Emerging Infectious Diseases
[Volume 6 No.1 / January - February 2000]

Perspectives

Malaria in England in the Little Ice Age, P. Reiter
Could a Tuberculosis Epidemic Occur in London as it
  Did in New York? A.C. Hayward
Control of Japanese Encephalitis through
    Immunization in Korea, Y.M. Sohn

Synopses

Coccidioidomycosis in New York State, V. Chaturvedi
Dengue Surveillance in Florida, 19971998, J. Gill

Research

Norwalk-Like Calicivirus Genes in Farm Animals,
    W.H.M. van der Poel

Dispatches

Genetics of a Novel Morbillivirus in a Long-Finned
  Pilot Whale, J.K. Taubenberger
First Cases of Candida dubliniensis Fungemia in
  North America, M.E. Brandt
Campylobacter spp. of Human and Animal Origin, B.
  Lucey
Outbreak Strain of B. pseudomallei Traced to Water
  Treatment Plant, T.J.J. Inglis
Typing of Resistant Salmonella Blockley Outbreak
  Isolates from Greece, P.T. Tassios
Risk for Neisseria meningitidis during an Outbreak
  in Wales, P.E. Fitzpatrick
Salmonellosis in the Republic of Georgia, A.
  Sulakvelidze
Report of Q Fever in Oman, E.M. Scrimgeour

Another Dimension

Philosophic Origins of Science and Evolution of the
  Two Cultures, N.C. Myrianthopoulos

Letters

Isolation of a Dengue Type 1 Virus in West Africa
  (Cte d'Ivoire), J.P. Durand
Carbapenem-Hydrolyzing Metallo--Lactamase from an
  Isolate of P. aeruginosa, L. Poirel
Population-Based Study on Invasive Kingella kingae
  Infections, P. Yagupsky
Involving Ornithologists in Surveillance of
  Vancomycin-Resistant Enterococci, M. Sellin

News and Notes

2nd International Conference on AIDS in India,
  December 1999
Workshop on Micronutrients and Infectious Diseases
Third Annual Conference on Vaccine Research, April
  2000
Dangerous Pathogens 2000 Conference, September 2000

Emerging Infectious Diseases
[Volume 6 No.1 / January - February 2000]

Perspectives

From Shakespeare to Defoe: Malaria in England in the
Little Ice Age

Paul Reiter
Centers for Disease Control and Prevention, San Juan,
Puerto Rico

--------------------------------------------------------

Present global temperatures are in a
warming phase that began 200 to 300 years
ago. Some climate models suggest that human
activities may have exacerbated this phase
by raising the atmospheric concentration of
carbon dioxide and other greenhouse gases.
Discussions of the potential effects of the
weather include predictions that malaria
will emerge from the tropics and become
established in Europe and North America.
The complex ecology and transmission
dynamics of the disease, as well as
accounts of its early history, refute such
predictions. Until the second half of the
20th century, malaria was endemic and
widespread in many temperate regions, with
major epidemics as far north as the Arctic
Circle. From 1564 to the 1730sthe coldest
period of the Little Ice Agemalaria was an
important cause of illness and death in
several parts of England. Transmission
began to decline only in the 19th century,
when the present warming trend was well
under way. The history of the disease in
England underscores the role of factors
other than temperature in malaria
transmission.

The earth's climate has always been in a state of
change. The past 250 to 300 years have seen a fairly
steady warming trend. Average temperatures are now
approaching those at the height of the Medieval Warm
Period, near the end of the 12th century. The
intervening centuries included a much colder period,
the Little Ice Age, by far the most important climatic
fluctuation in recent history (1). Such fluctuations,
spanning several generations, are natural phenomena
that have recurred several times in the past 10,000
years. They take place against a backdrop of episodes
of longer duration and greater impact, such as the last
Ice Age (1,600,000 to 10,000 years ago). In recent
years, there has been growing concern that human
activities may be modifying the natural climate. A
decline in temperatures from the 1940s to the late
1970s gave rise to warnings that industrial pollutants
were causing global cooling (2,3). Subsequent warming
has been attributed to increased concentrations of
atmospheric carbon dioxide produced by burning fossil
fuels, and other greenhouse gases (4). Climate models
suggest that this trend could accelerate in the coming
century, although the contribution of human-induced
greenhouse gases to global temperatures is far from
clear (4-6).

Discussions of the potential impact of human-induced
global warming frequently include malaria, a disease
widely perceived as tropical. Articles in the popular
and scientific press have predicted that warmer
temperatures will result in malaria transmission in
Europe and North America (7-12). Such predictions,
often based on simple computer models, overlook
malaria's history; until recently, malaria was endemic
and common in many temperate regions, and major
epidemics extended as far north as the Arctic Circle
(13). Despite the disappearance of the disease from
most of these regions, the indigenous mosquitoes that
transmitted it were never eliminated and remain common
in some areas. Thus, although temperature is important
in the transmission dynamics of malaria, many other
variables are of equal or greater importance. This
article reviews the history of the disease in a
nontropical countryEnglandduring the coldest years of
the Little Ice Age.

The Little Ice Age

The 16th century is the first period for which we have
a reliable history of climate and weather. Private
diaries, ships' logs, accounts of military campaigns,
and similar sources give descriptions of wind
direction, wind speed, cloud formations, and other
weather indicators. Precisely dated annals, chronicles,
audited accounts, agricultural records, tax ledgers,
and other archival material provide indirect
information, particularly on extreme weather events,
such as droughts, floods, or unusual cold. Additional
evidence is available from glacial moraines, lake and
ocean sediments, pollen strata, deposits of insects,
tree rings, coral structure, radiometric analysis of
ice cores, archaeologic sites, and many other sources.
All this information can be combined to reconstruct
past climates (Figure 1).

The first half of the 16th century
appears to have been much warmer
than the previous 150 years, which
had seen a fairly steady decline
in temperatures after the Medieval
Warm Period. However, the new
genial climate was not to last. In
the middle of the century, after a
decade or so of exceptionally warm
yearswarm enough for people to
bathe in the Rhine in Januarya
drastically sharp change occurred.
The winter 1564-65 was long and
bitterly cold and was the first of
many that brought great hardship
and strife throughout Europe.
Climatologists regard the next 150
to 200 years the climax of the
Little Ice Age. On average,
temperatures were probably lower
than in any similar episode since
the end of the last major Ice Age
(14).

The impact of this sudden change
of climate was dramatic and global
(1). Glaciers advanced rapidly in
Greenland, Iceland, Scandinavia, and the Alps. The
Arctic pack ice extended so far south that there are
six records of Eskimos landing their kayaks in
Scotland. Large tracts of land, particularly at higher
latitudes and altitudes, had to be abandoned. In many
years, snowfall was much heavier than recorded before
or since, and the snow lay on the ground for many
months longer than it does today. Many springs and
summers were outstandingly cold and wet, although there
was great variability between years and groups of
years. Crop practices throughout Europe had to be
altered to adapt to the shortened, less reliable
growing season, and there were many years of dearth and
famine. Violent storms caused massive flooding and loss
of life. Some of these resulted in permanent losses of
large tracts of land from the Danish, German, and Dutch
coasts. The dramatic cooling was captured in the
paintings of the Flemish artist Pieter Bruegel
(15251569), who initiated a new genre by completing at
least seven winter landscapes in 2 years (Figure 2).

Figure 1. Estimated mean annual temperatures (C) prevailing
in central England since A.D. 800. These are 50-year
averages, based on a variety of proxy data (see
text). Dotted lines indicate the range of
apparent uncertainty of the derived values. (Figures not
available in ascii)

Climate changes were equally striking in other
parts of the world. In Ethiopia and Mauritania,
permanent snow was reported on mountain peaks
at levels where it does not occur today.
Timbuktu, an important city on the
trans-Saharan caravan route, was flooded at
least 13 times by the Niger River; there are no
records of similar flooding before or since. In
China, warm weather crops, such as oranges,
were abandoned in Kiangsi Province, where they
had been grown for centuries. In North America,
the early European settlers also reported
exceptionally severe winters. For example, in
1607-8 ice persisted on Lake Superior until
June (1).

Ague

The identity of diseases described in early literature is often uncertain, but fevers with a periodicity described as tertian or quartan are highly suggestive of malaria. Descriptors in the context of such accounts are often supportive. For example, in temperate regions, epidemic transmission of malaria tended to occur in late
summer and autumn, giving rise to the common terms aestivo-autumnal or harvest fever for tertians and quartans.

During the Medieval Warm Period, mention of malarialike illness was common in the European literature from Christian Russia to caliphate Spain: "As one who has the shivering of the quartan so near,/ that he has his nails already pale/ and trembles all, still keeping the shade,/ such I became when those words were uttered." (The Inferno, Dante [1265-1321]).

The English word for malaria was ague, a term that remained in common usage until the 19th century. The Medieval Warm Period was already on the wane when Geoffrey Chaucer (1342-1400) wrote, in the Nun's Priest's Tale, "You are so very choleric of complexion./ Beware the mounting sun and all dejection,/ Nor get yourself with sudden humours hot;/ For if you do, I dare well lay a groat/ That you shall have the tertian fever's pain,/ Or some ague that may well be your bane."

Such mention of agues did not disappear when the coldest years of the Little Ice Age began. In 16th century England, many marshlands were notorious for their ague-stricken populations and remained so well into the 19th century. William Shakespeare (15641616), who was born in the autumn of Bruegel's first fierce winter, mentioned ague in eight of his plays. For example, in The Tempest (Act II, Scene
II), the slave Caliban curses Prosper, his master: "All the
infections that the sun sucks up/ From bogs, fens, flats, on Prosper fall and make him / By inch-meal a disease!" Later, Caliban is terrified by the appearance of Stephano, who, mistaking his trembling and apparent delirium for an attack of malaria, tries to cure the symptoms with alcohol: ". . . (he) hath got, as I take it, an ague . . . he's in his fit now and does not talk after the wisest. He shall taste of my bottle: if he have never drunk wine afore it will go near to remove his fit . . . Open your mouth: this will shake your shaking . . . if all the wine in my bottle will recover him, I will help his ague."

Alcohol and opium were commonly used to suppress the rigors of the first stage of the malarial paroxysm (15). We can assume that Shakespeare's audience was familiar with such details, although the play, one of his last, was probably not presented until 1611, long after the change to a colder climate.

Geographic Distribution: the Marsh Parishes

Five indigenous species of Anopheles mosquito are capable of
transmitting malaria in England. The most competent, An. atroparvus, prefers to breed in brackish water along river estuaries. Contemporary accounts of the distribution of ague in 16th and 17th century England reflect the ecology and distribution of this species. For example, the anaerobic bacterial flora of saline mud produces a strong and distinctive sulfurous odor. This was widely perceived to be the actual cause of agues in salt marsh areasShakespeare's "unwholesome fens"hence the Italian term mala aria (bad air).

The history of malaria in England and its demographic,
epidemiologic, and social impact have been described in detail (16). Descriptions by 17th century topographers of the airs and waters surrounding settlements along the River Medway (a tributary of the Thames estuary) alluded to this association (17). Thus, Upchurch, a town on the open estuary: "lies in a most unhealthy situation, close
to the marshes . . . the noxious vapours arising from which subject the inhabitants to continued intermittents." In nearby Iwade: " . . . in summer dry weather, the stench of the mud in the ponds and ditches. . . contribute so much to its unwholesomeness, that almost everyone is terrified to live in it . . ." In Burnham, further upstream, the air was: "rather more healthy . . . owing to the marshes being fewer and less offensive than those lower down the river. . ." and finally, the freshwater reaches were "far more healthy . . . instead of the noisesome smells, arising from the salt
marshes . . . the river here is encompassed with a range of pleasant fertile meadows, greatly conducive both to health and pride."

Even vicars of the Church were afraid of living in the estuarine areas. Dobson (15) reviewed several questionnaire surveys conducted by their bishops that asked "Do you reside personally upon your Cure, and in your Parsonage House? If not, where do you reside? What is the reason for your nonresidence?"

Nearly all the vicars of coastal "marsh parishes" answered no to the first question (Dobson lists 28 such parishes from the counties of Essex and Kent), and gave reasons such as "the place is so very Agueish," "frequently taken with agues and fevers," "the Thames having a very foul shore in this parish . . . attacked by so many repeated agues," "so violently afflicted with the worst of agues and
languishing so long under it till our constitutions were almost broke," and "so unhealthy a situation as to be absolutely unfit for any curate."

Demographic data give striking justification
for the vicars' fears (16) (Figures 3 and 4,
Table). Descriptions of the marsh inhabitants
resemble those of malaria-endemic area
populations in the tropics today. Visitors
commented on the swollen bellies of the
children and their sallow, sickly faces,
suggestive of anemia. An enlarged spleen was
called ague cake. Nevertheless, despite the
coldness of the climate, there is no indication
that there was any major change in these death
rates during the 17th century.

Not all the summers of the 17th century were
equally cool. The diarist Samuel Pepys
(1633-1703), who himself had ague, wrote that
the summers of 1661, 1665, and 1666 were dry
and remarkably hot. This may well have enhanced
transmission rates, for Thomas Sydenham
described an epidemic of tertian and some
quartan fevers in 1661, which "was doing
frightful mischief" by August. Drought malaria,
common in many parts of the world, arises when
rivers and ponds are reduced to the smaller
pools and puddles that anopheline mosquitoes
prefer for breeding. Dobson (15) found a
positive correlation between warm, dry summers
in 1660 to 1810 and seasonal burial rates in
Bradwell-juxta-Mare, a marsh parish in Essex.
Macdonald (18) showed a strong relationship
between high summer temperatures and cases of
ague in Kent in the mid-19th century. High was
defined as days when the temperature rose above
16C, as measured at the Royal Greenwich Observatory. Such hot weather, though clearly not tropical, could certainly have increased the probability of transmission by shortening the extrinsic incubation period (the time required for the mosquito to become infective after feeding on an infected person). Nevertheless, agues were not restricted to the warmer years.

Clinical Descriptions

Some accounts of intermittent fevers during the Little Ice Age were by physicians whose emphasis on precise methods, detailed observations, and accurate records leave us in no doubt that they were referring to malaria. William Harvey (1578-1657) made careful pre- and postmortem observations of cases of ague in London hospitals (St. Bartholomew's and St. Thomas').1 His interest was probably sharpened because a persistent ague had caused him to miss much of his final year (1598-99) at the University of Cambridge. In
his treatise on the circulation of the blood Exercitatio Anatomica de Motu Cordis et Sanguinis in Animalibus (1628), he gave a detailed description of the clinical pathologic features of the febrile episodes: "In tertian fever . . . in the first instance . . . the patient (is) short-winded, disposed to sighing, and indisposed to exertion . . . the blood (is) forced into the lungs and rendered thick. It does not pass through them (as I have myself seen in opening the bodies of those who had died in the beginning of the
attack), when the pulse is always frequent, small, and occasionally irregular; but the heat increasing . . . and the transit made, the whole body begins to rise in temperature, and the pulse becomes fuller and stronger. The febrile paroxysm is fully formed . . ."

Obstruction of circulationcommonly termed sludgingis mainly seen in Plasmodium falciparum infections. It results from complex changes in the consistency of the blood (19) and is manifest in many organs, although most often cited in the context of cerebral malaria.  As Harvey noted, this dangerous condition often occurs suddenly, soon after the initial symptoms of a paroxysm.

Figure 2. Hunters in the Snow by the Flemish painter
Pieter Bruegel the Elder 15251569). (Figures not available
in ascii)

Figure 3. Average crude burial rates in Kent and Essex
parishes, 1551-1750. (Figures not available in ascii)

Figure 4. Age-specific death rates (per 1,000) in Essex
parishes, c. 1800. Drawn from Dobson (15).
(Figures not available in ascii)

Table. Deaths per 1,000 per year in late 18th century England (17) and 20th century Nigeria (39)
----------------------------------------------------------
		Essex, England		Garki, Nigeria
Age		Nonmarsh	Marsha	Age	Savanna
----------------------------------------------------------
5 44   	95.3				1-4	154
6-10		6.3   	9.4		5-8	15
11-15		6.8		10.8		9-18	10
16-20		8.4	   	12.7		19-28	6
----------------------------------------------------------
aThe high death rates in the marsh parishes are attributable to malaria and were actually lower than in the previous century during the peak of the Little Ice Age. Moreover, the Essex data are skewed because many of the marsh communities included predominantly men. The rate was similar to that measured in the 1970s by the World Health Organization in a malaria-endemic area of sub-Saharan Africa. 

Thomas Sydenham (1624-1689) is widely regarded as a founder of clinical medicine and epidemiology. His book on fevers (1666), which was later expanded into Observationes Medicae (1676), a standard text for 2 centuries, includes extensive details of agues, with descriptions of the course of paroxysms, the periodicity of tertians versus quartans, and the seasonality of the disease. As with Harvey,
there can be no doubt that his descriptions refer to malaria; moreover, he states, "When insects do swarm extraordinarily and when . . . agues (especially quartans) appear early as about midsummer, then autumn proves very sickly." We cannot assume that insects referred to mosquitoes, but the statement that an early appearance
of quartans was a premonition of a high incidence of illness
(presumably agues?) has the distinct ring of modern epidemiology. Nevertheless, it is clear from these and other sources that the disease remained prevalent, even in the coldest years. For example, Harvey's ague began in the fall of 1597, yet the years 1594 to 1597 had been so cold and wet that wheat harvests were a disaster (1). Similarly, in 1657 to 1658, snow lay on the ground for 102 daysindicating exceptionally cold weather even for the times. The
summer crops were a disaster, yet Oliver Cromwell (1599-1658) died of a tertian ague in September 1658, just as another severe winter was setting in.

Malaria Species

P. vivax and P. falciparum both have a tertian periodicity, so it is unclear which species was responsible for tertiary ague in England. P. vivax can persist for many years as a dormant hypnozoite in the liver, giving rise to occasional clinical relapses (caused by production of the blood stages of the parasite), whereas there is no evidence of hypnozoites in P. falciparum. Twentieth-century studies
in Russia and Holland showed that some strains of P. vivax from the northern hemispheregiven the subspecific name P. vivax hibernansdid not produce clinical symptoms until 8-9 months after the infective bite. Both features would clearly have survival value for P. vivax in a temperate climate, enabling it to cope with long winters and episodes of successive cold summers. On the other hand, P. vivax is generally a more benign pathogen than P. falciparum, but
the death rate in the English marsh parishes suggests that
infections were far from benign. This may indicate that P.
falciparum was also present, and Harvey's observations on the consistency of blood in fatal cases support this possibility.

Laboratory studies have shown that tropical strains of P. falciparum do not multiply in European mosquitoes. However, European strains did cause major epidemics in Russia and Poland in the 1920s, with high death rates as far north as Archangel, Russia. In Holland, mosquitoes sheltering in the warmth of stables and human habitations continued to feed and transmit malaria throughout the winter and during colder summers. Thus, the dormancy and delayed onset of P.
vivax were probably not essential for transmission, and it is conceivable that P. falciparum was endemic in England during the Little Ice Age. Alternatively, a more virulent strain of vivax may have been present. The question may never be resolved because the strains involved are now extinct.

The Cure

The strongest evidence that ague was indeed malaria is the identity of its cure. Until the mid-20th century, the only effective remedy for malariaat least in western medicinewas an extract of cinchona powder, obtained from the bark of several tree species native to the
Andes, in South America. The principal active ingredient in this bark is quinine, a drug that has probably benefited more people than any other in the combat of infectious disease. Quinine is still used today, but few people know that its effective use for malaria therapy was first developed in tests with ague patients living in
the salt marshes of Essex, less than 50 km from the center of London. What is more, the field experimentation that led to this English remedy was conducted during the coldest years of the Little Ice Age.

The first prescription of cinchona powder in England is attributed to Robert Brady in 1660. Thomas Sydenham advocated its use in his Methodus curandi febres in 1666. By that time, the "Jesuit's Powder" was already widely known in Europe, but in Protestant England many orthodox physicians were prejudiced against its usepartly because
its export from Peru and Bolivia was in the hands of Catholics and its use had not been mentioned in the classical medical texts of Galen and partly because a reliable prescription had not been developed. In the end, popularization of the drug came in a highly unorthodox manner: a relatively untutored man, Robert Talbor,
abandoned his apprenticeship to an apothecary to develop a safe dosage and an effective treatment regimen: "I planted myself in Essex near the sea side, in a place where agues are the epidemical diseases, where you will find but few persons but either are, or have been afflicted with a tedious quartan." After several years of study and testing, he developed what we would now call a patent medicine, a secret formulation that was essentially an infusion of
cinchona powder in white wine. 

In 1672, Talbor popularized his remedy by publishing Pyretologia: a Rational Account of the Cause and Cures of Agues. The success of his treatments became widely known and brought him rapid fame and fortune. Charles II appointed him Physician Royal in 1672. He was knighted in 1678, after he cured the King of an ague (20). Sir Robert Talbor then traveled to France, where he cured the son of Louis XIV. With the additional title of Chevalier Talbot, he became
famous throughout Europe, curing Louis XIV, Louisa Maria, Queen of Spain, and hundreds of other royal and aristocratic persons (Figure 5).

Figure 5. The Talbor's English Remedy: Wonderful
Secret for Curing of Agues and Feavers (1682)....
(Figures not available in ascii)

Talbor's rise to fame has been told in many 
publications (21,22), yet none of these mention
that the entire story took place in a period
when temperatures were probably colder than in
any other period in the past 10,000 years.  
Records for central England from 1670 to 1700
suggest that snow lay on the ground for an   
average of 20 to 30 days (in some years more than 100 days) as opposed to 2 to 10 days in the present century. In the winter 1683-84, the ground was frozen to more than 1 m. Belts of sea ice 5 km wide were present along the coast 
in the English Channel and are believed to have   
been 30 to 40 km wide off the coast of the Netherlands. The average summer growing season  was approximately 5 weeks shorter than in the 20th century, and in some years the difference  may have been more than 2 months (1).
Nevertheless, the fact that the European 
aristocracy was eager to pour money and honors
on an untutored commoner suggests that malaria
continued to be a serious problem.

