). Group-B resultswere from 40 men who made collections in the U.K.in late June (group Bl) and early August (group B2).Although group A moved from an environmental

temperature of 13-7°C to one of 25-7°C, a rise of12°C, they were only exposed to an extra 1’73 hoursof sunlight per day. Group B, however, were exposedto a rise of 19.5°C and an extra 5-22 hours per dayof sunlight. It was not until group A had been inthe Gulf for several months that they too were exposedto a similar increase in sunlight.The mean 24-hour excretion of calcium and mag-

nesium and the mean magnesium/calcium X 100 ratioare shown in table n. In group A there was a smallbut significant fall in the urine calcium excretion

(p<0’OO5) ten days after the soldiers had arrived in

TABLE I-TIMES, PLACES, AND METEOROLOGICAL DATA DURING THE FIVE COLLECTION PERIODS