Figure 6. Map of the county of Essen in 1594. The extensive
coastal salt marshes provided high-quality grazing for sheep but were also a favored habital for An. atroparvus, a high effective malaria vector. The disease was a major cause of illness and death in the area until the end of the 18th
century. Reproduced by courtesy of Essex Record Office.
(Figures not available in ascii)

Defoe and Beyond

Robinson Crusoe was shocked to discover that a 
footprint in the sand was not his own: "so that 
I shook with cold, like one in an ague." His the
creator, Daniel Defoe (1660-1731), had traveled 
extensively in southern England in 1685 to 1690. In A Tour through the Whole Island of Great Britain (23), he described the  depredations of ague in the Dengie marshes of
Essex (Figure 6), an area 70 km east of London 
that remained notorious for the disease until
the end of the 18th century (15): ". . . our 
London men of pleasure . . . go from London on
purpose for the pleasure of shooting [the
abundant wildfowl] but those gentlemen who . . 
. go so far for it, often return with an Essex
ague on their backs, which they find a heavier
load than the fowls they have shot." These sportsmen may have been wealthy enough to buy Talbor's remedy, but ordinary people suffered greatly in these unhealthy marshes. They risked living there because, as the land was excellent for rearing high-quality sheep century. and cattle, landlords were willing to pay high wages: " . . . for it being a place where every body cannot live, those that venture it, will have encouragement, and indeed it is but
reasonable they should."

However, as in the malarious tropics today, the nonimmune were in great danger. Thus Defoe observed: "a strange decay of the [female] sex here . . . it was very frequent to meet with men that had had from five to six, to fourteen or fifteen wives . . . the reason . . . was this; that they [the men] being bred in the marshes themselves, and seasoned to the place, did pretty well with it; but that they always went into the hilly country . . . for a wife: that
when they took the young lasses out of the wholesome and fresh air, they were healthy, fresh and clear, and well; but when they came out of their native aire into the marshes . . . they presently changed their complexion, got an ague or two, and seldom held it above half a year, or a year at most; and then . . . [the men] would go to the uplands again, and fetch another; so that marrying of wives was
reckoned a kind of good farm to them."

During the 18th century, temperatures began to return to
pre-mid-16th century levels. However, the wide variability of the Little Ice Age continued for at least 150 years. In the 1770s, much as today, alarm that the climate was becoming increasingly erratic prompted new emphasis on the recording of weather variables. 

There are numerous accounts of malaria in all the northern European countries in the 18th and early 19th centuries. The wealth of records in this period confirms that the disease was common at many coastal sites in England and in some parts of Scotland, with occasional transmission as far north as Inverness (5720'). The northern limit (24) was roughly along the 15C July isothermnot the 15C winter isotherm, as stated by some authors (8,10). Thus, there was endemic transmission in southern Sweden and Finland, with
occasional devastating epidemics that extended to the northern end of the Gulf of Bothnia, close to the Arctic Circle (25,26). In North America transmission occurred throughout most of the United States (27) and in some parts of Canada (28).

In 1827, John Macculloch (29) wrote "We may take the average of life among ourselves, in round numbers, at fifty with sufficient safety for this purpose. In Holland it is twenty-five; the half of human life is cut off at one blow, and the executioner is malaria, for there is no other cause for the superior mortality of that country." 

The same was probably true of the coastal marshes of England, although the incidence appears to have decreased over the century. After the 1880s, transmission dropped precipitously, and the disease became relatively rare, except during a short period following World War I. This decline cannot be attributed to climate change, for it
occurred during a warming phase, when temperatures were already much higher than in the Little Ice Age. Factors contributing to the decline include the following: Mosquito habitat was eliminated by improved drainage and extensive land reclamation. Root crops, such as turnips and mangel-wurzels, were introduced as winter fodder, enabling farmers to maintain much greater numbers of animals throughout the year; this diverted An. atroparvus, a zoophilic species, from feeding on humans. Rural populations declined as
manual labor was replaced by machinery, further reducing the
availability of humans as hosts for both mosquitoes and parasites. New building materials and improved methods of construction made houses more mosquito-proof, especially in winter, thus reducing the risk for contact with mosquitoes. Greater access to medical care and a rapid drop in the cost of quinine reduced the survival rate of the malaria parasite in its human host.

A similar decline occurred in the more prosperous countries of EuropeNorway, Sweden, Denmark, Germany, Holland, Belgium, France, and northern Italy. However, malaria maintained a much firmer grip on Eastern EuropeFinland, Poland and Russia, and the countries bordering the Black Sea and the eastern Mediterranean. It was not until the advent of DDT, after World War II, that a concerted attempt could be made to eradicate the disease from the entire continent (30). At the same time, the Communicable Disease Center
(forerunner of the Centers for Disease Control and Prevention) was set up in Atlanta to eliminate malaria from the United States, where it was still endemic in 36 states (27), including Washington, Oregon, Idaho, Montana, North Dakota, Minnesota, Wisconsin, Iowa, Illinois, Michigan, Indiana, Ohio, New York, Pennsylvania, and New Jersey.

Conclusion

In 1975, the World Health Organization declared that Europe was free of malaria. The last indigenous case in England had been in the 1950s and in Bruegel's Holland in 1961. Results were equally spectacular on other continents. By 1977, 83% of the world's population was living in regions from which malaria had been eradicated or control activities were in progress. The only areas in which the control effort was limited were those of high endemic stability, particularly sub-Saharan Africa.

However, this momentum could not be sustained, and the goal of worldwide eradication was abandoned. Today, the disease is again common in many parts of Central America, the northern half of South America, much of tropical and subtropical Asia, some Mediterranean countries and many of the republics once part of the Union of Soviet Socialist Republics. This rapid recrudescence has been attributed to
population increase, forest clearance, irrigation and other
agricultural activities, ecologic change, movement of people, urbanization, deterioration of public health services, resistance to insecticides and antimalarial drugs, deterioration of vector control operations, and disruptions from war, civil strife, and natural disasters. Claims that malaria resurgence is due to climate change ignore these realities and disregard history. For example, the many
statements that recent climate change has caused malaria to ascend to new altitudes (10,31,32) are contradicted by records of its distribution in 1880 to 1945 (33-35).

With the return of malaria, there has been an exponential rise in international travel. Tens of thousands of cases are imported into Europe and North America each year. As was anticipated 30 years ago (36), a few of these cases give rise to autochthonous transmission by indigenous mosquitoes.

In much of Western Europe, the likelihood that malaria will become reestablished is probably small (37). However, in countries (e.g., in the Balkans) where malaria control has ceased but the probability of transmission remains high, reintroduction could threaten the public health. Indigenous transmission associated with imported cases has recently been reported in Kazakhstan, Kyrgyzstan, Turkmenistan, Uzbekistan, Bulgaria, the Republic of Moldova,
Romania, Italy, and Corsica, and the malaria-free status of Europe may be in jeopardy (38). Public concern should focus on ways to deal with the realities of malaria transmission, rather than on the weather.

Acknowledgments

I am grateful to Routledge PLC for permission to include Figure 1; the Kunsthistorisches Museum, Vienna, for permission to include Figure 2; the Wellcome Institute for the History of Medicine for the use of their facilities and permission to include Figure 5; and Kevin Bruce and the Essex Record Office for Figure 6.

Dr. Reiter is chief of the Entomology Section, Dengue Branch, CDC, San Juan, Puerto Rico. His research interests include the physiology, behavior, and ecology of mosquitoes, the transmission dynamics and epidemiology of the diseases they transmit, and improved methods for their control.

Address for correspondence: Paul Reiter, Dengue Branch, Centers for Disease Control and Prevention, 2 Calle Casia, San Juan, Puerto Rico 00921-3200, USA; fax: 787-766-6596; e-mail: ipr1@cdc.gov.s

References

  1. Lamb HH. Climate, history and the modern world. London:
Routledge; 1995. 
  2. Rasool SI, Schneider SH. Atmospheric carbon dioxide and
aerosols: effects of large increases on global climate. Science 1971;175:138-41.
  3. Calder N. The weather machine. New York: Viking; 1974.
  4. Houghton JT, Meira Filho LG, Callander BA, Harris N, Kattenberg A, Maskell K, editors. The science of climate change: contribution of Working Group I to the second assessmet of the Intergovernmental Panel on Climate Change. Cambridge: University Press; 1995.
  5. Lindzen RS. Can increasing carbon dioxide cause climate change? Proc Natl Acad Sci U S A 1997;94:8335-42.
  6. Soon W, Baliunas SL, Robinson AB, Zachary WR. Environmental effects of increased atmospheric carbon dioxide. Climate Research 1999;13:149-64.
  7. Watson RT, Zinyowera MC, Moss RH, editors. Impacts, adaptations and mitigation of climate change: scientific-technical analyses. Contribution of Working Group II to the Second Assessment of the Intergovernmental Panel on Climate Change. Cambridge: University Press; 1995.
  8. Patz JA, Epstein PR, Burke TA, Balbus JM. Global climate change and emerging infectious diseases. JAMA 1996;275:217-23.
  9. Martens WJM, Slooff R, Jackson EK. Climate change, human health, and sustainable development. Bull World Health Organ 1997;75:583-8.
 10. Epstein PR, Diaz HF, Elias S, Grabherr G, Graham NE, Martens WJM, et al. Biological and physical signs of climate change: focus on mosquito-borne diseases. Bulletin of the American Meteorological Society 1998;79:409-17.
 11. Houghton JT, Meira Filho LG, Callander BA, Harris N, Kattenberg A, Maskell K, editors. The regional impacts of climate change. Cambridge: University Press; 1998.
 12. Martens WJM. Health impacts of climate change and ozone
depletion: an ecoepidemiologic modeling approach. Environ
Health Perspect 1998;106 Suppl;241-51.
 13. Bruce-Chwatt LJ, de Zulueta J. The rise and fall of malaria in Europe, a historico-epidemiological study. Oxford: Oxford University Press; 1980.
 14. Grove JM. Little ice age. London: Routledge Keegan and Paul; 1988.
 15. Dobson MJ. "Marsh Fever" the geography of malaria in England. Journal of Historical Geography 1980;6:357-89.
 16. Dobson MJ. Contours of death and disease in early modern England. Cambridge: Cambridge University Press; 1997.
 17. Dobson MJ. Malaria in England: a geographical and historical perspective. Parassitologia 1994;36:35-60.
 18. Macdonald A. On the relation of temperature to malaria in England. Journal of the Royal Army Medical Corps
1920;35:99-119.
 19. Bruce-Chwatt LJ. Bruce-Chwatt's essential malariology. Gilles HM, Warrell DA, editors. London: Edward Arnold; 1993.
 20. Dock G. Robert Talbor, Madame de Svign, and the introduction of cinchona: an episode illustrating the influence of women in medicine. Annals of Medical History 1927;4:241-7.
 21. Siegel RE, Poynter FNL. Robert Talbor, Charles II and cinchona: a contemporary document. Medical History 1962;6:82-5.
 22. Dobson MJ. Bitter-sweet solutions for malaria: exploring natural remedies from the past. Parassitologia 1998;40:69-81.
 23. Defoe D. A tour through the whole island of Great Britain. London: Penguin; 1986.
 24. Russell PF. World-wide malaria distribution, prevalence, and control. Am J Trop Med Hyg 1956;5:937-65.
 25. Ekblom T. Les races Sudoises de l'Anopheles maculipennis et leur role pidmiologique. Bull Soc Pathol Exot 1938;31:647-55.
 26. Renkonen KO. ber das Vorkommen von Malaria in Finnland. Acta Medica Scandinavica 1944;119:261-75.
 27. Faust EC. The distribution of malaria in North America, Mexico, Central America and the West Indies. In: A symposium on human malaria, with special reference to North America and the Caribbean Region. Washington: American Association for the Advancement of Science; 1941. p. 8-18.
 28. Fisk GH. Malaria and the Anopheles mosquito in Canada. Can Med Assoc J 1931;Dec:679-83.
 29. Macculloch J. Malaria: an essay on the production and
propagation of this poison and on the nature and localities of the places by which it is produced: with an enumeration of the diseases caused by it, and to the means of preventing or diminishing them, both at home and in the naval and military service. London: Longman & Co; 1827.
 30. Russell PF. Man's mastery of malaria. London: Oxford University Press; 1955.
 31. Loevinsohn ME. Climatic warming and increased malaria incidence in Rwanda. Lancet 1994;343:714-8.
 32. McMichael AJ, Patz J, Kovats RS. Impacts of global
environmental change on future health and health care in
tropical countries. Br Med Bull 1998;54:475-88.
 33. Hackett LW. The malaria of the Andean region of South America. Revista del Instituto de Salubridad y Enfermedades Tropicales 1945;6:239-52.
 34. Reiter P. Global warming and vector-borne disease in temperate regions and at high altitude. Lancet 1998;351:839-40.
 35. Mouchet J, Manguin S, Sircoulon J, Laventure S, Faye O, Onapa AW, et al. Evolution of malaria in Africa for the past 40 years: impact of climatic and human factors. J Am Mosq Control Assoc 1998;14:121-30.
 36. Zulueta J. Malaria eradication in Europe. J Trop Med Hyg 1973;76:279-82.
 37. Rodhain F, Charmot G. Evaluation des risques de reprise de transmission du paludisme en France. Mdicine et Maladies
Infectieuses 1982;12:231-6.
 38. Sabatinelli G. Malaria situation and implementation of the global malaria control strategy in the WHO European region. WHO Expert Committee on Malaria 1998;MAL/EC20/98.9.
 39. Molineaux L, Gramiccia G. The Garki project. Geneva: World Health Organization; 1980.

1St. Thomas' Hospital (1213), in the Borough of Lambeth, was on the edge of the River Thames, surrounded by tidal marshes. Parliament met in two buildings at a similar site in the Borough of Westminster, directly across the river. Both areas were notoriously malarious. Centuries later, the American Founding Fathers followed British parliamentary procedure in choosing a site for their new nation's capital at the edge of a malarious swamp, later referred to as "A Mud-hole Equal to the Great Serbonian Bog." The Serbonian Bog probably refers to the vast flood plains of the Danube that border northern Serbia and Bulgaria. The Balkan region was the last major stronghold of malaria in Europe. Malaria was finally eliminated there in 1975.

Perspectives

Could a Tuberculosis Epidemic Occur in London as It Did
in New York?

Andrew C. Hayward* and Richard J. Coker
*University of Nottingham, Nottingham, United Kingdom;
and St. Mary's Hospital, London, United Kingdom

--------------------------------------------------------

 In early 1999, more than 160 senior
 physicians, public health officials, and
 nurses met to discuss London's tuberculosis
 (TB) control program. The program was
 examined against the public health response
 of New York City's Bureau of Tuberculosis
 Control during a 1988-1992 epidemic. This
 article outlines TB epidemiology and
 control in New York City 10 years ago and
 in London today to assess whether the kind
 of epidemic that occurred in New York could
 occur in London.

Worldwide, tuberculosis (TB) kills more people than any
other infection (1). Prevalence of the disease has been
increasing in many countries since the mid-1980s, and
more people are ill with TB now than at any other time
in history (1). Although most cases occur in the
developing world, some major urban areas in
industrialized countries have also had a resurgence of
the disease (1).

In the late 1980s and early 1990s, New York City had a
major epidemic of TB, with rates tripling in 15 years
and outbreaks of multidrug-resistant TB in many
hospitals. Massive reinvestment in TB services to
control the epidemic (2,3) resulted in a 59% decrease
in cases, from a peak of >3,700 in 1992 to <1,000 in
1998. The incidence of multidrug-resistant TB decreased
by 91% over the same period (4).

In the United Kingdom, TB rates began to increase in
1988 but have since leveled off (5), except in London,
where the increase has been much more dramatic and has
not subsided (6). This article compares the current
epidemiology and control of TB in London with the
situation in New York City in the late 1980s. Urgent
action is needed to strengthen TB control in London if
an epidemic like that in New York City is to be
avoided.

Epidemiology of TB in the Two Cities

The increase in TB notifications
in London is similar to that seen
during the first 10 years of the
epidemic in New York City (Figure)
(7). As in New York, rates of
disease differ considerably in
different parts of the city, with
the highest rates in areas of low
socioeconomic conditions and large
immigrant populations (2,3,6). In
New York, the highest rates were
in central Harlem (79 per 100,000
in 1980 to 170 in 1989) (2). In
London, the highest rates are in
Newham, Tower Hamlets, and Brent
(77-79 per 100,000 population) (6). Rates in several
London boroughs have increased two- to threefold in 10
years (6). In both cities, the increase in case reports
has been mainly among young adults (15-24 years of age)
(3,6).

In both cities, most cases are in nonwhite residents.
In New York City in 1994, 50% of TB patients were
classified as black, non-Hispanic; 26% as Hispanic; 12%
as white, non-Hispanic; and 12% as other/unknown (8).
In London in 1993, 40% were classified under Indian
Subcontinent, 31% white, and 29% "other nonwhite" (9).
From 1988 to 1993, London rates increased most in the
"other nonwhite" category (mainly black Africans) but
also increased among whites and nonwhites who were born
in the United Kingdom. (The rate among those of Indian
Subcontinent background who were born in the United
Kingdom rose from 12 per 100,000 in 1988 to 41 per
100,000 in 1993 [9]). Therefore, importation of disease
is not the only factor causing the increase in London.

In London (as in New York at the beginning of the
epidemic), data on patient origins are not routinely
collected. Preliminary data suggest that at least 55%
of cases are among foreign-born persons (H. Maguire,
pers. comm.). In New York in 1993-94, approximately 28%
of TB cases were among immigrants (3). Thus, in both
cities a substantial proportion of cases is among
immigrants, but this disparity is greater in London.

Early in the epidemic in New York, 38% of persons with
culture-confirmed cases of TB were known to be HIV
infected (2). Data on HIV infection in TB patients are
not routinely collected in the United Kingdom, but a
study in 1993 estimated that at least 7% of TB cases in
London were among HIV-positive persons (10). A more
recent study of 157 patients starting treatment for TB
at an inner-city London hospital in 1996 and 1997
showed that 25% were coinfected with HIV (44%
Europeans, 49% Africans, and 3% Asians). These data
suggest that, in some ethnic groups in some areas of
London, HIV infection is strongly affecting the
epidemiology of TB (11).

Data on drug resistance were not routinely collected
early in the epidemic in New York City. In the early
1990s, 19% of patients with culture-confirmed TB had
multidrug-resistant TB, and in 26% of patients TB was
resistant to isoniazid (12). These figures had declined
to 4.1% and 4.4% by 1998 (4). In 1994-96, 2.6% of
isolates in London were multidrug resistant, compared
with 1% in the rest of the country; 8% were resistant
to isoniazid, compared with 4.7% in the rest of the
country (13). The current levels of drug resistance in
London are lower than those during the epidemic in New
York. However, data from New York demonstrate that
transmission of resistant strains in hospitals and
other settings can lead to a rapid increase in rates of
resistance (14). Several outbreaks of TB, including two
involving multidrug-resistant TB, have already occurred
in London, principally affecting HIV-positive patients
in teaching hospitals (15-17).

 Figure. Tuberculosis rates in London, 1982-1997,
 compared with those in New York, 1972-1994. (Figures
not available in ascii)

Recent transmission appears to be a substantial problem in both cities. Preliminary results from a molecular study to identify recent transmission of TB in inner London suggest that approximately 27% of cases are clustered (10). In New York City, an analysis of the molecular epidemiologic links of TB isolates obtained in April 1991 showed that, of 344 patients, 126 (37%) belonged to one of 31 clusters, and clustering was more frequent in patients who had
multidrug-resistant TB (involved in 53% of clusters), were black (44%), and were homeless (49%) (14).

Both London and New York City have large populations of homeless people (86,000 in New York in 1990 compared with 50,000 in London in 1992) (18,19). In New York City in the 1970s and 1980s, approximately 6% to 7% of homeless persons had active TB (20,21). By 1992, up to 25% of the city's cases of TB occurred in the homeless (22). Fewer data are available for TB rates in London's homeless, but information suggests very high levels of TB. Screening at a
shelter in London in 1993 identified active TB in 1.5% of those screened. Another 3.5% had chest X-rays suggestive of active disease, but the patients were lost to follow-up before further investigation could take place (23). At least 5% of TB patients in London have a history of residence in a hostel for the homeless (H. Maguire, pers. comm.). In both cities, poor compliance with treatment and transmission of disease are major problems in the homeless (18,21).

TB transmission in state correctional facilities was a major problem in New York (3). No outbreaks of TB have yet been documented in prisons in London.

Control in London and New York City

The loss of government funding of TB programs in the 1970s and 1980s in New York City made access to treatment more difficult for poorer sectors of the population (often nonwhite patients) (3), likely contributing to increases in disease in these groups. In the United Kingdom, all patients have free access to National Health Service treatment, but new immigrants, asylum seekers, refugees, and illegal
immigrants may have difficulty accessing health services because of cultural and linguistic barriers (24). Some may have difficulty in obtaining free prescriptions because of lack of necessary documents (24). In addition, immigration policies differ: in the United States, immigrants are screened and treated for active disease before entering the country, and skin-test-positive new entrants are
usually treated for latent infection (25). In the United Kingdom, a few entrants are screened at airports, with the rest being screened at the local level. In some areas, only a few new entrants are screened, and chemoprophylaxis is rarely used except in children (24,26). Thus, disease prevalence among nonwhite patients may be related to inadequate screening in the United Kingdom.

Treatment completion rates in some areas of New York were low. In 1989, for example, 40% of patients with TB did not complete treatment in the city (27), and in some locales, up to 90% of patients did not complete treatment (2). Early in the epidemic, directly observed therapy was rarely used. In London, the number of patients who complete treatment is not known, and directly observed therapy is rarely used (24). An audit in a London hospital showed that 19% of patients were lost to follow-up before completing treatment. In another 15%, patient records were unavailable (A. Pearson, pers. comm.). A study of TB among the homeless in London
found that 43% of suspected cases were lost to follow-up even before the diagnosis could be confirmed (23). Currently, treatment completion rates are not routinely monitored in London.

In the United Kingdom, section 37 of the Public Health (Control of Disease) Act of 1984 allows detention of patients who pose a serious risk for infection to others. In contrast to New York City, where coercion was considered by many to be an important element of TB control (28), this power is rarely used in London (24). Use of detention is difficult to justify when the infrastructure to allow
less coercive adherence-support methods (such as directly observed therapy) is not in place (29). Increasing use of incentives and flexible provision of directly observed therapy in New York City has greatly decreased the use of detention (28). Directly observed therapy to improve compliance is rarely used in London, and for the
least compliant patients it is almost impossible to provide (24).

Early in the epidemic in New York, there were insufficient measures to prevent spread of TB in hospitals, but by 1997 the city had 400 negative-pressure isolation facilities (30). In 1995 in London, there were 103 negative-pressure isolation facilities, only 17 of which had continuous air-pressure monitoring and 49 were considered adequate for housing patients with infectious multidrug-resistant TB
(A. Hayward, unpub. obs.). Guidelines have recently been published to improve infection control in hospitals (31), but adherence will be difficult without an increase in the availability of isolation facilities.

Inappropriate treatment of isoniazid-resistant patients (e.g., failing to use an initial regimen including at least four drugs) is thought to have contributed to the development of multidrug-resistant TB in New York (12), but the explosive rise in rates of multidrug-resistant disease was mainly due to transmission of disease among hospitalized HIV-infected patients (14). It is not known what proportion of patients in London are initially treated with a four-drug regimen. The high rates of isoniazid resistance in
London indicate the potential for rates of multidrug-resistant TB to increase if such regimens are not used. Hospital transmission could then lead to further rapid increases.

At the beginning of the epidemic in New York, surveillance did not provide routine data on drug resistance, ethnicity, country of origin, HIV-related TB, or treatment completion rates. The lack of information delayed recognition of the problem, and assumptions that TB was an immigrant problem proved not to be accurate (32). Similarly, surveillance in London has failed to collect routine data. Drug resistance levels have been monitored routinely only since 1994, and information (such as knowledge of previous treatment) that would be of value in interpreting routine data is
often not available. An enhanced surveillance system has been launched in the United Kingdom, but many London districts are having difficulties in obtaining the additional information required (24). The new system does not currently collect data on treatment completion rates, but collecting such data is proposed for the next
stage of development (J. Watson, pers. comm.).

During the early part of the TB epidemic in New York City, TB control was underfunded and highly fragmented (3). Underfunding of TB services is also a major barrier to improving TB control in London (24), and the system is also highly fragmented (24). For example, in London in 1993, TB was treated by 250 doctors working in 45 different hospitals (5). In the United Kingdom, district-based "Consultants in Communicable Disease Control" are responsible for
control of TB and other infectious diseases in the community. Unlike TB coordinators in the United States, they have no direct authority to design treatment programs or institute directly observed therapy.

Conclusions

The epidemiology and control of TB in London now differ in several ways from those in New York City early in the epidemic: London has a lower proportion of cases in HIV-infected patients, a higher proportion of cases among immigrants, and lower levels of multidrug resistance. However, similarities in epidemiology and control
include similar numbers of cases; a similar increase in disease prevalence; very high prevalence in areas of lower socioeconomic conditions and large ethnic populations; concentrations of disease in the homeless, the HIV infected, and immigrants; transmission of multidrug-resistant strains in hospitals; inadequate isolation facilities; unknown treatment completion rates; high levels of loss to follow-up; and no overall coordination among providers of TB
services. All these factors suggest that if TB control in London is not improved, the city could experience an epidemic of similar proportions to that in New York.

Although need for action was recognized in New York City in 1987, a clear plan for action was not implemented until 1992. By 1995, the epidemic had cost more than $1 billion (3). London needs to learn from the New York epidemic and take prompt action to improve control by developing solutions based on the local epidemiology of the
disease.

Acknowledgment

This study was conducted at the Public Health Laboratory Service, Communicable Disease Surveillance Centre, London Region, United Kingdom.

Dr. Hayward is a Lecturer in Public Health and Epidemiology at the University of Nottingham. His research interests are the epidemiology and control of infectious diseases, particularly tuberculosis.

Dr. Coker is affiliated with the Imperial College School of Science, Technology and Medicine in London as a Senior Lecturer. His book, "From Chaos to Coercion: Detention and the Control of Tuberculosis," is to be published in January 2000, by St. Martin's Press of New York.

Address for correspondence: Andrew C. Hayward, Division of Public Health and Epidemiology, University Hospital, Queen's Medical Centre, Nottingham, NG7 2UH, United Kingdom; fax: 441-115-970-9312; e-mail: Andrew.Hayward@Nottingham.ac.uk

References

  1. World Health Organization. Tuberculosisa global emergency. WHO report on the TB epidemic. Geneva: The Organization; 1994. 
  2. Brudney K, Dobkin J. Resurgent tuberculosis in New York City: human immunodeficiency virus, homelessness and the decline of tuberculosis control programs. American Review of Respiratory Disease 1991;144:745-9.
  3. Frieden TR, Fujiwara PI, Washko RM, Hamburg MA. Tuberculosis in New York Cityturning the tide. N Engl J Med 1995;333:229-33.
  4. Bureau of Tuberculosis Control Information Summary 1998. New York: New York City Department of Health; 1999.
  5. Hayward AC, Watson JM. Tuberculosis in England and Wales 1982-1993: notifications exceeded predictions. Commun Dis Rep Wkly 1995;5:RR29-33.
  6. Hayward A. Tuberculosis control in London: the need for change. London: NHS Executive; 1998.
  7. Tuberculosis in New York City, 1988. New York: New York City Department of Health; 1990.
  8. Information summary. New York City: Bureau of Tuberculosis Control, New York City Department of Health, 1995.
  9. Ormerod LP, Charlett A, Gilham C, Darbyshire JH, Watson JM. Geographical distribution of tuberculosis in national surveys of England and Wales in 1998 and 1993: report of the Public Health Laboratory Service, British Thoracic Society, Department of Health Collaborative Group. Thorax  1998;53:176-81.
 10. Hayward AC, Goss S, Drobniewski F, Saunders N, Goyal M, Shaw R, et al. Molecular epidemiology of tuberculosis in London. British Thoracic Society Winter Conference, December 1997. 
 11. Marshall BG, Mitchell DM, Shaw RJ, Marais F, Watkins RM, Coker RJ. HIV and tuberculosis coinfection in an inner London Hospitalprospective anonymized seroprevalence study. Journal of Infection 1999:38:162-6.
 12. Frieden TR, Sterling T, Pablos-Mendez A, Kilburn JO, Cauthen GM, Dooley SW. The emergence of drug-resistant tuberculosis in New York City. N Engl J Med 1993;328:521-6.
 13. Irish C, Herbert J, Bennett D, Gilham C, Drobniewski F,
Williams R, et al. Database study of antibiotic resistant
tuberculosis in the United Kingdom, 1994-6. BMJ  1999;318:497-8.
 14. Frieden TR, Woodley CL, Crawford JT, Lew D, Dooley SM. The molecular epidemiology of tuberculosis in New York City: the importance of nosocomial transmission and laboratory error. Tuber Lung Dis 1996;77:407-13.
 15. Outbreak of hospital acquired multidrug-resistant tuberculosis. Commun Dis Rep CDR Wkly 1994;4:1.
 16. Breathnach AS, de Ruiter A, Holdsworth GM, Bateman NT,
O'Sullivan DG, Rees PJ, et al. An outbreak of multidrug
resistant tuberculosis in a London teaching hospital. J Hosp
Infect 1998;39:111-7.
 17. Kent PJ, Uttley AHC, Stoker NG, Miller R, Pozniak AL.
Transmission of tuberculosis in British centres for patients
infected with HIV. BMJ 1994;309:639-40.
 18. Citron KM, Southern A, Dixon M. Out of the shadow. London: CRISIS; 1995.
 19. Dugger CW. Study says shelter turnover hides scope of
homelessness. The New York Times; 1993 November 16:A1.
 20. Sherman MN, Brickner MS, Schwartz MS. Tuberculosis in
single-room occupancy hotel residents: a persisting focus of
disease. New York Medical Journal 1980;2:39-41.
 21. McAdam JM, Brickner PW, Scharer LL, Crocco JA, Duff AE. The spectrum of tuberculosis in a New York City men's shelter clinic (1982-1988). Chest 1990;97:798-805.
 22. Tuberculosis in New York City, 1992. New York City: New York City Department of Health; 1992.
 23. Kumar D, Citron KM, Leese J, Watson JM. Tuberculosis among the homeless at a temporary shelter in London: report of a chest X-ray screening programme. J Epidemiol Community Health 1995;49:629-33.
 24. Results of pan London multidisciplinary workshops on
tuberculosis control. Report to the Director of Public Health for London; 1999.
 25. American Thoracic Society. Control of tuberculosis in the United States. Am Rev Respir Dis 1992;146:1623-33.
 26. Hardie RM, Watson JM. Screening immigrants at risk of
tuberculosis. BMJ 1993;307:1539-40.
 27. Tuberculosis. City Health Information (CHI) 1996;15:12-3. 
 28. Gasner MR, Maw KL, Feldman GE, Fujiwara PI, Frieden TR. The use of legal action in New York City to ensure treatment of tuberculosis. N Engl J Med 1999;340:359-66.
 29. Coker RJ. Public health, civil liberties, and tuberculosis. BMJ 1999;318:1434-5.
 30. Perales, CA. Memorandum to Mayor David N. Dinkins. Update on tuberculosis control activities. New York: Office of the Mayor; 1998.
 31. The Interdepartmental Working Group on Tuberculosis. The prevention and control of tuberculosis in the United Kingdom. Prevention of transmission of drug resistant tuberculosis and tuberculosis in HIV infected patients. London: Her Majesty's Stationery Office; 1999.
 32. Bellin E. Failure of tuberculosis control: a prescription for change. JAMA 1994;271:708-9.

Perspectives

Japanese Encephalitis Immunization in South Korea:
Past, Present, and Future

Young Mo Sohn
Yonsei University College of Medicine, Seoul, South
Korea

--------------------------------------------------------

 Japanese encephalitis (JE), once a major
 public health problem in South Korea, has
 declined since the 1980s, as a result of
 improved living conditions, a mosquito
 eradication program, and a national JE
 vaccination program, which includes annual
 booster vaccine for all children < 15 years
 of age. Increased immunity has greatly
 reduced illness and death; however, vaccine
 adverse effects are increasing, and a
 National Compensation Program for Vaccine
 Injury was begun in 1995. This article
 reviews past successes, current problems,
 and future direction of the JE vaccination
 program in South Korea.

Epidemiology of Japanese Encephalitis (JE) in South
Korea

Historically, JE occurred seasonally in South Korea,
causing high rates of illness and death. Although
public health records concerning the incidence of the
disease before 1933 are not available, outbreaks of
"summer encephalitis" were recognized. During summer
1934-35, an outbreak was reported of suspected
encephalitis, with acute onset of spiking fever and
headache, as well as neurologic abnormalities and
mental changes. Although these outbreaks were described
as meningococcal meningitis by Korean health
authorities, some Japanese workers reported that many
of the cases were encephalitis (1,2). In 1946, Sabin et
al. (3) isolated JE virus from an American soldier
stationed in South Korea. Deuel et al. (4) conducted
the first extensive serologic survey by neutralizing
test in four areas of South Korea and reported that JE
virus was widely disseminated. In 1947, JE was added to
the list of first-class emergency diseases in South
Korea.

In 1949, when JE became notifiable, 5,548 cases of
clinically suspected disease were reported with 2,429
deaths (49% case-fatality rate). The criteria for
reporting were high spiking fever, headache, and mental
changes with any symptom of central nervous system
involvement. Hullinghorst et al. (1), who described the
1949 epidemic in South Korea, reviewed >600 hospital
records. Results of bacterial cultures of cerebrospinal
fluid (CSF) were reviewed for 567 cases, and the
complement fixation and neutralization tests were
performed in selected cases. Pathology findings and JE
virus isolation from a case, negative results of CSF
bacterial cultures, and a relatively uniform clinical
picture, including acute high fever, headache, and
various neurologic abnormalities, suggested that the
outbreak in 1949 was JE (1,5). Thereafter, several
outbreaks of thousands of JE cases were reported every
2 to 3 years, and JE was recognized as a recurring
public health problem in South Korea (6,7).

In 1954, when the Communicable
Disease Control Act (Reg. 308) was
enacted, JE was classified as a
first-class notifiable
communicable disease. The largest
recorded JE epidemic, involving
6,897 reported cases, occurred in
1958 (7). In 1963, JE was
reclassified as a second-class
notifiable disease, along with
polio, pertussis, measles, mumps,
and other childhood diseases.
Since 1964, seroepidemiologic
studies with the hemagglutination
inhibition test (HI) have been
conducted routinely for clinically
suspected cases. In 1964, 108
paired and 1,636 acute-phase sera
were tested (8). The results
indicated that 86.7% of
acute-phase sera were negative by
the HI test and 54% of paired sera
were positive in their second
sample. Several thousand cases
were reported annually until 1968,
with the exception of 1963, when
only 19 cases were reported; Chang
et al. (9) provide evidence for
underreporting in that year
(Figure 1). Case reports sharply
declined to <100 cases in 1969;
<1,000 cases were reported
annually until 1973. The last
large epidemic was recorded in
1982, when 1,197 cases and 40
deaths were reported. This rapid
decline in reported cases may be
related to the extensive use of
insecticides in rice farming,
which led to a marked decrease of
Culex tritaeniorhynchus in rural
habitats (10,11).

The highest incidence of JE was recorded in Seoul and
Kyonggi Province during the first officially reported
outbreak in 1949 (Figure 2A). In the epidemics during
1965 to 1984, a higher incidence was recorded in the
southwestern than in the northeastern provinces (Figure
2B). The southwestern provinces are mainly under rice
cultivation with large flooded rice paddies, while the
northeastern provinces are mountainous. Vector mosquito
populations were more prevalent in the southwest than
in the northeast (8), leading to greater risk for
enzootic viral transmission in the southwestern
provinces. The high incidence in Seoul in the 1949
outbreak may be accounted for by the rapidly increasing
susceptible population in the capital and its suburbs
since 1945 (7). Approximately 90% of the cases occurred
from mid-August to mid-September; more than 80% were
among children 3 to 15 years of age (9,10).

Factors affecting the size of JE epidemics include the
risk for being infected by the virus when bitten by a
vector mosquito and the JE seropositive rate in the
population. In 1965, before the national vaccination
program was implemented, Chang et al. (9) estimated the
natural seroconversion rate of HI antibody in children
<15 years of age at approximately 9%, indicating that
the estimated incidence of JE cases during epidemics
would have been higher without intervention. Several
studies comparing seroprevalence rates before and after
the epidemic season indicated that the seropositivity
rate was higher in South Choongchung Province (86.4%
before the epidemic season vs ~100% after the epidemic
season in 1984) than in Seoul (56% before the epidemic
season vs ~72% after the epidemic season in 1987)
(Figure 3) (12-15).

Figure 1. Cases of Japanese encephalitis by
liters of vaccine distributed, 19361998, South
Korea.  (Figures not available in ascii)

Figure 2. A. Incidence of Japanese encephalitis
(JE) per 100,000, by province, South Korea, 1949 and
1958 (7). B. Incidence of JE per 100,000, by
province, South Korea, 1965 to 1998 (modified
from 8-11,24). (Figures not available in ascii)

From 1985 to 1998, only 21 cases of JE were
serologically confirmed. Factors leading to
this decrease include the National Immunization
Program (NIP), initiated in 1983, which has
conferred protective immunity on children at
risk. The national socioeconomic growth for the
past 3 decades has allowed improved standards
of living and environmental conditions. The
modernization of agricultural practices led to
changes in the epidemiology of JE.

Epidemic Forecast Program

Since 1975, an epidemic forecast program to
alert the public against JE has been
implemented by the health authorities every
year before the epidemic season (16) (Table 1).
To predict the possibility of an outbreak, the
health authorities have attempted to isolate
the virus from the vector mosquito, C.
tritaeniorhynchus; estimate population density
of C. tritaeniorhynchus among captured
mosquitoes; and determine JE antibody levels in
pigs. Monitoring for C. tritaeniorhynchus
begins in early May. The population density of
C. tritaeniorhynchus in mosquitos captured in seven urban and rural areas was 76.1% in 1982, when the most recent epidemic occurred (11). Because outbreaks in humans seem closely related to a rapid rise of HI antibody titers in pigs, the blood of young pigs (<6 months old) is screened weekly at slaughterhouses for immunoglobulin (Ig) M antibody. In areas where epidemics have occurred, vector
mosquitos collected are homogenized and injected into suckling mice; JE virus is identified by HI and, recently, by polymerase chain reaction in the brains of mice manifesting JE symptoms.

When the JE vector mosquito is first detected, health authorities issue an alert that encourages JE vaccination (Table 1). If JE IgM antibody titers increase in pigs or JE virus is isolated, aggressive mosquito control is implemented.

Active JE Immunization in the Spring Mass Immunization Campaign 

A small amount of the mouse brain-derived formalin-inactivated Nakayama vaccine from Japan was imported in 1967 (17,18). In the early 1970s, the Nakayama vaccine began to be produced locally. Despite an active campaign with the Nakayama vaccine, the annual number of doses distributed remained under 500,000 in the 1970s, for a vaccine coverage rate of <5% (11). However, the vaccine coverage
rate increased to 16.8% in 1981. The most recent epidemics1,197 reported cases in 1982 and 139 in 1983prompted a mass immunization program for children at risk. Children 3 to 15 years of age were vaccinated at state public health centers, and simultaneous vaccination in the private sector was encouraged; as a result, the immunization rate increased to 60% in 1983, when a JE epidemic
occurred. In 1986, 12 million doses were distributed in the public and private sectors, which was more than the estimated number of children <15 years of age. From 1985 to 1992, almost 10 million doses of Nakayama vaccine were distributed each year, enough to vaccinate 97% of the nation's children (Figure 1).

Figure 3. Seropositivity of HI antibody before and after
the Japanese encephalitis epidemic. (A), (B), (C) before
the implementation of vaccination program; (D), (E)
after the implementation of vaccination program (modified
from 13-16). (Figures not available in ascii).

 Table 1. The Japanese encephalitis
 (JE) epidemic forecast program, South
 Korea (Table not available in ascii)
 
In 1983, the age of initial dose of JE vaccine was recommended by the health authorities at 3 years of age, except for areas in which the disease had recently become endemic, where it could be given earlier. The series consisted of three doses of primary vaccination,
with annual boosters until 15 years of age. Because the vaccination rate could not be maintained above 90% by individual immunization during the spring campaign (May to July), mass immunization at schools and public health centers was used.

Since 1984, when JE vaccine coverage reached almost 90%, only a few sporadic cases occurred in young adults or the elderly. The incidence of JE was reduced from >18.5/105 in 1964 to <0.02/105 over the past 3 decades. Several factors contributed to the rapid decline. In addition to improved living conditions, public health, the widespread use of agricultural herbicides and insecticides, a decrease in the population density of vector mosquitos by the removal of mosquito habitats, and the modernization of agricultural
practices, such as improved sanitary conditions on pig farms in rural areas, may have decreased the risk for human infection. In South Korea, C. tritaeniorhyncus usually appears in May and multiplies rapidly in July and August. From 1982 to 1985, mosquitos were collected in seven areas from May to late October. The number of mosquitos gradually decreased, from 11,278 per day (76% C. tritaeniorhyncus) in 1982; to 4,334 (59.2%) in 1983; 2,150 (40.3%) in 1984; and 1,212 (34.4%) in 1985 (11). The virus infestation rate
of the vector mosquito has been consistently <1% since 1990.
Consequently, the risk of being exposed to the vector mosquito and infected by JE virus has decreased.

Another contributing factor is the policy of maintaining high levels of immunity through the expansion of the routine childhood immunization program. An immunization coverage rate >97% has significantly contributed to the prevention of JE. In 1969, a JE vaccine efficacy trial was performed among schoolchildren in southwestern provinces in South Korea; approximately 45,000 children were vaccinated and another 45,000 were controls. Although no epidemic occurred during this period (19,20) because of vaccination, the HI seropositive rate of 11.6% in prevaccination sera increased
to 75% in postvaccination sera after two doses of mouse
brain-derived Nakayama vaccine. The seropositivity rate by
neutralizing antibody test was 99% postvaccination.

At present, visitors to South Korea on business or vacation are considered at low risk for JE; routine vaccination with Nakayama vaccine may not be necessary for these travelers. However, JE immunization is recommended for visitors to disease-endemic areas during transmission season. Vaccine is advised especially for travelers who stay for an extended time (>30 days) or are at high risk for exposure to vector mosquitos through activities or housing. 

Vaccine Manufacture and Supply

In 1972, the manufacture of Nakayama JE vaccine was privatized and transferred to the South Korea Green Cross Company. Currently, seven pharmaceutical companies manufacture JE vaccine. Before 1995, when boosters were given annually, the total amount of vaccine needed for
the population at risk was 8,500 to 9,000 liters per year. Vaccine production is projected more than 1 year in advance; the amount to be used the next year is usually determined in February. Then the injection of JE virus into mice, recovery of the virus from mouse brain tissue, and production of bulk vaccine begin. Vaccine is ready in October, and after a thorough testing in January of the following year, is distributed in March. 

Vaccine Adverse Effects

Although the mass immunization campaign was successful in attaining high rates of vaccination coverage, JE vaccine-related adverse effects may have been overlooked. A reporting system was not available until 1995, so the incidence of JE vaccine-related adverse effects among South Korean children is not known. However, in 1994, six cases of severe systemic illness after JE vaccination were reported (21) (Table 2). Two sudden deaths were attributed to anaphylactic reaction to the vaccine, and four cases of severe neurologic illness, including encephalopathy and acute disseminated encephalomyelitis, were reported. Since 1994, seven cases have been filed with the Advisory Committee of the National Immunization Program, according to the National Compensation Program for Vaccine
Injury (Table 2). 

Table 2. Cases of adverse reactions to Japanese encephalitis vaccine reported to the National Compensation Program for Vaccine Injury, South Korea, 1994 (Table not available in
ascii)

The Future of the Immunization Program

Although the cost-effectiveness of the JE immunization program, including annual boosters, has not been evaluated, JE has been successfully controlled in South Korea for the past 2 decades. Questions remain about the optimum immunization schedule, including appropriate ages for initial vaccination and boosters. The state health authority recommends two doses 4 weeks apart with a booster 1
year later as a primary immunization, and annual boosters thereafter up to 15 years of age. Since the longevity of neutralizing antibody after primary vaccination was not known and seemed short lived, frequent boosters appeared necessary. However, without sound evidence, the total of 14 doses of vaccine until 15 years of age led to concerns about excessive vaccination and increased risk for vaccine adverse effects.

The 1994 report of the cluster of severe adverse systemic effects after JE vaccination prompted medical and social debate (22,23). A causal association between JE vaccine and the cluster of severe adverse effects has not been clearly established; however, empiric evidence, including the timing of events, characteristics of the adverse reactions, susceptibility, and lack of alternative etiologic
agents, indicates a causal relationship. Public concern over adverse reactions led to refusal of the vaccination and a consequent decrease in coverage rate. In response, the National Compensation Program for Vaccine Injury was begun in 1995.

During public debate about the JE immunization schedule, the safety and quality of the domestically produced JE vaccines were questioned. The Advisory Committee of the National Immunization Program announced that the annual booster immunization schedule had become impractical because of the inappropriate administration of the vaccine during the mass immunization in the spring, the need to define the long-term immunity in the elderly (> 65 years of age), poor recognition of adverse effects, the need to guarantee the
quality of the vaccine manufactured in South Korea, and public reluctance to receive JE vaccination. The committee agreed that the JE immunization program should be improved with regard to the schedule, age of initial vaccination, booster schedule, and vaccine strain. The annual booster schedule was changed to once every 2 years, and single-dose rather than multidose vials were used, as recommended by the South Korean Society of Pediatricians.

In 1996, a seroprevalence study for plaque-reduction neutralizing antibody (PRNT) was carried out to indirectly evaluate the efficacy of booster vaccination in 311 schoolchildren (24). The neutralizing antibody titers were found to decrease gradually as the interval between boosters increased (Figure 4A). The seropositivity rate of
98.1%, 99%, and 95.6% at 6, 18, and 30 months, respectively, had declined to 71.4% by 42 months (Figure 4B).

To replace the inactivated mouse brain-derived
vaccine, a clinical trial with a live
attenuated SA14-14-2 vaccine, which has been
used safely and effectively in >100 million
children in China since 1988 (25), was
conducted in South Korean children in 1997. A
preliminary immunogenicity study among South
Korean children 1 to 3 years of age indicated a
96% rate of seroconversion in PRNT antibody,
with a geometric mean titer (GMT) of 188 after
a single primary immunization dose. In children
who had been immunized with two or three doses
of inactivated Nakayama vaccine, the booster
administration of SA14-14-2 vaccine produced an
anamnestic response with a GMT of 3,378 in all
cases without virus-specific IgM response
(Figure 5) (26).

Figure 4. A. Plaque-reduction neutralizing antibody titers
(PRNT) according to interval since last booster injection with Nakayama vaccine among 311 South Korean children, 1996. B. PRNT antibody seropositivity rate according to the interval since last booster injection.
(Figures not available in ascii)

Figure 5. Primary or booster neutralizing antibody responses to one dose of Japanese encephalitis SA14-14-2 vaccine
in South Korean children, 1997 (26).
(Figures not available in ascii)

During the last 10 years, JE has occurred more
frequently in adults than in childrentwo-thirds
of Korean JE patients were middle-aged adults.
Adults are not protected by the current
immunization program, probably because of
waning immunity. Long-term immunity must be
maintained to prevent secondary vaccine
failure. A single booster with the
live-attenuated SA14-14-2 vaccine might confer
long-term immunity for adults and decrease the
frequency of vaccination, as well as the risk
for vaccine adverse effects.

The national immunization program against JE in
children should be continued according to
established schedules in South Korea; however,
the booster schedule should be adjusted.
Surveillance for JE and vaccine adverse
reactions should be strengthened to better
assess the number of cases and reactions
associated with immunization. A new, more
advanced vaccine, such as the live attenuated
SA14-14-2 vaccine, should be adopted and
integrated into the pediatric immunization
schedule.

This work was supported in part by grants from
the Advisory Committee of the National
Immunization Program, Department of Health and
Welfare Grant no. HMP-98-P-0008.

Dr. Sohn is professor of pediatric infectious
disease at Yonsei University and a member of the Advisory Committee of the National Immunization Program. He was a member of a working group for the improvement of the national immunization program and of an ad hoc committee for assessing JE vaccine adverse effects. He also participated in the clinical trial of SA14-14-2 vaccine in South Korea.

Address for correspondence: Young Mo Sohn, Department of Pediatrics, Yongdong Severnace Hospital, Yonsei University College of Medicine, Youngdong P.O. Box 1217, Seoul, 135-270 South Korea; fax: 82-2-3461-9473; e-mail:  youngmo@yumc.yonsei.ac.kr

References

  1. Hullinghorst RL, Burns KF, Choi YT, Whatley LR. Japanese B encephalitis in Korea. The epidemic of 1949. JAMA
1951;145:460-6.
  2. Shiba Y, Chun CH. On epidemic encephalitis or so-called summer encephalitis occurring in Seoul last summer. Journal of Mansen No Ikai 1935;180:1.
  3. Sabin AB, Schlesinger RW, Ginder WR, Matsumoto M. Japanese B encephalitis in an American soldier in Korea. American Journal of Hygiene 1947;46:356-75.
  4. Deuel RE, Bawell MB, Matumoto M, Sabin AB. Status and
significance of inapparent infection with virus of Japanese B encephalitis in Korea and Okinawa in 1946. American Journal of Hygiene 1950;51:13-20.
  5. Lee SY, Kim UY. Studies on the epidemic of Japanese B
encephalitis in Korea 1949. Part 1: Studies on the virus
isolation. Bulletin of National Institute for Prevention of
Infectious Disease 1950;2:54-69.
  6. Chang KS, Han JS, Lee BH. Epidemiological observation of Japanese B encephalitis in Korea, 1958. The New Medical Journal 1959;2:75-82.
  7. Chang IJ. Epidemiology of Japanese encephalitis in Korea. Korean Medical Journal 1959;4:35-50.
  8. Kim KH, Paik SB, Whang CH, Kim HD. Seroepidemiology of Japanese encephalitis in Korea, 1964. Report of National Institute of Health, Korea 1965;2:63-9.
  9. Chang IC, Hong KW, Whang KS. Japanese encephalitis in Korea, 1963. Human serology. Korean Medical Journal 1965;10:77-92.
 10. Kim KH. Epidemiological features of Japanese encephalitis in the Republic of Korea. Korean Journal of Virology 1979;9:43-55.
 11. Lee CW, Oh DK. Epidemiological trend of Japanese encephalitis in Korea. Korean Journal of Preventive Medicine 1987;20:137-46.
 12. Lee YT, Youm BJ. A hemagglutination inhibition antibody test for Japanese encephalitis virus among the Koreans, 1984-1985.
Journal of Korean Society of Virology 1986;15:1-9.
 13. Lee YT, Cho KB, Kang PW, Hong SH, Park CH, Youm BJ.
Distribution of hemagglutination inhibition antibody to
Japanese encephalitis virus in Koreans 1987. Journal of Korean Society of Virology 1989;19:41-7.
 14. Lee CH, Lee YT, Ko KK, Moon KS, Kim OJ. The distribution of hemagglutination inhibition antibody for Japanese encephalitis virus in Koreans, 1979. Korean Journal of Virology 1980;10:65-9.
 15. Lee YT, Lee CH. The distribution of hemagglutination inhibition antibody for Japanese encephalitis virus in Koreans, 1976. Journal of Korean Society of Microbiology 1977;10:65-9.
 16. Kim KH, Shin HK, Ree HI. Survey for the prediction of the occurrence of Japanese encephalitis epidemic in Korea, 1978. Journal of the Korean Society of Virology 1978;8:37-43.
 17. Kim KH, Paik SB, Chang KS. Epidemiology of Japanese
encephalitis in the Republic of Korea, 1967. Annual Report of National Institute of Health, Korea 1967;1:55-72.
 18. Kim KH, Paik SB. Studies on the preparation of JE vaccine with mouse brain. Annual Report of National Institute of Health, Korea 1968;1:31-6.
 19. Kim KH. Japanese encephalitis vaccination. Journal of Korean Medical Association 1982;25:812-7.
 20. Kim KH. Japanese encephalitis vaccine and its efficacy.
Postgraduate Medical Digest 1979;7:148-55.
 21. Sohn YM, Lee DH, Choi KW, Sohn KC, Lee JB, Sshin HK, et al. Cluster of adverse events after Japanese encephalitis
vaccination in Korea. In: Program and Abstract of the 1994
Annual Meeting of Korean Society of Infectious Disease; Seoul, Korea; 1994 Nov 25; [abstract 14]. Korean Journal of Infectious Disease 1994;26;449.
 22. Sohn YM, Pyen BY, Hong YJ, Kang JW, Lee WJ, Oh SH, et al. Antibody response to Japanese encephalitis virus after
immunization. Journal of Korean Pediatrics 1995;38:730-1.
 23. Sohn YM. Standards for immunization practices and vaccine injury compensation. Korean Journal of Infectious Disease 1995;27:247-53.
 24. Sohn YM, Choi BY, Kim CW, Kang JH, Kim JA, et al. Evaluation for the improvement of efficiency of immunization program in Korea; Japanese encephalitis. Report of Evaluation of National Vaccination Program. Washigton: National Institute of Health; 1999, p. 205-6.
 25. Tsai TF, Chan GJ, Yu YX. Japanese encephalitis vaccines. In: Plotkin SE, Orenstein E, editors. Vaccines. 3rd ed. Philadeplhia: W.B. Saunders Company; 1999. p. 672-710.
 26. Sohn YM, Park MS, Rho HO, Chandler LJ, Shope RE, et al. Primary and booster immune response to SA14-14-2 Japanese encephalitis vaccine in Korean infants. Vaccine 1999;17:2259-64.
Synopses

Coccidioidomycosis in New York State

Vishnu Chaturvedi,* Rama Ramani,* Sally Gromadzki,*
Birgit Rodeghier,* Hwa-Gan Chang, and Dale L. Morse*

New York State Department of Health, Albany, New York,
USA; and School of Public Health, University at
Albany, SUNY, Albany, New York, USA

--------------------------------------------------------

      Coccidioidomycosis, a systemic fungal
      disease caused by Coccidioides immitis, is
      endemic in the southwestern United States
      and in parts of Mexico and Central and
      South America. Only sporadic cases have
      been reported in areas (including New York)
      where the disease is not endemic. We used
      hospital discharge records and state
      mycology laboratory data to investigate the
      characteristics of C. immitis infections
      among New York State residents. From 1992
      to 1997, 161 persons had hospital discharge
      diagnoses of coccidioidomycosis (ICD9 Code
      114.0 - 114.5, 114.9). From 1989 to 1997,
      49 cultures from patients were confirmed as
      C. immitis; 26 of these patients had
      traveled to disease-endemic areas. Fourteen
      of 16 isolates had multilocus genotypes
      similar to those of Arizona isolates, which
      corroborates the travel-related acquisition
      of the disease. Our results indicate that
      coccidioidomycosis may be more common in
      New York residents than previously
      recognized. Increased awareness among
      health-care providers should improve timely
      diagnosis of coccidioidomycosis and
      prevention of associated illnesses and
      deaths among patients in nondisease-endemic
      areas.

Coccidioidomycosis, a systemic disease caused by the
dimorphic fungus Coccidioides immitis, is endemic in
the southwestern United States and parts of Mexico and
in Central and South America (1,2). The incidence of
this systemic mycosis in disease-endemic areas
increased during the 1990s (3-5). An estimated 100,000
infections occur in the United States annually, and 1
in 200 infections progresses to disseminated disease
(1,6). Sporadic cases of coccidioidomycosis have been
reported among visitors to the Southwest, and one
earlier report recognized coccidioidomycosis as a
serious travel hazard for visitors to that region
(7-9).

C. immitis, a soil fungus, inhabits a unique ecologic
niche in the topsoil of the lower Sonoran life zone
(10). The infectious propagules are arthroconidia,
single-cell fragments of mycelial threads, which become
easily airborne to cause inhalation exposure. In the
alveoli, arthroconidia undergo dimorphic transition to
spherules, which fragment into endospores. When
released from the spherule, each endospore can act as a
new infectious unit in vivo (1). C. immitis, one of the
most virulent and infectious fungal pathogens, poses a
serious occupational hazard for laboratory personnel,
especially in areas where the disease is not endemic
and workers are less likely to practice biohazard
safety level (BSL)-3 containment, which is required for
the handling of this pathogen. The serious biohazard
potential of C. immitis has led to its inclusion among
the biological agents covered under the recently
enacted Anti-Terrorist and Effective Death Penalty Act,
which regulates interstate transport of infectious
materials (11).

The extent and source of C. immitis infections have not
been thoroughly investigated in areas where the disease
is not endemic. In this study we summarize discharge
data from persons with coccidioidomycosis hospitalized
in New York, a nondisease-endemic area with a single
coccidioidomycosis case report (7). In addition, we
tested a proposed set of molecular markers for
multilocus genotyping of C. immitis to determine the
geographic origins of fungal isolates obtained in this
study.

The Study

Data on hospitalization for coccidioidomycosis in the
state of New York from 1992 to 1997 were obtained from
the New York Statewide Planning and Research
Cooperative System, which compiles uniform inpatient
data from all general acute-care hospitals (all public
and private hospitals and hospital-based and
free-standing ambulatory surgery facilities).

Patients were selected if their hospitalizations listed
a primary or secondary discharge diagnosis of
coccidioidomycosis (ICD9 114.0-114.5, 114.9) during
1992 to 1997. Information was collected from hospital
discharge data abstracts and uniform billing forms for
age at admission, sex, race, ethnicity, county of
residence, insurance status, seasonality, disposition
of patient, hospital length of stay, and total hospital
cost.

Hospital medical records were requested from the 16
patients with positive C. immitis cultures available
for multilocus genotyping (1994-1997). These records
were reviewed for travel histories.

Laboratory Methods

All suspect C. immitis isolates were transported and processed with full safety precautions in a BSL-3 facility. The isolates were subcultured on modified Sabouraud's agar and Mycosel agar, morphologic and microscopic features were examined, and cultures were processed for specific nucleic acid detection. The AccuPROBE C. immitis culture identification test (Gen-Probe Inc., San Diego, CA) was used to confirm the fungal identity. Before 1993, most culture identifications were confirmed by using a C. immitis exoantigen test kit (Scott Laboratories Inc., Fiskeville, RI).

The fungal DNA was extracted from mycelial cultures according to the method of Pan et al. (12). PCR-RFLP was done as described by Burt et al. (13), with minor modifications. We studied five of the 10 loci originally used for each C. immitis isolate because analysis showed
these loci to be most useful for strain differentiation (13). The loci/restriction enzymes used included a1/BsrI, bg/DdeI, bl/DdeI, bq/HinFI, and e1/BsmI. Accordingly, California isolates were expected to show polymorphism at e1, and Arizona isolates could be either positive or negative at each locus, while isolates from Texas would be
polymorphic only for the a1, bq, and e1 loci.

Findings

Hospital discharge data from 1992 to 1997 in New York showed 181 hospitalizations with coccidioidomycosis as the primary or secondary discharge diagnoses (Table 1). The yearly distribution of hospitalizations was 30 (1992), 28 (1993), 30 (1994), 33 (1995), and 32 (1996), with no discernible seasonal pattern. Coccidioidomycosis was the primary diagnosis in 75 hospitalizations and the secondary
diagnosis in 106. The clinical diagnoses according to ICD9 Codes included 105 hospitalizations for pulmonary coccidioidomycosis (114.0, 114.5), one with extrapulmonary coccidioidomycosis (114.1), five with coccidioidal meningitis (114.2), 18 with other forms of
coocidioidomycosis (114.3), 19 with chronic pulmonary
coccidioidomycosis (114.4), and 33 with unspecified coccidioidomycosis (114.9). The mean length of stay was 16 days, with an average hospital cost of $22,516.00.

Table 1. Coccidioidomycosis hospital
discharge data, New York State,
19921997
-------------------------------------
Characteristic	N (%)
Total
hospitalizations  181
Total patients    161
Male 			93(58)
Female  		68(42)
Race
White   		128    (79)
Black   		18(11)
Asian/Pacific
Islander 		2 (1)
Other   		 9 (6)
Unknown  		4 (3)
Ethnicity
Hispanic 		4 (2)
Non-hispanic 	151    (94)
Unknown  		6 (4)
Coccidioidomycosis
diagnosis
Primary 		5(41)
Secondary   	106    (59)
Concurrent diagnosis
HIV			32(20)
Cancer  		16(10)
Died while in
hospital		18(11)
Mean length of stay
(days)  16

Age range (yrs)  0-93  (median
58)
Mean hospital charge $22,516.00
-------------------------------------

One hundred sixty-one patients were hospitalized, of whom five had two hospitalizations, two had three, one had four, and one had nine (Table 1). Thirty-two (20%) patients had HIV, 16 patients (10%) had cancer, and 18 patients (11%) died while hospitalized. Overall, 120 (75%) of these 161 patients were either >54 years of age, had cancer, or were
HIV infected.

Table 2. Correlation among clinical history, travel, and multilocus genotypes of Coccidioides immitis isolates from 16 patients, New York, 19941997 (Table not available
in ascii)

Mean and median ages for HIV-infected patients were 36 and 33 years, compared with 66 and 64 years for cancer patients and 59 and 63 years for others. Further comparison of HIV-infected patients with patients who had neither cancer nor HIV infection showed differences in percentages with a primary diagnosis of coccidioidomycosis (22% vs 50%), male sex (81% vs 50%), black race (31% vs 5%), and death during
hospitalization (31% vs 6%).

Forty-nine C. immitis cultures were identifiedFigure
at the state mycology laboratory from 1989 to 1997 (Figure). 
(Figure not available in ascii)

Sixteen patient isolates were available for
multilocus genotyping. The hospital records of
these patients were reviewed to determine the
likely source of infection. All had a history of
travel to the Southwest, with 12 of 16 patients traveling to Arizona. However, date(s) of travel in New York, could not be correlated with onset of the disease. The typing of five alleles allowed unambiguous matches with Arizona genotypes for eight isolates from patients whose histories 
supported this conclusion (Table 2). It was also 
possible to type the respective C. immitis
isolates to Arizona in five instances in which 
patients gave a history of travel to more than 
one disease-endemic area (four with travel to
Arizona). The Arizona genotypes of three
isolates (474-97, 639-97, and 779-97) did not match, as these patients had a history of travel to California, California and Mexico, and California, respectively.

Conclusions

Before 1994, the Centers for Disease Control and Prevention (CDC) definition for coccidioidomycosis relied solely on a physician's clinical diagnosis (5). The current Council of State and Territorial Epidemiologists/CDC surveillance case definition requires the presence of both clinically compatible symptoms and laboratory evidence of
infection (5). The laboratory criteria include a positive culture, positive histopathologic results, molecular evidence of C. immitis, a positive serologic test, or a positive skin test. The retrospective nature of this study and our exclusive reliance on hospital discharge summary data precluded detailed evaluation of diagnostic criteria.
Nevertheless, the average of 30 hospital discharges per year suggests that coccidioidomycosis may be more common in New York residents than previously recognized. The largest number of positive cases was reported from New York City, but the rate was not increased, and cases were reported throughout the state. Furthermore, while information on
travel history was limited, all 16 patients from whom information was obtained had traveled to disease-endemic areas before becoming ill.

Immunocompromised persons, infants, and non-Caucasians are considered at increased risk for coccidioidomycosis (1). The mean age of patients in this series was 54 years, which is in agreement with >40-year age-group most commonly diagnosed in disease-endemic areas (5,6). As expected, a large proportion of patients had other diagnoses consistent with underlying immunosuppression, including a primary diagnosis of HIV in 20% and cancer in 10%. Overall, 75% were 55 years
of age, had cancer, or were HIV infected. The availability of C. immitis cultures from only 10% of cases precluded any definitive investigations of geographic areas in which New Yorkers are at greater risk for infection.

The discovery of recombination in clinical isolates of C. immitis (14) has shown genetic evidence of two sexually distinct taxa in this pathogen, which was previously thought to reproduce asexually (15). A corollary of these investigations was the development of a multilocus
genotyping scheme for determining the geographic origin of clinical isolates. Although the discharge data available for this study did not include travel information, we were able to delineate Arizona isolates in a subpopulation of our study for which cultures and medical record travel data were available. Thus, a patient's travel history was correlated with disease acquisition in a particular area by independently demonstrating the geographic pattern of the patient's isolate in 13 (81%) of 16 patients evaluated. However, the typing scheme could not determine the geographic origin of two C. immitis isolates, which came from patients thought to have traveled to both
California and Mexico. This discrepancy may have resulted from the limited number of alleles used for typing, incomplete travel histories, shortcomings of the typing scheme for California isolates, or the possibility that the two isolates belonged to an outlier group of strains. Further testing is needed on a larger set of isolates from
different geographic areas to evaluate this typing scheme.

As a result of this study, laboratories participating in the New York State Clinical Laboratory Evaluation Program (Mycology) were alerted about the hazards posed by C. immitis. A safe procedure for initial examination of suspect cultures was recommended. The laboratories were also provided with guidelines for safe transport of C. immitis to the state mycology laboratory for confirmation and characterization.

The diagnosis and clinical management of coccidioidomycosis in areas where the disease is not endemic pose unique challenges. The clinical symptoms of the disease mimic those of other infectious diseases, which may result in misdiagnosis and inappropriate treatment (1,16).
Additionally, C. immitis cultures could be easily confused with a number of nonpathogenic fungi by unsuspecting laboratory personnel, who are at risk for infection. Increased awareness among health-care providers is likely to help in the timely diagnosis of coccidioidomycosis and the prevention of associated illness and death among patients in nondisease-endemic areas.

Acknowledgments

This study was partially supported by Centers for Control and Prevention Emerging Pathogens Program funds (DLM) and an unrestricted educational grant from Pfizer, Inc. (VC), and was presented as a poster at the First International Conference on Emerging Infectious Diseases, Atlanta, Georgia, March 8-11, 1998.

Dr. Chaturvedi is director of the Mycology Laboratory, Wadsworth Center, New York State Department of Health, and assistant professor of Biomedical Sciences, School of Public Health, SUNY, Albany. His research interests are molecular epidemiology, fungal pathogenesis, and antifungal drug discovery.

Address for correspondence: Vishnu Chaturvedi, Mycology Laboratory, Wadsworth Center, New York State Department of Health, 120 New Scotland Ave., Albany, NY 12208-2002, USA; fax 518-486-7971; e-mail: vishnu@wadsworth.org.

References

  1. Stevens DA. Current concepts: coccidioidomycosis. N Engl J Med 1995;332:1077-82.
  2. Kirkland TM, Fierer J. Coccidioidomycosis: a reemerging
infectious disease. Emerg Infect Dis 1996;2:192-9.
  3. Centers for Disease Control and Prevention.
CoccidioidomycosisUnited States, 1991-1992. MMWR Morb Mortal Wkly Rep 1993;42:21-4.
  4. Pappagianis D. Marked increase in cases of coccidiodomycosis in California: 1991, 1992, and 1993. Clin Infect Dis 1994;19 Suppl 1:S14-8.
  5. Centers for Disease Control and Prevention.
CoccidioidomycosisArizona, 1990-1995. MMWR Morb Mortal Wkly Rep 1996;49:1069-73.
  6. Schneider E, Hajjeh RA, Spiegel RA, Jibson RW, Harp EL, Marshall Ga, et al. Coccidioidomycosis outbreak following the Northridge, California, earthquake. JAMA 1997;277:904-8.
  7. Smith MA, Anderson AE, Kostroff K. An unusual case of
coccidioidomycosis. J Clin Microbiol 1994;32:1063-4.
  8. Ogiso A, Ito M, Koyama M, Yamaoka H, Hotchi M, McGinnis MR. Pulmonary coccidioidomycosis in Japan: case report and review. Clin Infect Dis 1997;25:1260-1.
  9. Harrell ER, Honeycut WM. Coccidiodomycosis: a traveling fungus disease. Arch Dermatol 1963;87:98-106.
 10. Fiese MJ. Coccidioidomycosis. Springfield (IL): Charles C. Thomas Publ.; 1958. p. 79-80.
 11. Antiterrorism and Effective Death Penalty Act of 1996, Pub. L. No. 104-132, 110 Stat. 1214 (codified as amended in scattered sections of the U.S.C.).
 12. Pan S, Sigler L, Cole GT. Evidence for a phylogenetic connection between Coccidioides immitis and Uncinocarpus reesii (Onygenaceae). Microbiology 1994;140:1481-94.
 13. Burt A, Dechairo BM, Koening GL, Carter DA, White TJ, Taylor JW. Molecular markers reveal differentiation among isolates of Coccidioides immitis from California, Arizona and Texas. Mol Ecol 1997;6:781-6.
 14. Burt A, Carter DA, Koenig GL, White TJ, Taylor JW. Molecular markers reveal cryptic sex in the human pathogen Coccidioides immitis. Proc Natl Acad Sci U S A 1996;93:770-3.
 15. Koufopanou V, Burt A, Taylor JW. Concordance of gene genealogies reveals reproductive isolation in the pathogenic fungus Coccidioides immitis. Proc Natl Acad Sci U S A 1997;94:5478-82.
 16. Rippon JW. Medical mycology: the pathogenic fungi and the pathogenic actinomycetes. 3rd ed. Philadelphia (PA): W.B. Saunders Co.; 1988.

Perspectives

Dengue Surveillance in Florida, 1997-98

Julia Gill,* Lillian M. Stark,* and Gary G. Clark
Florida Department of Health, Bureau of Laboratories
and University of South Florida, College of Public
Health, Tampa, Florida, USA; and Centers for Disease
Control and Prevention, San Juan, Puerto Rico, US

--------------------------------------------------------

 Recent dengue outbreaks in the Caribbean
 and Central and South America and the
 presence of competent mosquito vectors
 increase the likelihood of future
 autochthonous transmission in Florida.
 During April 1997 to March 1998, a
 laboratory-based active surveillance
 program detected 18 cases of dengue
 involving all four dengue serotypes. All
 patients reported recent travel to
 countries with indigenous dengue
 transmission. These results demonstrate
 that dengue infections are imported into
 Florida at a much higher rate than
 reflected by previous passive surveillance;
 therefore, the risk for local dengue
 transmission may be increasing.

Dengue is a mosquito-borne viral disease caused by one
of four antigenically distinct dengue flaviviruses:
DEN-1, DEN-2, DEN-3, and DEN-4. Primary infection with
any serotype may lead to acute illness defined as fever
with two or more of the following symptoms: headache,
retroorbital pain, myalgia, arthralgia, rash, and
hemorrhagic manifestations (1,2). Fever and other
symptoms may subside after 3 or 4 days, and the patient
may recover completely, or the fever may return with a
rash within 1 to 3 days (3). Secondary exposure to the
same serotype generally does not produce illness
because of pre-existing antibodies. However, secondary
exposure to a different serotype may lead to another
dengue fever episode, and the patient may be at risk
for more serious forms of infection, dengue hemorrhagic
fever (DHF) or dengue shock syndrome (DSS) (3). Dengue
virus infection may also cause a nonspecific febrile
illness that can be easily confused with measles or
influenza. Therefore, laboratory testing is essential
to clinical diagnosis and public health reporting.

Dengue-viremic persons are usually infectious to the
mosquito vector 1 day before the onset of the febrile
period and remain so for 6-7 days. When a mosquito
ingests virus in a blood meal, the virus replicates
during an extrinsic incubation period of 8 to 12 days,
after which the mosquito remains infective for life
(4,5). The life span of Aedes aegypti, the primary
vector of dengue in the Americas, is usually 21 days,
although life span and incubation periods depend on
temperature and rainfall (6). Both A. aegypti and A.
albopictus, a recently introduced vector species (7),
have been found throughout Florida, and A. aegypti
breeds year-round in south Florida (8).

The last dengue epidemics in Florida, in the Tampa and
Miami areas in 1934-35 (9,10), affected an estimated
15,000 of the 135,000 population of Miami. The last
recorded epidemic in the southeastern states was in
Louisiana in 1945 (11). Most cases of dengue reported
in the United States since the 1940s have been
imported; however, indigenous transmission of dengue
occurred in Texas in 1986 and 1995 (8,12-14). In
response to an outbreak of dengue in Mexico in 1995,
the Texas Department of Health initiated an active
surveillance program that detected 29 confirmed cases,
including seven in persons with no recent history of
travel outside Texas (14,15). Thirteen imported dengue
cases (0 to 4 cases per year) were reported in Florida
from 1985 to 1995 (16).

The recent introduction of DEN-3 in Mexico and Central
America is of public health importance because most of
the population in the tropical Americas is susceptible
to infection with this serotype (17,18). The presence
of the vector, the rapid spread of the virus, and
increased air travel and immigration contribute to the
possibility of future dengue transmission in the
continental United States (19-21). A serosurvey
conducted after the first confirmed dengue outbreak in
Peru in 1990 clearly demonstrated earlier undetected
dengue transmission (22). Silent transmission of dengue
was also demonstrated in 1992 in an area of Taiwan
believed free of the disease (23). In both cases, an
early warning system based on immunoglobulin (Ig)M
antibody-capture enzyme-linked immunosorbent assay
(MAC-ELISA) laboratory tests was recommended for
disease monitoring. Active surveillance, an essential
component of an early warning system for detection of
dengue, provides information vital to defining
epidemiologic aspects of cases and enabling educational
and mosquito control efforts (24-27).

Recent outbreaks of dengue in nearby Caribbean and
Central and South American countries may increase the
likelihood of future autochthonous transmission in
Florida (15). Mosquito vectors are widely distributed
in the state, and travelers returning from
dengue-endemic areas place at risk the resident
population, which has minimal (if any) immunity to
dengue viruses. Because physicians' awareness of dengue
is low and specialized laboratory diagnostic methods
are not available locally, low-level dengue
transmission may go undetected. Imported dengue may
thus be underreported in Florida, which has relied on
passive surveillance. We used an educational campaign
for county epidemiologists and health-care providers
and an active laboratory-based surveillance program
that facilitated prompt, accurate diagnosis of dengue
to assess the risk for local dengue fever transmission
in Florida.

The Study

The first phase of the surveillance program was the design of a dengue information packet for all 67 county health department epidemiologists in Florida, to be distributed to hospital emergency rooms, clinics, health departments, and infectious disease physicians in the county. The letter included information on case reporting, the dengue case definition, specimen requirements and transport instructions, and a dengue case investigation form.

Under cooperative agreements with two Florida commercial clinical laboratories (national reference clinical laboratories), specimens from patients with suspected dengue were forwarded to the state laboratory for free testing. In cases where specimens were tested at commercial laboratories only, dengue antibody-positive results were forwarded to county health departments and to the state laboratory
for inclusion in this study. In Florida, dengue testing is offered only by the state laboratory and some commercial clinical laboratories.

Before this study, the hemagglutination inhibition (HAI) assay was the only serologic test for dengue offered at the state laboratory. Laboratory capabilities were enhanced to include testing for IgM antibodies to dengue. Acute- and convalescent-phase serum specimens were tested for dengue antibodies by both HAI assay and MAC-ELISA, using a DEN1-4 serotype cocktail (28-30). Available specimens positive for IgM antibodies to dengue, tested at the Florida state
laboratory, were forwarded to CDC's Dengue Branch laboratory for virus isolation, serotyping, and confirmation of serologic results.

Cases were classified as DHF if all the following were present: fever, hemorrhagic tendencies, thrombocytopenia (100,000/mm3 or less), and evidence of plasma leakage (hematocrit level increased by > 20%) or other objective evidence of increased capillary permeability (31). If all the above were present, plus hypotension or pulse pressure < 20 mm Hg, the case was classified as DSS.

In this study, a case was classified as presumptive dengue on the basis of serologic evidence of an HAI titer > 1:1280, an equivalent IgG titer, or a positive dengue IgM antibody test on a single serum sample. A confirmed dengue case required a fourfold rise in HAI, IgG, or IgM antibody titers between acute- and convalescent-phase serum specimens; isolation of virus; or detection of viral antigen by immunohistochemistry, immunofluorescence, or viral nucleic acid detection. Confirmed or presumptive dengue cases are referred to as laboratory-diagnosed cases.

A case was classified as undetermined if sufficient information was not available on the timing of specimen collection in relation to onset of symptoms or a convalescent-phase serum was not available to demonstrate a fourfold rise in antibody titers. A case was also
considered undetermined if the acute-phase serum was negative for antibodies and a convalescent-phase serum was not available.

Epidemiologic data were obtained from dengue case investigation forms that accompanied the patients' specimens. Suspected as well as confirmed cases of dengue are reportable in Florida (32). County health departments were notified of suspected cases, and a convalescent-phase serum was requested. 

We used the Epi Info Software package for data analysis (33). Comparisons were made with historical data on reported cases of dengue (9,16,34-36).

From April 1, 1997, to March 31, 1998, 83 suspected cases of dengue were studied. Commercial clinical laboratories referred specimens for analysis for 36 (43%) of these cases. The rest were referred through county health departments, hospital laboratories, infection control practitioners, or physicians. Recent dengue infection was laboratory diagnosed in 18 (22%) of these cases. Twelve (67%) of the 18 confirmed dengue specimens were referred by commercial clinical
laboratories. Virus isolation or polymerase chain reaction of five cases yielded all four dengue serotypes. Dengue was ruled out as the etiologic agent in 24 (29%) cases. The remaining 41 (49%) cases were undetermined because convalescent-phase serum samples were not available (Table 1).

Table 1. Characteristics of 83 suspected
cases of Dengue investigated in Florida,
April 1997March 1998

----------------------------------------------
						Undeter-
Characteristic		Yes	No	mined
----------------------------------------------
History of recent		41    14 	28
travel to
  to dengue-endemic	(49%)(17%)  (34%)
area
Fits dengue fever case   30    8	45
  definition  		(36%)(10%) (54%)
Flavivirus antibody 	41    42
  detected    		(49%)(51%)
Convalescent-phase  	25    58
  specimen provided	(30%)(70%)
Laboratory confirmation 18    24 	41
  of recent dengue 	(22%)(29%)  (49%)
  infection
----------------------------------------------

Most (65%) of suspected-dengue patients were male (chi-square goodness of fit test p value = 0.006). Among suspected cases the mean age was 41 years (1 day to 79 years). Forty-one (49%) initially tested positive for anti-flavivirus antibodies. Convalescent-phase serum was obtained from 25 (30%) of the cases. The average age of patients with confirmed dengue cases was 37 years (8 to 69); 14
(78%) of the 18 patients were male. 

Laboratory-diagnosed cases were identified 
from five counties in central and extreme
southeastern Florida (Figure 1). Cases were
confirmed in persons residing in the following
counties: Dade (8), Hillsborough (4), Orange
(3), Palm Beach (2), and Broward (1). Table 2
lists Florida counties with
laboratory-diagnosed dengue cases, case travel

Figure 1. County of residence for 18 laboratory-
diagnosed dengue cases detected between April 1997-
March 1998. (Figures not available in ascii)

history, and dengue virus serotypes detected.
All 18 laboratory-diagnosed dengue cases were
in persons who had recently traveled to 
dengue-endemic areas and were therefore 
classified as imported. We included
out-of-state cases in our analysis because the 
acute phase of their illness occurred while
they were in Florida. Current county health
department policy dictates that only cases in Florida residents are reported to the state epidemiologist for recording in the weekly and yearly morbidity statistics. Other case reports are forwarded to the county and state of primary residence of the patient.

Table 2. Laboratory-diagnosed dengue cases in
Florida by county, area of travel, and serotype, April 1997March 1998 
----------------------------------------------
			Area of travel  number
   			of cases (dengue serotype)
County(n = 18)
----------------------------------------------
Broward    		Barbados  1
Dade  		Colombia  1
   			Haiti  3
   			Puerto Rico  1 (DEN-2)
   			Venezuela  2 (DEN-1)
   			Unknown  1
Hillsborough    	Colombia  1 (DEN-2)
   			Nicaragua  1 (DEN-3)
   			Thailand  1
   			Unknown  1
Orange		Haiti  2 (DEN-4)
   			Puerto Rico  1
Palm Beach 		Haiti  1
   			Puerto Rico  1
----------------------------------------------

Hemorrhagic manifestations were reported in 7 (39%) of the 18 confirmed cases; one met the DHF case definition; however, it was not possible to classify the remaining six cases with hemorrhage because information on hemoconcentration and plasma leakage was incomplete. Encephalopathy was present in one case. Antibody titers
suggested secondary dengue infections in 10 (56%) of the 18 cases. Only 2 (11%) of the 18 cases appeared to be primary infections. Laboratory tests necessary to determine infection status (primary vs. secondary) were not available in the other six cases. A woman with acute secondary dengue infection with hemorrhagic manifestations gave birth to a healthy uninfected baby.

Conclusions

During the year of active surveillance, 18 laboratory-diagnosed cases of dengue were detected. On the basis of the previous 10-year mean of 1.3 cases per year (Figure 2), the probability of detecting 18 cases was virtually 0% (Poisson distribution rare event vs. standard test). These cases were identified in Florida counties with high rates of international travel and large immigrant populations,
as well as year-round breeding of A. albopictus and A. aegypti mosquitoes. According to Florida Department of Commerce statistics, of the 6 million international visitors to Florida in 1995, 38.4% traveled from South and Central America, the Caribbean, Mexico, Asia, and other tropical areas (37) in which dengue is endemic.

All four dengue serotypes were detected in five 
specimens during this study. Improved specimen
handling should increase the rate of virus  
isolation. The serotype of the infecting dengue
virus was identified in only five cases for the
entire United States in 1995, when 79 
laboratory-diagnosed dengue cases were
documented (12). In the same year, 22 imported

Figure 2. Number of reported dengue cases in Florida,
1987-1997. (*1997 = study year April 1, 1997March 31,

and seven indigenous cases were detected in 
Texas (15). In 1996, the infecting dengue
serotype was identified in 5 of the 43  
laboratory-diagnosed cases of imported dengue
in the United States (3 cases of DEN-1 and 2 of DEN-2) (38). 
This study found multiple problems with routine 1998)
clinical laboratory confirmation and follow-up of dengue infections: Tests requested by physicians and performed at
clinical laboratories were not always optimal for identifying a current dengue infection. Even though the dengue IgM test is the most appropriate assay for determining current infection, it is not routinely performed at commercial laboratories and may not be readily available if requested. Test results are frequently misinterpreted, e.g., a single positive indirect fluorescent antibody test performed at a commercial laboratory may be interpreted as positive for current dengue infection when it only
indicates infection with a flavivirus (e.g., dengue, St. Louis encephalitis, Japanese encephalitis) or vaccination (e.g., yellow fever) at an undetermined time in the past. In addition, cases are rarely investigated, and the convalescent-phase serum samples needed for confirmation are rarely requested. When an investigation indicates need for further testing, specimens may have already been discarded. Finally, positive test results are often not forwarded to
the county and state epidemiologists in a timely manner. In cases tested only at commercial laboratories, delays of 2 to 4 months before positive cases were reported to the state Bureau of Epidemiology preclude prompt follow-up.

Three of the confirmed dengue cases in this study tested at
commercial laboratories had not been reported to the state
epidemiologist by the county health departments because the patients were primary residents of other states, although they became ill while in Florida.

This study indicates that surveillance efforts should be
concentrated in densely populated counties with large numbers of international travelers (Dade, Palm Beach, Orange, and Hillsborough), especially during dengue season in the Caribbean (July to November). As a part of the epidemiologic investigation of imported dengue cases, an attempt should be made to identify secondary cases.

Acknowledgment

The authors thank the Association for Public Health Laboratories and CDC for supporting the laboratory training for this program through the Emerging Infectious Diseases Advanced Laboratory Training Fellowship and the staff of the Florida Department of Health, Bureaus of Laboratories and Epidemiology, and CDC's Dengue Branch for their collaboration in this program.

Dr. Gill is on the staff of the Florida Department of Health, Pinellas County Health Department in the Disease Control Division. She is also a Courtesy Assistant Professor in the Department of Environmental and Occupational Health, College of Public Health, University of South Florida. Her research interests include laboratory-based surveillance programs for hepatitis, dengue, and other arboviral diseases.

Address for correspondence: Julia Gill, Florida Department of Health, Pinellas County Health Department, Disease Control Division, 500 7th Avenue South, St. Petersburg, FL, 33701, USA; fax: 727-893-1681; e-mail: Julia_Gill2@doh.state.fl.us

References

  1. Centers for Disease Control and Prevention. Case definitions for infectious conditions under public health surveillance. MMWR Morb Mortal Wkly Rep 1997;46(RR-10):45-6.
  2. Pan American Health Organization. Dengue and dengue hemorrhagic fever in the Americas: guidelines for prevention and control. Washington: The Organization (Scientific Pub. No. 548); 1994. 
  3. Hayes EB, Gubler DJ. Dengue and dengue hemorrhagic fever. Pediatr Infect Dis J 1992;11:311-7.
  4. Benenson AS, editor. Dengue and dengue hemorrhagic fever. In: Control of communicable diseases manual. 16th ed. Washington: American Public Health Association; 1995.
  5. Kuno G. Review of the factors modulating dengue transmission. Epidemiol Rev 1995;17:321-35.
  6. Focks DA, Daniels E, Hailem DG, Keesling JE. A simulation model of the epidemiology of urban dengue fever: literature analysis, model development, preliminary validation, and samples of simulation results. Am J Trop Med Hyg 1995;53:489-506.
  7. Hawley WA, Reiter P, Copeland RS, Pumpuni CB, Craig GB. Aedes albopictus in North America: probable introduction in used tires from Northern Asia. Science 1987;236:1114-6.
  8. Clark GG. Dengue and dengue hemorrhagic fever. Journal of the Florida Mosquito Control Association 1992;63:48-53.
  9. Griffitts THD. Dengue in Florida 1934, and its significance. J Fla Med Assoc 1935;21:395-7.
 10. MacDonnell GN. The dengue epidemic in Miami. J Fla Med Assoc 1935;21:392-8.
 11. Ehrenkranz NJ, Ventura AK, Cuadrado RR, Pond WL, Porter JE. Pandemic dengue in Caribbean countries and the southern United Statespast, present and potential problems. N Engl J Med 1971;285:1460-9.
 12. Centers for Disease Control and Prevention. Imported
dengueUnited States, 1995. MMWR Morb Mortal Wkly Rep
1996;45:988-91.
 13. Centers for Disease Control and Prevention. Dengue activity in the Americas, 1994. MMWR Morb Mortal Wkly Rep 1995;44.
 14. Centers for Disease Control and Prevention. Dengue fever at the U.S.-Mexico Border, 1995-1996. MMWR Morb Mortal Wkly Rep 1996;45:841-4.
 15. Rawlings JA, Hendricks KA, Burgess CR, Campman RM, Clark GG,Tabony LJ, et al. Dengue surveillance in Texas, 1995. Am J Trop Med Hyg 1998;59:95-9.
 16. Florida Department of Health. Florida Morbidity Statistics, 1995. Tallahassee (FL): Florida Department of Health, Bureau of Epidemiology; 1995.
 17. Briseo-Garcia B, Gomez-Dantes H, Argott-Ramirez E, Montesano R, Vazquez-Martinez A-L, et al. Potential risk for dengue hemorrhagic fever: the isolation of serotype Dengue-3 in Mexico. Emerg Infect Dis 1996;2:133-5.
 18. Centers for Disease Control and Prevention. Dengue-3 in Central America. Dengue Surveillance Summary 1995;70.
 19. Pan American Health Organization. Re-emergence of dengue in the Americas. Epidemiol Bull 1997;18:1-10.
 20. Gubler DJ, Clark GG. Dengue/dengue hemorrhagic fever: the emergence of a global health problem. Emerg Infect Dis
1995;1:55-7.
 21. Pan American Health Organization. Dengue and dengue hemorrhagic fever, 1996. Epidemiol Bull 1996;17:12-4.
 22. Hayes CG, Phillips IA, Callahan JD, Griebenow WF, Hyams KC, Wu S-J, et al. The epidemiology of dengue virus infection among urban, jungle, and rural populations in the Amazon region of Peru. Am J Trop Med Hyg 1996;55:459-63.
 23. Chen W-J, Chen S-L, Chien L-J, Chen C-C, King C-C, Harn M-R, et al. Silent transmission of the dengue virus in Southern Taiwan. Am J Trop Med Hyg 1996;55:12-6.
 24. Gubler DJ. Aedes aegypti and Aedes aegypti-borne disease control in the 1990s: top down or bottom up. Am J Trop Med Hyg 1989;40:571-8.
 25. Gubler DJ. Surveillance for dengue and dengue hemorrhagic fever. Bulletin of the Pan American Health Organization 1989;23:397-404.
 26. Gubler DJ. The global resurgence of arboviral diseases. Trans R Soc Trop Med Hyg 1996;90:449-51.
 27. Ruangturakit S, Rojanasuphot S, Srijuggravanvong A, Duangchanda S, Nuangplee S, Igarashi A. Storage and stability of dengue IgM and IgG antibodies in whole blood and serum dried on filter paper strips detected by ELISA. Southeast Asian J Trop Med Public Health 1994;25:560-4.
 28. Gubler DJ, Sather GE. Laboratory diagnosis of dengue and dengue hemorrhagic fever. Proceedings of the International Symposium on Yellow Fever and Dengue. Rio de Janeiro, Brazil; 1988 May 15-19. Bio-Manguinhos; 1988. p. 291-322.
 29. Kuno G, Gomez I, Gubler DJ. Detecting artificial anti-dengue IgM immune complexes using an enzyme-linked immunosorbent assay. Am J Trop Med Hyg 1987;36:153-9.
 30. Kuno G, Gomez I, Gubler D. An ELISA procedure for the diagnosis of dengue infections. J Virol Methods 1991;33:101-13.
 31. World Health Organization. Dengue hemorrhagic fever: diagnosis, treatment and control. Geneva: The Organization; 1986.
 32. Florida Department of Health and Rehabilitative Services. Florida Statutes, 1996 (Supplement 1996). Chapter 381: Public Health; General Provisions. Rule 10D-3.062.
 33. Centers for Disease Control. Epi Info Software Package, 1991. Version 5.0.
 34. Florida Department of Health and Rehabilitative Services. Florida Morbidity Statistics, 1981. Tallahassee (FL): Health Program Office; 1981.
 35. Florida Department of Health and Rehabilitative Services. Florida Morbidity Statistics, 1985. Tallahassee (FL): Health Program Office; 1985.
 36. Rigau-Perez JG, Gubler DJ, Vorndam AV, Clark GG. Dengue
surveillanceUnited States, 1986-1992. MMWR Morb Mortal Wkly
Rep 1994;43(SS-2):7-19.
 37. Florida Department of Commerce. 1995 Florida visitor study. Tallahassee (FL): Bureau of Economic Analysis; 1995.
 38. Centers for Disease Control and Prevention. Imported
dengueUnited States, 1996. MMWR Morb Mortal Wkly Rep
1998;47:544-7.

Research

Norwalk-Like Calicivirus Genes in Farm Animals

Wim H.M. van der Poel,* Jan Vinj,* Reina van der
Heide,* Maria-Inmaculada Herrera, Amparo Vivo, and
Marion P.G. Koopmans*
*National Institute of Public Health and the
Environment, Bilthoven, the Netherlands; and Instituto
de Salud Carlos III, Majadahonda, Madrid, Spain

--------------------------------------------------------

 Viruses closely related to Norwalk-like
 viruses (NLVs) were recently found in
 stored stool samples from two calves
 (United Kingdom and Germany) and four pigs
 (Japan), sparking discussions about the
 potential for zoonotic transmission. To
 investigate if NLVs are commonly present in
 farm animals, pooled stool samples from 100
 pig farms, 48 chicken farms, 43 dairy cow
 herds, and 75 veal calf farms from the
 Netherlands were assayed by reverse
 transcription-polymerase chain reaction
 amplification, using primers specific for
 the detection of NLVs from humans. NLV RNA
 was detected in 33 (44%) of the specimens
 from veal calf farms and two (2%) specimens
 from pig farms. Our data show that NLV
 infectionsuntil recently thought to be
 restricted to humansoccur often in calves
 and sometimes in pigs. While zoonotic
 transmission has not been proven, these
 findings suggest that calves and pigs may
 be reservoir hosts of NLVs.

Caliciviruses infect animals and humans (1). Within the
family Caliciviridae, four genera have been
distinguished: vesivirus, lagovirus, Norwalk-like
viruses (NLV), and Sapporo-like viruses (SLV) (2). The
genera vesivirus and lagovirus contain a broad range of
animal caliciviruses, but viruses in the NLV and SLV
genera until recently have been found only in humans.
In recent years NLVs, also known as small
round-structured viruses, have emerged as a common
cause of infectious gastroenteritis in all age groups
and the main cause of outbreaks of gastroenteritis in
restaurants and institutions such as nursing homes and
hospitals (3-5).

Genetically, the human caliciviruses separate into at
least three genetic clusters, called genogroups (GG),
i.e., GGI and GGII NLV and the GGIII SLV (6). Each of
these genogroups comprises genomically and
antigenically diverse strains (7,8). This high degree
of diversity results in at least 13 distinct genotypes
for the NLVs (3,5,8-10). Many types of NLV cocirculate
in the general population, causing sporadic cases and
outbreaks. However, occasionally epidemics occur in
which most outbreaks are caused by a single genotype
(e.g., Lordsdale-like virus in the Netherlands in 1996)
(5,11). Hypotheses for the mechanisms behind the
emergence of epidemic types range from large-scale
foodborne transmission of a single strain to
introduction from a nonhuman reservoir. An indication
for the latter was a recent report from Japan of the
finding of NLV-like sequences in stool specimens from
pigs (12). Enteric caliciviruses have also been
detected in other animal species (calves, dogs, and
chickens) (13-16). However, except for canine
calicivirus (17), these viruses lack definitive
sequence evidence linking them to the Caliciviridae
(18). Molecular characterization of three calicivirus
strains from cattle has recently been reported. The
first virus (Tillamook virus; BCV-Bos1), described by
Neill et al. (19), caused respiratory symptoms and was
phylogenetically related to San Miguel sea lion virus
and vesicular exanthema of swine virus, both within the
genus vesivirus. Viruses in this genus have a broader
host range (20). Two other viruses closely related to
GGI NLVs (Jena virus 117/80 and Newbury agent type 2)
were detected in feces from newborn calves with
diarrhea (21,22). The similarity of the porcine and
bovine sequences with those of NLVs found in humans
suggests that a reservoir for these human pathogens may
exist in farm animals.

We studied enteric caliciviruses in recently collected
fecal samples from cattle, chicken, and swine in the
Netherlands by using polymerase chain reaction (PCR)
assays specific for NLVs found in humans. In addition,
we analyzed (by sequence analysis) the genetic
variation of detected calicivirus strains and compared
it with that of prototype GGI and GGII NLV strains.

Materials and Methods

Fecal Specimens

Stool specimens from animals were collected at farms in
the Netherlands as part of ongoing surveillance for
potential zoonotic microorganisms associated with
gastroenteritis in humans. From October 10, 1998, to
April 21, 1999, samples were collected from 3- to
9-month-old fattening pigs at 100 pig farms of 22 to
1600 pigs. From January 1 to April 1, 1998, stool
samples were collected from 75 veal calf farms of 38 to
930 calves and 43 dairy cattle herds of 25 to 180 cows.
The age of the calves was 1 to 52 weeks (average 12
weeks), and of dairy cattle herds 4 to 6 years. From
January 6 to April 21, 1998, samples were collected
from < 6-week-old chickens at 48 broiler farms, with
5,000 to 70,000 per farm.

Sampling

The sampling strategy allowed monitoring for pathogens
in a large number of animals and detection of
microorganisms at farm level with a prevalence of 5%
and 95% confidence (23). Fecal samples from calves,
pigs, and chickens (20 to 60 per farm) were collected
from animals housed in one randomly chosen farm
building. Dairy cattle of one farm were regarded as a
single herd, and stool samples were collected from
randomly selected cows. Until tested, fecal samples
were stored at -70C in 15 g/L of Trypton Soya broth
(TSB) (Oxoid CM 129) and 10% glycerol.

Molecular Detection of NLVs by RT-PCR

For extraction of viral RNA, stool samples were resuspended in HBBS Hanks (Gibco BRL, Breda, Netherlands) to a final concentration of approximately 10%. These suspensions were centrifuged at 3,000 x g for 20 minutes, and 100 l was used for RNA extraction by addition of a high-molarity solution of guanidinium isothiocyanate (GuSCN). Bound RNA was washed and eluted as previously described (24).

To reduce the risk for contamination, specimens from different species were analyzed separately, and one negative control sample was included for every two stool specimens. A human stool sample positive for NLV by electron microscopy was included as positive control. 

We used a single-round reverse transcription (RT)-PCR assay with a broadly reactive primer pair, which had been developed for the detection of NLVs in stool specimens from humans (5). For RT, 5 l RNA was mixed with 4 l 50 pmol JV13 primer. The solution was heated to 94C for 2 minutes, cooled, and 6 l RT buffer was added. The RT reaction was performed in a final volume of 15 l, consisting of 10
mM Tris-HCl (pH 8.3), 50 mM KCl, 3 mM MgCl2, 1 mM each of
deoxynucleoside triphosphates (dNTPs), and 5 units of avian
myeloblastoma virus RT (Boehringer Mannheim, Almere, Netherlands). The mixture was incubated for 1 hour at 42C, heated for 5 minutes at 94C to denature the enzyme, and then cooled. Five l of the RT mixture was added to the PCR mix, containing 10 mM Tris-HCl (pH 9.2), 75 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTPs, 2.5 units AmpliTaq (Perkin Elmer, Nieuwerkerk a/d IJssel, Netherlands), and 15 pmol primer JV12. Mineral oil was added, and 40 amplification cycles, 1 minute at 94C, 1.5 minute at 37C, and 1 minute at 74C each, were performed. The amplification products were analyzed by 2% agarose gel electrophoresis and visualized with UV after ethidium bromide staining.

For Southern blots, the RT-PCR products in the agarose gel were denatured by incubating in 0.5 M NaOH for 30 minutes and transferred to a positively charged nylon membrane (Boehringer, Almere, Netherlands) by vacuum blotting (Millipore, Etten-Leur, Netherlands). Hybridization of NLV RT-PCR products was performed as described previously (5).

Sequence and Phylogenetic Analysis

The NLV RT-PCR products of expected size (326 bp) and 21 products of smaller size of the calf herd samples were excised from a 2% agarose gel and purified with a Qiaquick gel extraction kit (Qiagen, Hilden, Germany). Purified RT-PCR products were sequenced with the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer Applied
Biosystems, Foster City, CA) by using PCR primers. Nucleotide sequences were edited by using SeqEd (V1.03, Applied Biosystems), aligned by Geneworks (V2.5, Intelligenetics, Mountain View, CA), and imported into the Treecon software package (25). The confidence
values of the internal nodes were calculated by performing 100 bootstrap analyses. Evolutionary trees for nucleotide sequences were drawn by using the Neighbor-joining method.

Electron Microscopy

Electron microscopy was performed as recommended by Flewett (26) and Doane and Anderson (27), with model Philips 400T (Philips, Eindhoven, Netherlands) at 80 kV. Identification of virus particles was based on morphologic criteria, i.e., the size and characteristic surface morphology (28).

Figure 1. Results of ethidium bromide staining (Panel A) and corresponding Southern hybridization (panels B and C) of
RT-PCR products of eight calf herd (CH) samples....
(Figures not available in ascii)

Results

Twenty-five (33%) of 75 veal calf farm samples 
and 2 (2.0%) of 100 pig farm samples showed
visible products of the expected size (326 bp).
The gel electrophoresis and hybridization
results are shown for eight strains (Figure 1).
Hybridization with a set of probes used for 
detection of NLVs in humans yielded no positive
reactions (Figure 1B); therefore, 46 products 
(25 of the expected size and 21 of a smaller
size [Figure 1A, lane 7]) were sequenced. All 25 
RT-PCR products of the expected size containedthe GLPSG amino acid motif characteristic of 
viral RNA polymerases (29). The 21 products of
smaller size revealed no viral sequences. The
NLV-RT-PCR was negative for all pooled specimens
from 43 dairy herds and 48 chicken farms.
By phylogenetic analysis, all calf calicivirus
sequences formed a tight cluster closely related
to NLVs from GGI with the highest similarity
with the Newbury calf calicivirus (Figure 2)
(22). Nucleotide sequence identities between the
calf NLVs and GGI NLVs were 63% to 70% (75% to
77% amino acids). The swine virus sequences
clustered as a separate lineage within GGII
NLVs, with 69% to 71% nucleotide and 79% to 83%
amino acid identity. In addition, the swine
sequences strongly resemble (94% nucleotides, 
100% amino acids) the swine calicivirus 
sequences from Japan. Partial polymerase 
sequences of Bo/NLV/176/1998/NET and

Figure 2. Phylogenetic relationships among human and
animal NLVs clustering in genogroup (GG) I and II, based on a 145-bp nucleotide sequence withing RNA polymerase gene....
(Figures not available in ascii)

Sw/NLV/34/1998/NET have been assigned GenBank Phylogenetic
accession numbers AF194183 and AF194184, relationships
respectively.  Since the calf and pig strains did not hybridize to the probe mixture for detection of NLVs from    
humans, specific probes were developed based on 
the consensus sequence of RT-PCR products of 10 
calf herd samples and on the pig farm samples,
respectively. The probe sequences were:  
RH1(calf) (5'-GGATGTGGTGCAGGCAA AC-3') and
RH2(pig) (5'-TCCGCATCTCTATCGT GG-3').
Hybridizations with the calf probe (RH1)
confirmed all RT-PCR-positive calf samples after
15 minutes exposure to an ECL hyperfilm (Figure
1C). Overnight exposure to ECL hyperfilm
revealed eight more positives, for 33 (44%)
positive calf farm samples.

Figure 3. Electron microscopy showing NLV particles in
a calf herd sample (CH176), negatively stained
with 2% K-PTA, pH 7.0. Bar - 40 nm.
(Figures not available in ascii)

All veal calf farm samples were screened by
electron microscopy for viruses (Table).
Particles with NLV morphologic features were
found in only one specimen (Figure 3), in which
the presence of calicivirus sequence was
confirmed by RT-PCR and hybridization (Table).

Conclusions

Until recently, humans were considered the sole 
host of NLVs. Recent studies in Japan and the
United Kingdom, however, demonstrated
calicivirus sequences in the caecum of pigs and
in stored calf stool samples containing
calicivirus-like particles by electron
microscopy (12,21,22). Molecular characterization of calf enteric caliciviruses, named Newbury agent (22) and Jena virus (21), revealed that they were genetically related
and more closely associated with GGI NLVs than with any other known calicivirus. In this study, we detected NLV nucleotide sequences in 33 (44%) of 75 of the pooled samples from veal calf farms.

Phylogenetic analysis of the detected sequences showed that they formed a tight cluster with the Newbury sequence (22) and were closely related to NLVs from GGI. Bridger et al. (30) demonstrated that calves can be infected with Newbury calicivirus. The detection of Newbury-like NLVs in pooled veal calf samples of 33 farms from different regions suggests that the species is a normal NLV host.
Similarly, the close genetic relationship between calicivirus sequences detected in the pig samples in our study and those from Japan suggests that these viruses commonly infect pigs. The close similarity of porcine and bovine sequences to the NLVs infecting humans indicates the possibility of an animal reservoir for human infection. However, these findings by no means prove zoonotic
transmission. Genetically related viruses are commonly found in different species without resulting in apparent widespread interspecies transmission (e.g., rotavirus). On the other hand, zoonotic transmission cannot be excluded. The genetic distances between the animal and human NLVs are similar to the distances between the GGI and GGII strains, and epidemic spread of some NLV strains (5) led to the widespreadpossibly globalemergence of a single
predominant strain in the human population in 1995-96 (11). This epidemiologic observation resembles early descriptions of the vesicular exanthema of swine virus (VESV) epidemics (31). These viruses, belonging to another genus within the Caliciviridae, the vesiviruses, are known for their broad host range (20). The occasional epidemic spread of VESV was later linked to introduction of caliciviruses from an ocean reservoir (20). Therefore, interspecies transmission of NLVs is possible; the occasional widespread NLV epidemics caused by a single strain may result from introductions of new NLV strains from an animal reservoir. 

Table. Viruses and virus particles detected by electron
 microscopy and corresponding Norwalk-like virus (NLV)
 RT-PCR results (including hybridization with a calf-NLV
 specific probe) in 16 of the 75 calf herd samples
(Table not available in ascii)

Reynolds et al. reported electron microscopy detection of
caliciviruses in calf diarrhea outbreaks (32), and the bovine caliciviruses Newbury agent (22) and Jena virus (21) were pathogenic for calves under experimental conditions and in field studies (33). In our study, we did not record disease or death on the surveyed farms and therefore cannot determine whether these caliciviruses were associated with disease. Despite repeated attempts, experimental infection of different animal species with the prototype Norwalk
virus has not been successful, with the exception of infection in chimpanzees (3). Our findings may lead to the development of an animal model for the NLVs of humans; for example, a pig model would enable studies of (mucosal) immunity following NLV infection (1).

The low prevalence of Norwalk-like calicivirus in swine farms (2 per 100) is in agreement with the findings reported from Japan (12). Electron microscopy confirmed the calicivirus origin of the detected NLV sequences in only one calf sample. This low number of positives by electron microscopy was not surprising, given the greater sensitivity of RT-PCR and the use of pooled specimens for screening.
NLVs in humans typically are shed at relatively low levels.

The absence of NLV sequences in all 43 dairy herd specimens may be explained by use of an inappropriate primer pair, which was optimized for detection of NLVs of humans (4) or by an acquired strain-specific immunity in older animals that prevents repeated infections. A third possibility is that levels of shedding in adult animals are very low,
precluding virus detection in pooled specimens. The swine farm samples were all from fattening pigs at least 3 months old. We may have found a higher prevalence in recently weaned pigs, as the prevalence of other enteric pathogens is highest in young piglets (34).

Our findings raise important questions about the host range of NLVs. It is unclear if animal NLVs form genetically distinct stable lineages or are part of a common pool of viruses circulating between animals and humans. If calves or swine indeed are reservoirs, the prevalence of calicivirus should be determined in both healthy and diseased cattle and swine and methods to detect such interspecies transmissions at an early stage should be developed, in collaborative
research by public health and veterinary sciences.

Acknowledgments

We thank A.W. van de Giessen and ing. W.D.C. Deisz for volunteering fecal specimens from the monitoring study for zoonotic enteric pathogens, and A.M. Henken and A.M. de Roda Husman for critically reading the manuscript.

This research was financially supported and approved by the Dutch Inspectorate for Health Protection, Commodities and Veterinary Public Health.

Dr. van der Poel is a veterinary virologist in the Microbiological Laboratory for Health Protection, National Institute of Public Health and the Environment, the Netherlands. His research involves viral zoonoses and foodborne virus infections.

Address for correspondence: Wim H.M. van der Poel, Microbiological Laboratory for Health Protection, National Institute of Public Health and the Environment, Antonie van Leeuwenhoeklaan 9, 3720 BA Bilthoven, the Netherlands; fax: 31-30-274-4434; e-mail: Wim.van.der.Poel@rivm.nl.

References

  1. Kapikian AZ, Estes MK, Chanock M. Norwalk group of viruses. In: Fields BN, Knipe DM, Howley PM, Channock RM, Melnick JL, Monath TP, et al., editors. Fields virology. 3rd ed. Vol. 1. Philadelphia (PA): Lippincott-Raven; 1996. p. 783-810.
  2. Pringle CR. Virus taxonomySan Diego 1998. Arch Virol
143:1449-59. 
  3. Green KY. The role of human caliciviruses in epidemic
gastroenteritis. Arch Virol Suppl 1997;13:153-65.
  4. Vinj J, Koopmans MPG. Molecular detection and epidemiology of small round structured viruses in outbreaks of gastroenteritis in the Netherlands. J Infect Dis 1996;174:610-5.
  5. Vinj J, Altena SA, Koopmans MPG. The incidence and genetic variability of small round-structured viruses in outbreaks of gastroenteritis in the Netherlands. J Infect Dis 1997;176:1374-8.
  6. Matson DO, Zhong WM, Nakata S, Numata K, Jiang X, Pickering LK, et al. Molecular characterization of a human calicivirus with sequence relationships closer to animal caliciviruses. J Med Virol 1995;45:215-22.
  7. Ando T, Mulders MN, Lewis DC, Estes MK, Monroe SS, Glass RI. Comparison of the polymerase region of small round-structured virus strains previously classified in three serotypes by solid-phase immune electron microscopy. Arch Virol 1994;135:217-26.
  8. Jiang X, Cubitt WD, Berke T, Zhong W, Dai X, Nakata S, et al. Sapporo-like human caliciviruses are genetically and
antigenically diverse. Arch Virol 1997;142:1813-27.
  9. Green J, Vinj J, Gallimore C, Koopmans MPG, Hale A, Brown D. Capsid protein diversity among Norwalk-like caliciviruses In press 2000.
 10. Vinj J, Deijl H, van de Heide R, Lewis D, Hedlund K-O, Svensson L, et al. Molecular detection and epidemiology of Sapporo-like viruses. J Clin Microbiol. In press 2000.
 11. Noel JS, Fankhauser RL, Ando T, Monroe SS, Glass RI.
Identification of a distinct common strain of Norwalk-like
viruses having a global distribution. J Infect Dis
1999;179:1334-44.
 12. Sugieda M, Nagaoka H, Kakishima Y, Ohshita T, Nakamura S, Nakajima S. Detection of Norwalk-like virus genes in the caecum contents of pigs. Arch Virol 1998;143:1215-21.
 13. Granzow H, Schirmeier H. Identification of 32 nm viruses in faeces of diarrhoeic calves by electron microscopy. Monatshafte Veterinrmedizin 1985;40:228-9.
 14. Herbst W, Lange H, Krauss H. Elektronenmikroskopischer Nachweis von calicivirus-hnlichen Partikeln im Kot durchfallkranker Klber. Deutsche Tierrztliche Wochenschrift 1987;94:381-440.
 15. Mochizuki M, Kawanishi A, Sakamoto S, Tashiro S, Fujimoto R, Ohwaki M. A calicivirus isolated from a dog with fatal diarrhoea. Vet Rec 1993;132:221-2.
 16. Reckling KF. Use of electron microscopy for observation of small round viruses in faecal samples collected from calves and animals with diarrhoea. Monatshafte Veterinrmedizin 1987;42:272-5.
 17. Hashimoto M, Rierink F, Tohya Y, Mochizuki M. Genetic analysis of the RNA polymerase gene of caliciviruses from dogs and cats. J Vet Med Sci 1999;61:603-8.
 18. Cubitt D, Bradley MJ, Carter S, Chiba S, Estes MK, Saif LJ, et al. Viral taxonomy, classification and nomenclature; sixth report of the committee on the taxonomy of viruses. Arch Virol Suppl 1997;ESVV 77-82, Reading, United Kingdom.
 19. Neill JD, Meyer RF, Seal BS. Genetic relatedness of the
caliciviruses: San Miguel sea lion and vesicular exanthema of swine viruses constitute a single genotype within the
Caliciviridae. J Virol 1995;69:4484-8.
 20. Smith AW, Skilling DE, Cherry N, Mead JH, Matson DO. Calicivirus emergence from ocean reservoirs: zoonotic and interspecies movements. Emerg Infect Dis 1998;4:13-20.
 21. Liu BL, Lambden PR, Gunther H, Otto P, Elscher M, Clarke IN. Molecular characterization of a bovine enteric calicivirus: relationship to the Norwalk-like viruses. J Virol 1999;73:819-25.
 22. Dastjerdi AM, Green J, Gallimore CI, Brown DWG, Bridger J. The bovine newbury agent-2 is genetically more closely related to human SRSVs than to animal caliciviruses. Virology 1999;254:1-5.
  23. Giessen van de AW, Frankena K, Leeuwen van WJ, Notermans SHW. An approach for monitoring salmonella serotypes in farm animals. Proceedings of the Symposium "Salmonella and salmonellosis." Ploufragan 1992:375-85.
 24. Boom R, Sol CJA, Salimans MMM, Jansen CL, Wertheim-van Dillen, van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol 1990;28:495-503.
 25. Van der Peer Y, De Wachter R. TREECON for windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Computer Applications in Bioscience 1994;10:569-70.
 26. Flewett TH. Electron microscopy in the diagnosis of infectious diarrhea. J Am Vet Med Assoc 1978;173:538-43.
 27. Doane FW, Anderson N. Pretreatment of clinical specimens and viral isolates. In: Electron microscopy in diagnostic virology. Cambridge: Cambridge University Press; 1987. p. 4-10.
 28. Caul EO, Appleton H. The electron microscopical and physical characteristics of small round human fecal viruses: an interim scheme for classification. J Med Virol 1982;9:257-65.
 29. Bruenn JA. Relationships among the positive strand and
double-strand RNA viruses as viewed through their RNA-dependent RNA polymerases. Nucleic Acids Res 1991;25:217-26.
 30. Bridger JC, Hall GA, Brown JF. Characterization of a calici-like virus (Newbury agent) found in association with astrovirus in bovine diarrhea. Infect Immun 1984;43:133-8.
 31. Smith AW, Akers TG. Vesicular exanthema of swine. J Am Vet Med Assoc 1976;169:700-3.
 32. Reynolds DJ, Morgan JH, Chanter N, Jones PW, Bridger JC, Debney TG, et al. Microbiology of calf diarrhoea in southern Britain. Vet Rec 1986;119:34-9.
 33. Gnther H, Otto P, Heilman P. Studies into diarrhoea of young calves. Sixth communication: detection and determination of pathogenicity of a bovine corona virus and an undefined icosahedric virus. Archiven Experimenteller Veterinrmedizin (Leipzeig) 1984;38:781-92.
 34. Will LA, Paul PS, Proescholdt TA, Aktar SN, Flaming KP, Janke BH, et al. Evaluation of rotavirus infection and diarrhea in Iowa commercial pigs based on epidemiologic study of a population represented by diagnostic laboratory cases. J Vet Diagn Invest 1994;6:416-22.

Dispatches

Molecular Genetic Evidence of a Novel Morbillivirus in
a Long-Finned Pilot Whale (Globicephalus melas)

Jeffery K. Taubenberger,* Mark M. Tsai,* T. Joy Atkin,*
Thomas G. Fanning,* Amy E. Krafft,* R.B. Moeller,* S.E.
Kodsi, M.G. Mense, and Thomas P. Lipscomb*
*Armed Forces Institute of Pathology, Washington, D.C.,
USA; and Walter Reed Army Institute of Research,
Washington, D.C., USA

--------------------------------------------------------

 A long-finned pilot whale with
 morbilliviral disease was stranded in New
 Jersey. An immunohistochemical stain
 demonstrated morbilliviral antigen. Reverse
 transcriptase-polymerase chain reaction for
 morbillivirus P and N genes was positive.
 Novel sequences most closely related to,
 but distinct from, those of dolphin and
 porpoise morbilliviruses suggest that this
 virus may represent a third member of the
 cetacean morbillivirus group.

In the last 12 years, newly recognized members of the
morbillivirus family have caused many deaths among
marine mammals, specifically cetaceans and pinnipeds
(1). The first recognized marine mammal morbilliviral
epizootic occurred in 1987-88 along the Atlantic coast
of the United States (2,3). More than half the in-shore
population of bottlenose dolphins (Tursiops truncatus)
may have died. In 1988, thousands of harbor seals
(Phoca vitulina) (4) and small numbers of harbor
porpoises (Phocoena phocoena) (5) died in morbilliviral
epizootics in northwestern Europe. A separate epizootic
claimed thousands of striped dolphins (Stenella
coeruleoalba) in the western Mediterranean (6). In
1993-94, another bottlenose dolphin epizootic occurred
in the Gulf of Mexico (7,8). Recently, morbilliviral
infection has been reported in cetaceans in the Pacific
(9).

Viruses have been cultured from animals from some of
the epizootics, and novel marine mammal morbilliviruses
have been recognized. Two cetacean morbilliviruses have
been identified and named porpoise morbillivirus (PMV)
and dolphin morbillivirus (DMV). PMV was isolated from
harbor porpoises that died along the Irish coast. DMV
was first identified in striped dolphins from the
Mediterranean (10). Since the characterization of these
viruses, genetic identification has also been possible
by reverse transcriptase-polymerase chain reaction
(RT-PCR) and DNA sequence analysis in specimens from
epizootics where virus was not cultured (11). These
analyses have shown that both cetacean morbilliviruses
(DMV and PMV) were present in the first recognized
cetacean epizootic in bottlenose dolphins in 1987.
While not species-specific in that study, only DMV was
recovered in the striped dolphin epizootic in the
Mediterranean, and only PMV sequences were noted from
bottlenose dolphins in the subsequent Gulf of Mexico
epizootic (11). Common dolphins (Delphinus delphis)
stranded off the California coast from 1995 to 1997
were shown to have morbilliviral infection by virus
neutralization and enzyme-linked immunosorbent assay
(ELISA), as well as RT-PCR (9). Sequence analysis has
shown that these animals were infected with PMV
(Taubenberger et al., unpub. data).

We report a case of lethal morbilliviral infection in a
long-finned pilot whale (G. melas). Sequence analysis
of fragments of the morbillivirus phosphoprotein (P)
and nucleoprotein (N) genes suggests that this is a
novel morbillivirus, phylogenetically related to, but
distinct from, the other cetacean morbilliviruses, PMV
and DMV.

A female long-finned pilot whale (G. melas) was
stranded below the deck of a beach home on the Delaware
Bay, New Jersey. The whale was inactive and appeared
near death. The likelihood of recovery was remote, and
euthanasia was considered appropriate. The animal was
transported by truck to a veterinary diagnostic
laboratory, where it died shortly after arrival.

At necropsy, body fat was found
to be depleted. The lungs were
dark red and congested. Nematodes
were observed in the second
stomach chamber. Significant
histologic lesions were present
in many organs. In the lung,
alveoli and airways contained
numerous neutrophils,
histiocytes, and multinucleate
cells, interspersed with cellular
debris. Multifocal type 2
pneumocyte hyperplasia was
observed. Many multinucleate
cells and some bronchiolar
epithelial cells contained
eosinophilic intracytoplasmic and
intranuclear inclusion bodies. An
immunohistochemical stain
demonstrated morbilliviral
antigen (8) in bronchiolar
epithelium, multinucleate
(syncytial) cells, and type 2
pneumocytes (Figure 1). Syncytial
cells, often containing
eosinophilic intracytoplasmic and
intranuclear inclusion bodies,
were also observed in spleen,
lymph nodes, and liver.
Additionally, eosinophilic intracytoplasmic inclusion
bodies were present in colonic and gastric epithelia
and in transitional epithelium of the kidney.
Intracytoplasmic and intranuclear inclusion bodies were
seen in glossal epithelium. Other histologic lesions
included nonsuppurative encephalomyelitis, necrotizing
hepatitis, erosive colitis, necrotizing gastritis,
ulcerative glossitis, and ulcerative esophagitis.

Figure 1. Lung of pilot whale. Positive
intracytoplasmic and intranuclear
immunoperoxidase staining of
morbilliviral antigen in
syncytial cell (center) and
bronchiolar epithelium (lower
left). Avidin-biotin-peroxidase
with Harris' hematoxylin
counterstain. Original
magnification 594x.
(Figures not available in ascii)

RT-PCR was performed on RNA isolated from formalin-fixed,
paraffin-embedded lung and brain tissues (7,11). A 378 nucleotide fragment of the P gene (GenBank accession number AF200817 and a 230 nucleotide fragment of the N gene (GenBank accession number AF200818 were amplified and sequenced. In both cases the sequences were novel
but related to the previously described cetacean morbillivirus sequences. We have tentatively named this virus "pilot whale morbillivirus" or PWMV.

Sequences of both the N and P gene fragments
were analyzed by parsimony and neighbor-joining
(Figure 2). Parsimony analyses (12) with Sendai
virus as outgroup placed PWMV in the cetacean
morbillivirus clade with PMV and DMV, with
moderate-to-high bootstrap values (N sequence =
70; P sequence = 100). Neighbor-joining (13)
also placed PWMV in the cetacean morbillivirus
clade with high bootstrap values (N sequence =
97; P sequence = 100). Thus PWMV is clearly
related to, but distinct from, PMV and DMV, and
the latter two are more closely related to one
another than either is to PWMV (Figure 2).

Both the origin of these cetacean
morbilliviruses and the mechanism of spread to
different parts of the world remain unknown.
Recently, serologic evidence of morbilliviral
infection was obtained from 11 of 15 species of
odontocete cetaceans of the western Atlantic
(14), suggesting that exposure to these viruses has been widespread. Serologic evidence of enzootic infection (with a seroprevalence of 86%) was also obtained from two species of pilot whales (G. melas and G. macrorhynchus) in the western Atlantic (15). Clinical disease consistent with morbilliviral pneumonia was detected in one G. melas
calf, although the morbillivirus in that case was not characterized. Duignan et al. (15) speculated that pilot whales may serve as vectors of morbillivirus infection to other odontocete cetaceans.

The results from this case suggest that pilot whales may have their own species-adapted morbillivirus. Given that this is a single case, that no morbilliviral epizootic has been described for pilot whale species, and that seroprevalence is consistent with enzootic morbilliviral infection (15), lethal infection might be rare in
these species. Nevertheless, it remains possible for pilot whales to serve as vectors by which immunologically nave species are exposed to morbillivirus infection. The viruses in these hosts may have undergone species-adaptive changes, reflecting the genetic differences between DMV, PMV, and PWMV. While only partial sequences have been obtained from two of the morbilliviral genes in this case, the fact that it is closer to the root of the cetacean morbillivirus
clade (Figure 2) suggests that PWMV resembles the common ancestor of the clade more closely than either DMV or PMV.

Figure 2. Neighbor-joining analyses of partial P and N gene sequences with branch distances as shown. Analyses were performed with MEGA, version 1.01 (13).... (Figures not available in ascii)

Acknowledgments

We thank the Marine Mammal Stranding Center, Brigantine, New Jersey, for collecting and transporting the whale; Sionagh H. Smith for performing the necropsy; and R.-A. V. Ferris for assistance with the photomicrograph.

This study was partially funded by the National Marine Fisheries Service.

Dr. Taubenberger is chief of the division of Molecular Pathology, department of Cellular Pathology, Armed Forces Institute of Pathology, Washington, D.C., U.S.A. His areas of expertise include molecular pathology, RNA viruses, and immunology. His research interests include genetic characterization of the 1918 influenza virus and marine mammal morbilliviruses, genetic changes in breast
cancer and lymphomas, and the development of B and T lymphocytes.

Address for correspondence: Jeffery K. Taubenberger, Armed Forces Institute of Pathology, Room G-137, 14th Street and Alaska Avenue, N.W., Washington, D.C. 20306-6000, USA; fax: 202-782-7623; e-mail: taubenbe@afip.osd.mil.

References

  1. Kennedy S. Morbillivirus infections in aquatic mammals. J Comp Pathol 1998;119:201-25.
  2. Lipscomb TP, Schulman FY, Moffett D, Kennedy S. Morbilliviral disease in Atlantic bottlenose dolphins (Tursiops truncatus) from the 1987-1988 epizootic. J Wildl Dis 1994;30:567-71.
  3. Schulman FY, Lipscomb TP, Moffett D, Krafft AE, Lichy JH, Tsai MM, et al. Reevaluation of the 1987-88 Atlantic coast bottlenose dolphin (Tursiops truncatus) mortality event with histologic, immunohistochemical, and molecular evidence for a morbilliviral etiology. Vet Pathol 1997;34:288-95.
  4. Osterhaus ADME, Groen J, Spijkers HEM, Broeders HWJ, UytdeHaag FGCM, de Vries P, et al. Mass mortality in seals caused by a newly discovered morbillivirus. Vet Microbiol 1990;23:343-50. 
  5. Kennedy S, Smyth JA, Cush PF, McAliskey M, McCullough SJ, Rima BK. Seven histopathologic and immunocytochemical studies of distemper in harbor porpoises. Vet Pathol 1991;28:1-7.
  6. Domingo M, Vilafranca M, Visa J, Prats N, Trudgett AL, Visser I. Evidence of chronic morbillivirus infection in the
Mediterranean striped dolphin (Stenella coeruleoalba). Vet
Microbiol 1995;44:229-39.
  7. Krafft AE, Lichy JH, Lipscomb TP, Klaunberg BA, Kennedy S, Taubenberger JK. Postmortem diagnosis of morbillivirus
infection in bottlenose dolphins (Tursiops truncatus) in the
Atlantic and Gulf of Mexico epizootics by a polymerase chain
reaction-based assay. J Wildl Dis 1995;31:410-5.
  8. Lipscomb TP, Kennedy S, Moffett D, Krafft AE, Klaunberg BA, Lichy JH, et al. Morbilliviral epizootic in Atlantic bottlenose dolphins of the Gulf of Mexico. J Vet Diagn Invest 1996;8:283-90.
  9. Reidarson TH, McBain J, House C, King DP, Stott JL, Krafft AE, et al. Morbillivirus infection in stranded common dolphins (Delphinus delphis) from the Pacific Ocean. J Wildl Dis 1998;34:771-6.
 10. Barrett T, Visser IKG, Mamaev L, Goatley L, van Bressem M-F, Osterhaus ADME. Dolphin and porpoise morbilliviruses are genetically distinct from phocine distemper virus. Virology 1993;193:1010-2.
 11. Taubenberger JK, Tsai MM, Krafft AE, Lichy JH, Reid AH,
Schulman FY, et al. Two different morbilliviruses implicated in bottlenose dolphin epizootics. Emerg Infect Dis 1996;2:213-6.
 12. Swofford DL. PAUP: Phylogenetic Analysis Using Parsimony, version 3.1.1 (Illinois Natural History Survey, Champaign, Illinois; 1991.
 13. Kumar S, Tamura K, Nei M. Molecular evolutionary genetics analysis, version 1.01. Pennsylvania State University, University Park, Pennsylvania; 1993.
 14. Duignan PJ, House C, Geraci JR, Duffy N, Rima BK, Walsh MT, et al. Morbillivirus infection in cetaceans of the western Atlantic. Vet Microbiol 1995;44:241-9.
 15. Duignan PJ, House C, Geraci JR, Early G, Copland HG, Walsh MT, et al. Morbillivirus infection in two species of pilot whales (Globicephala sp.) from the western Atlantic. Marine Mammal Science 1995;11:150-62.

Dispatches

Candida dubliniensis Fungemia: the First Four Cases in
North America

Mary E. Brandt,* Lee H. Harrison, Margaret Pass,
Andre N. Sofair, Sharon Huie, Ren-Kai Li,* Christine
J. Morrison,* David W. Warnock,* and Rana A. Hajjeh*1
*Centers for Disease Control and Prevention (CDC),
Atlanta, Georgia, USA; Johns Hopkins University
School of Hygiene and Public Health, Baltimore,
Maryland, USA; University of Pittsburgh Graduate
School of Public Health, Pittsburgh, Pennsylvania,
USA; and Yale University School of Medicine, New
Haven, Connecticut, USA.

-------------------------------------------------------

 We report the first four North American
 cases of Candida dubliniensis fungemia,
 including the first isolation of this
 organism from the bloodstream of an
 HIV-infected person. All isolates were
 susceptible in vitro to commonly used
 antifungal drugs. This report demonstrates
 that C. dubliniensis can cause bloodstream
 infection; however, the incidence of
 disease is not known.

In recent years, Candida species other than C.
albicans have emerged as causes of human candidiasis,
particularly in HIV-infected and other
immunocompromised persons (1). C. dubliniensis, a
recently described species closely related to C.
albicans (2), has been implicated as an agent of oral
candidiasis in HIV-positive persons (2-5) but has also
been recovered from HIV-negative persons with clinical
signs of oral candidiasis and from the genital tract
of some women with vaginitis (2,4). First isolated
from AIDS patients in Dublin, Ireland (2), C.
dubliniensis has a worldwide distribution (3-6). Most
isolates are susceptible to amphotericin B and the
azoles, but resistance has been shown in HIV-positive
patients on fluconazole for oral candidiasis (7). Its
potential to cause deep or disseminated candidiasis is
not known, largely because C. dubliniensis has rarely
been isolated from sterile body sites (6); however,
the phenotypic characteristics the organism shares
with C. albicans (producing germ tubes and
chlamydospores) suggest that some C. dubliniensis
isolates may have been misidentified as C. albicans.

Three cases of C. dubliniensis fungemia have been
reported from Europe, all in HIV-negative bone marrow
transplant recipients with chemotherapy-induced
neutropenia (8). We report the first isolation of C.
dubliniensis from the blood cultures of four patients
from the United States. All four patients had multiple
underlying conditions and at least one symptom of
septicemia (fever, hypotension, or multiple organ
system failure) at the time blood cultures were drawn.
These cultures were collected from October 1998 to
January 1999 through active, population-based
laboratory surveillance for candidemia in residents of
Connecticut and of Baltimore City or County, Maryland
(combined population, 4.8 million). An incident case
of candidemia was defined by the first isolation of
any Candida species from a blood culture from a
resident of one of the surveillance areas. Medical
records were reviewed to obtain demographic data,
clinical data on underlying medical conditions,
treatment, and outcome.

Case 1

A 74-year-old black man from Baltimore, with a history
of chronic lymphocytic leukemia, chronic obstructive
pulmonary disease, coronary artery disease and
hypertension, was hospitalized for fatigue and anemia
4 weeks after chemotherapy with chlorambucil. He
received multiple blood transfusions on day 1 of
hospitalization and quickly progressed to multiple
organ failure, including renal failure and hepatic and
cardiogenic shock, and was transferred to a medical
intensive care unit. Multiple indwelling catheters
were placed. A peripheral blood culture obtained 3
days after admission grew C. dubliniensis. The patient
died 1 day after this single positive blood culture
was drawn. No autopsy was performed.

Case 2

A 30-year-old black woman from Connecticut with a
history of end-stage liver disease was hospitalized
for gastrointestinal bleeding and refractory ascites.
The patient had a history of intravenous drug use and
alcoholism but was not infected with HIV. During
hospitalization, she required multiple transfusions;
acute renal failure requiring hemodialysis followed.
The patient was receiving many medications, including
vasopressors, antibiotics, and corticosteroids. Yeasts
were isolated from peripheral blood cultures collected
on days 11, 15, and 17 of hospitalization; four of the
isolates were later identified as C. dubliniensis. The
patient had a triple-lumen catheter placed on the day
before the initial isolation of the organism. She was
treated with intravenous fluconazole 200 mg/day for 5
days, starting 6 days after the first yeast isolation.
A blood culture collected on day 20 was negative, and
the patient died after 24 days of hospitalization. No
autopsy was performed.

Case 3

The third patient was a 39-year-old black man from Baltimore who was
admitted for complications of end-stage liver disease, including
acute renal failure and ascites, and diffuse lymphadenopathy of
unknown etiology. He also had a history of diabetes mellitus. A week
before hospitalization, the patient had been discharged from another
hospital, where he had been admitted because of pancreatitis and treated for Escherichia coli bacteremia and renal insufficiency. The patient had a peripheral intravenous catheter and a central venous catheter placed during this hospitalization. On day 2, yeasts were recovered from a peripheral blood culture; these were later identified as C. dubliniensis. By the time the result of the blood culture was reported, the patient's clinical status had deteriorated
because of worsening respiratory distress. He was treated with fluconazole 400 mg/day for 3 days but died 5 days after the blood culture was obtained. No autopsy was performed.

Case 4

The fourth case occurred in a 37-year-old white woman from
Baltimore, who had a history of intravenous drug use, chronic deep vein thrombosis, and valvular heart disease. She was also HIV-infected (CD4+ lymphocyte count was 779 cells/ml). She was hospitalized because of fever and chills, and blood cultures on day 1 of admission grew group A streptococci, for which she was treated with various antibiotics. On day 7, fever developed and peripheral
blood cultures grew C. dubliniensis and C. glabrata. She was treated with oral fluconazole 400 mg/day for 2 weeks and was discharged to a skilled-nursing facility 1 day after being started on fluconazole.

Microbiologic Results

All seven isolates were originally identified as C. albicans on the basis of their phenotypic characteristics. They were reexamined at the Fungus Reference Laboratory, Centers for Disease Control and Prevention, and reidentified as C. dubliniensis on the basis of biochemical and morphologic criteria (9). The identification was confirmed by reactivity of DNA with a polymerase chain reaction (PCR)-enzyme immunoassay (EIA) probe specific for this species (10)
and by PCR amplification of a region containing the novel C.
dubliniensis group I intron in the large ribosomal subunit (11).

Broth microdilution MICs were determined according to National Committee for Clinical Laboratory Standards document M27-A guidelines (12). All isolates were susceptible to commonly used antifungal agents. MICs of amphotericin B were 0.25 (one patient) to 0.5 g/ml (three patients); MICs of itraconazole were from <0.015 (two patients) to 0.03 g/ml (two patients): and MICs of fluconazole and flucytosine were <0.125 g/ml for all isolates.

Conclusions

The incidence of candidemia due to C. dubliniensis is not known, largely because of the difficulty in readily distinguishing this species from the morphologically similar C. albicans. However, in laboratory-based surveillance conducted in 1992-93 in two sites in the United States (population 5.8 million), we did not find C. dubliniensis as an agent of candidemia, even with the DNA-based dentification method used in this study (13). More recently, three cases of C. dubliniensis fungemia have been reported from Europe in patients with chemotherapy-induced immunosuppression and bone marrow transplantation (8). The four cases described here are the first reported in the United States.

The demonstration that C. dubliniensis has the potential to cause bloodstream infection provides information central to our understanding of its clinical relevance and pathogenic potential. As in the earlier European report (8), the patients in our study had multiple serious medical conditions. Two of the four patients had end-stage liver disease, which is a known risk factor for bloodstream infections with organisms that are part of the normal
gastrointestinal flora because of breakdown of the normal mucosal barrier (14). This strongly suggests that the gastrointestinal tract was the source of the C. dubliniensis in these patients. Odds et al. (6) have reported the reidentification as C. dubliniensis of a number of C. albicans isolates that were obtained from fecal surveillance cultures in hematologic patients.

The isolation of C. dubliniensis from mucosal sites in HIV-infected persons has been widely reported (3,5). Although not severely immunocompromised, our fourth patient was HIV-positive, which makes hers the first reported case of bloodstream infection with this organism in an HIV-infected person. The fact that C. dubliniensis is able to cause invasive disease in these patients is of clinical interest. However, it may be more significant that C. glabrata, a
recognized pathogen, was also isolated from blood cultures in our patient.

As our population-based surveillance for candidemia continues, we will be able to estimate more accurately the incidence of candidemia due to C. dubliniensis and define more clearly its clinical importance, epidemiologic characteristics, and outcome. The specific proportional impact of C. dubliniensis candidemia on outcome is
difficult to assess in these patients, all of whom had multiple underlying conditions. The organisms isolated from our patients were all fully susceptible to amphotericin B, flucytosine, fluconazole, and itraconazole. However, resistance has been shown to occur in HIV-positive patients given fluconazole treatment for oral infection with C. dubliniensis (7). As our knowledge about this emerging
pathogenic yeast increases and diagnostic tests are developed, prevention and better management of the disease will become possible.

Acknowledgment

We thank the laboratory participants who collected isolates for this study.

Dr. Brandt is a research microbiologist in the Mycotic Diseases Branch, Division of Bacterial and Mycotic Diseases, CDC. She and Dr. Hajjeh are members of the CDC Fungal Active Surveillance Group, which conducts active population-based surveillance for fungal diseases of public health importance.

Address for correspondence: Mary E. Brandt, Mycotic Diseases Branch, Centers for Disease Control and Prevention, 1600 Clifton Road, Mailstop G11, Atlanta, GA 30333, USA; fax: 404-639-3546; e-mail: mbb4@cdc.gov.

References

  1. Coleman DC, Rinaldi MG, Haynes KA, Rex JH, Summerbell RC, Anaissie EJ, et al. Importance of Candida species other than Candida albicans as opportunistic pathogens. Medical Mycology 1998;36 Suppl 1:156-65.
  2. Sullivan DJ, Westerneng TJ, Haynes KA, Bennett DE, Coleman DC. Candida dubliniensis sp. nov.: phenotypic and molecular characterization of a novel species associated with oral candidosis in HIV-infected individuals. Microbiology 1995;141:1507-21.
  3. Coleman DC, Sullivan DJ, Bennett DE, Moran GP, Barry HJ, Shanley DB. Candidiasis: the emergence of a novel species, Candida dubliniensis. AIDS 1997;11:557-67.
  4. Sullivan D, Coleman D. Candida dubliniensis: characteristics and identification. J Clin Microbiol 1998;36:329-34.
  5. Sullivan D, Haynes K, Bille J, Boerlin P, Rodero L, Lloyd S, et al. Widespread geographic distribution of oral Candida dubliniensis strains in human immunodeficiency virus-infected individuals. J Clin Microbiol 1997;35:960-4.
  6. Odds FC, Van Nuffel L, Dams G. Prevalence of Candida
dubliniensis isolates in a yeast stock culture collection. J
Clin Microbiol 1998;36:2869-73. 
  7. Moran GP, Sullivan DJ, Henman MC, McCreary CE, Harrington BJ, Shanley DB, et al. Antifungal drug susceptibilities of oral Candida dubliniensis isolates from human immunodeficiency virus (HIV)-infected and non-HIV-infected subjects and generation of stable fluconazole-resistant derivatives in vitro. Antimicrob Agents Chemother 1997;41:616-23.
  8. Meis JFGM, Ruhnke M, DePauw BE, Odds FC, Siegert W, Verweij PE. Candida dubliniensis candidemia in patients with
chemotherapy-induced neutropenia and bone marrow transplantation. Emerg Infect Dis 1999;5:150-3.
  9. Salkin IF, Pruitt WR, Padhye AA, Sullivan D, Coleman D, Pincus DH. Distinctive carbohydrate assimilation profiles used to identify the first clinical isolates of Candida dubliniensis recovered in the United States. J Clin Microbiol 1998;36:1467.
 10. Elie CM, Lott TJ, Reiss E, Morrison CJ. Rapid identification of Candida species with species-specific DNA probes. J Clin Microbiol 1998;36:3260-5.
 11. Boucher H, Mercure S, Montplaisir S, Lemay G. A novel group I intron in Candida dubliniensis is homologous to a Candida albicans intron. Gene 1996;180:189-96.
 12. National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved Standard M27-A. Wayne (PA): The Committee;1997.
 13. Elie CM, Kao AS, Hajjeh RA, Brandt ME, Pruitt WR, Reiss E, et al. Examination of bloodstream yeast isolates for the presence of the recently described Candida species, C. dubliniensis, as part of a prospective, population-based surveillance study. In: Abstracts of the Fifth Candida and Candidiasis Conference, 1999. Abstract C41, p. 60. Washington: American Society for Microbiology.
 14. Cole GT, Halawa AL, Anaissie EJ. The role of the
gastrointestinal tract in hematogenous candidiasis: from the
laboratory to the bedside. Clin Infect Dis 1996;22 Suppl
2:S73-88. 

1For the CDC Candidemia Surveillance Group, which includes W. Ruth Pruitt, Nathelia LeSane, Gabriel Ponce de Leon, Meral Ciblak, G. Marshall Lyon (CDC); Laurie Thomson Sanza (Maryland); Lily Yeo and Brian Wong (Connecticut).

Dispatches

Integronlike Structures in Campylobacter spp. of Human and Animal Origin

Brigid Lucey,* D. Crowley,* P. Moloney, B. Cryan, M. Daly,* F. O'Halloran,* E.J. Threlfall, and S. Fanning*
*Cork Institute of Technology, Bishopstown, Cork, Ireland; Cork University Hospital, Cork, Ireland; Cork Corporation Veterinary Department, Cork, Ireland; and PHLS Central Public Health Laboratory, London, United Kingdom

-----------------------------------------------------------

Resistance to antimicrobial agents used to treat severe
Campylobacter spp. gastroenteritis is increasing worldwide.
We assessed the antimicrobial resistance patterns of
Campylobacter spp. isolates of human and animal origin. More
than half (n = 32) were resistant to sulphonamide, a feature
known to be associated with the presence of integrons.
Analysis of these integrons will further our understanding
of Campylobacter spp. epidemiology.

Campylobacter spp. are isolated from animals and birds and from the environment, particularly surface water. Poultry have been implicated as a major source of sporadic infection (1). Thermophilic Campylobacter spp., particularly Campylobacter jejuni and C. coli, are recognized as one of the etiologic agents of acute diarrheal disease in humans worldwide (2,3). Antimicrobial chemotherapy is usually reserved for patients with advanced infection or patients prone to relapse. Erythromycin, fluoroquinolones, and
tetracycline are the antimicrobial drugs of choice.

Bacterial resistance to antimicrobial agents, which is increasing worldwide, is frequently caused by the acquisition of new genes rather than by mutation
(4,5). An efficient means of acquiring new genes is by mobile genetic elements such as resistance (R)-plasmids and transposons. Recently, a novel class of naturally occurring mobile genetic elements, integrons, have been
described as vehicles for the acquisition of antimicrobial resistance genes (5). Horizontal and vertical transfer can occur readily, as shown by the widespread acquisition of these gene cassettes among the Enterobacteriaceae
and Pseudomonas spp. Integrons comprise two conserved structural regions (5'CS and 3'CS) flanking an internal variable region containing one or more site-specific recombined gene cassettes. While most known cassette-associated genes located distal to the 5'CS region encode resistance to antimicrobial drugs, some cassettes may include one or more open reading frames whose
product(s) and corresponding function(s) remain to be defined (5). In the 3'CS downstream of the gene cassette are two genes, one of which encodes resistance to quaternary ammonia compounds (qacED1), while the other is the
sulphonamide resistance determinant (sul1). Antimicrobial resistance among Campylobacter spp. to drugs used in the treatment of human infection is increasing (6-8). This article reports the results of an investigation of a
collection of Irish thermophilic Campylobacter spp. cultured from clinical cases of gastroenteritis and from porcine and poultry sources. We studied a representative sample of 55 isolates (47 C. jejuni and eight C. coli isolated
between 1996 and 1998), cultured from intestinal tissue of animals at slaughter and from human fecal samples.

Antimicrobial agent susceptibility tests were performed by the agar diffusion method on IsoSensitest agar (Difco, Dublin, Ireland) with 5% horse blood (9).
Cultures were prepared by inoculating colonies from a fresh, pure, 24-hour culture into sterile distilled water to give an inoculum turbidity equivalent to a 0.5 McFarland turbidity standard. The McFarland standard was prepared by
adding 0.5 ml 0.048 M BaCl2 to 99.5 ml 0.18 M H2SO4 with constant stirring. Samples were swabbed evenly onto agar plates and allowed to dry. Twelve antimicrobial agents were tested on disks. Antimicrobial drugs tested,
together with their abbreviations and corresponding concentrations in parentheses, included ampicillin (Ap, 10 g/disc), chloramphenicol (C, 10 g/disc), ciprofloxacin (Cp, 5 g/disc), colistin (Ct, 25 g/disc), erythromycin (E, 5 g/disc), gentamicin (G, 10 g/disc), nalidixic acid (Na,
30 g/disc), spectinomycin (Sp, 10 g/disc), streptomycin (S, 25 g/disc), sulphafurazole (Su, 100 g/disc), tetracycline (T, 10 g/disc), and trimethoprim (Tm, 1.25 g/disc). The plates containing the antibiotic disks
were incubated at 37C under microaerophilic conditions for 24 hours. Inhibition zone sizes were recorded according to the guidelines of the National Committee for Clinical Laboratory Standards (10). Resistance profiles were further confirmed by E-Test (AB Biodisc, Solna, Sweden).

Table. Isolates of Campylobacter coli and C. jejuni from which gene cassettelike structures were amplified (Table not
available in ascii)

Briefly, 17% of all isolates were resistant to ampicillin, 3.8% to chloramphenicol, 1.9% to ciprofloxacin, 7.5% to colistin, 11.3% to erythromycin, 1.9% to gentamicin, 17% to nalidixic acid, 77.4% to spectinomycin, 20.8% to streptomycin, 62.3% to sulphonamide, and 24.5% to tetracycline. Many of the isolates tested (n = 42, 77%)
were resistant to three or more antimicrobial agents with part of the R-type, including SSpTm among others. Two strains, C. jejuni CIT-H17 (R-type: ApCtENaSSpSuTTm) and C. coli CIT-V6 (R-type: CCpENaSSpTTm), were particularly resistant (Table 1); both were resistant to nalidixic acid, and the latter was also resistant to ciprofloxacin. In reviewing the R-types in the sample, the presence
of sulphonamide resistance (in 62.3% of the sample) suggested that integron-like structures may exist in Campylobacter spp.

To test the latter hypothesis, genomic DNA was
purified from all isolates (11). Using the
oligonucleotide primers Int 1 F 5'-GGC ATC CAA
GCA CGA AG-3'and Int 1 B 5'-AAG CAG ACT TGA CCT
GA-3' designed to anneal to the 5'CS and 3'CS
flanking regions (12) of integrons, we tested
the Campylobacter spp. genome by polymerase
chain reaction (PCR) for putative gene
cassettes. Escherichia coli containing the
characterized plasmids R100.1 and R751 (13)
together with CIT-F 100, a Salmonella enterica
serotype Typhimurium DT104 strain cultured from
a contaminated food source (14), were included
as controls. Gene cassettes of 1.0-kb and 800
bp, respectively, from E. coli (data not shown)
and 1.0- and 1.1-kb (Figure 1a, lane 2), from
Salmonella Typhimurium were detected after
amplification. These amplicon profiles were
designated as integron pattern (IP)-groups A,
B, and C, respectively (Table). After
amplification and conventional agarose gel
analysis of all Campylobacter spp. isolates in
the study population, DNA amplicons of 230 bp
to 1.47 kb were detected. A total of 22 gene
cassette structures were identified (Figure 2).
The most commonly occurring amplified gene
cassette pattern was designated IP-group I,
consisting of four DNA fragments of 350 bp to
700 bp (Figure 2, lane 1 and Figure 1a, lane
1). This gene cassette pattern was present in
both poultry and clinically derived C. jejuni,
accounting for 38% of strains. IP-group II
(Figure 2, lane 2) accounted for 7% of all C.
jejuni isolates only. The IP-groups III (Figure
2, lane 3), XIV (Figure 2, lane 14), XVI
(Figure 2, lane 16), and XX1 (Figure 2, lane 21) each accounted for 6% of the collection, with IP-groups XIV and XVI being unique to C. coli. All other IP-groups (Table; Figure 2) were represented by single isolates. A 350-bp amplified DNA fragment was common to all isolates, with the exception of the poultry-derived C. coli CIT-P2 and a clinical isolate C. jejuni CIT-H3. Amplicons of 230 and 250 bp were conserved among C. coli isolates only.

Three putative gene cassettes of 243, 388, and 466 bp were cloned after amplification by using the Int 1 F and Int 1 B primers (4,12) as described above. All were sequenced by automated methods. Sequencing data showed a short, imperfect inverted repeat element at the 3' end of the cloned fragments which represented the 59 base element (5'-GTTRR-3'). This is the target for site-specific recombination involved in the insertion and excision of gene cassettes (4,5,15). Isolates were also tested for the 5' CS encoded
integrase (int) and the 3' CS encoded qacE 1 and sul1 genes by PCR. A DNA fragment of 225 bp was detected after amplification and agarose gel analysis (Figure 1b, lane 1) using primers qacE?1 F 5'-ATC GCA ATA GTT GGC GAA GT-3', and qacE?1 B 5'-CAA GCT TTT GCC CAT GAA GC-3' (13). The latter fragment corresponded with a similar sized amplicon in S. Typhimurium (Figure 1b, lane 2). The 3'-CS region of integrons, known to contain a sul1 gene, was similarly
tested with the primers sul1 F 5'-CTT CGA TGA GAG CCG GCG GC-3' and sul1 B 5'-GCA AGG CGG AAA CCC GCG CC-3' (13). When compared with an S. Typhimurium DT104 amplicon of 436 bp (Figure 1c, lane 2) (13), the Campylobacter spp. sul1-primer derived DNA fragment (Figure 1c, lane 1) appears smaller at approximately 360 bp. Nevertheless, the latter amplicon was consistently amplified from all Campylobacter spp. Smaller sul1 primer generated DNA fragments were also detected
in S. Typhimurium after PCR and gel analysis (14). These may derive from the partial sul1 genes recently located in a 14-kb gene cluster on the chromosome of S. Typhimurium (16). On probing the Campylobacter spp. sul1-primer derived amplicon (Figure 1c, lane 1) with the digoxigenin-labeled 436 bp S. Typhimurium sul1 DNA amplicon (Figure 1c, lane 2), no hybridizing signal was detected (data not shown). This result suggests that the Campylobacter spp. sul1 gene
is different when compared with S. Typhimurium. To investigate the 5'-CS region, primers (int1 F [Tn21]): 5'-GAA GAC GGC TGC ACT GAA CG-3' and int1 R [Tn21]: 5'-AAA ACC GCC ACT GCG CCG TTA-3') were designed to amplify a 1.2-kb DNA fragment from the integrase gene of Tn21 and were tested against Campylobacter spp. and S. Typhimurium (as a control) (Table). The predicted amplicon was detected in the
latter, together with two smaller amplimers of 270 bp and 450 bp. These latter PCR products (270 bp and 450 bp) were also identified in Campylobacter spp. (data not shown), consistent with a deleted form of a class 1 integrase gene in these isolates.

Figure 1. Agarose (1.5%) gel electrophoresis
in 1x TAE buffer containing 0.5 g/ml ethidium bromide....
(Figures not available in ascii)

Figure 2. Schematic representation of all amplified gene cassettes from Campylobacter spp. in the study population.... (Figures not available in ascii)

The DNA sequences from the amplified cassettes (of 243 bp, 388 bp, and 463 bp) above were also searched by using the BLAST search tool (17). GenBank accession numbers were assigned as follows: AF155357 (243-bp gene cassette); AF155356 (388-bp gene cassette), and AF152561 (463-bp gene cassette). The former applicant contains two open reading frames. No corresponding sequences were identified in
the database. The 388-bp amplicon also contained two open reading frames and did not match any sequences when subjected to a BLAST search of the current databases. Finally, the larger 463-bp amplicon contained two incomplete open reading frames. BLAST searches using the latter sequence identified glycyl-tRNA synthetase from the
genome of Helicobacter pylori matching 102 (85%) of 119 nucleotides. Further characterization of other gene cassettes is in progress, focusing on amplicons of 700 bp and greater. 

Drug selection may promote recombinational events between
Campylobacter spp., Enterobacteriaceae and other gram-negative organisms (15). A common habitat for these organisms is the human and animal gastrointestinal tract. Modern animal husbandry promotes the use of large animal housing facilities, thereby ensuring genetic interconnection between large populations of bacteria. Campylobacter
spp. have a natural ability for transformation (18), and in shared animal reservoirs, interspecies transfer of DNA, including antimicrobial resistance encoding genes and other unrelated genes, may occur by strategies analogous to site-specific recombination (19,20). Our findings may indicate a novel mechanism by which unrelated DNA becomes incorporated into cells (21). Detailed characterization of these integronlike structures is an essential step in understanding the role(s) of these novel genetic elements.
The existence of these structures may have interesting implications regarding the diversity of the Campylobacter spp. genome and the evolution of this species. Together with the corresponding DNA fingerprint profile (Lucey B., Fanning S., manuscript in preparation) the variation in genetic content and structure of these determinants may be used as a potential tool in elucidating the epidemiology of these pathogens (22,23).

Acknowledgments

The authors thank Helen O'Shea, Alessandra Carattoli, Fred Angulo, and colleagues at the Department of Medical Microbiology, Cork University Hospital, for valuable comments on the manuscript; Michael Betts and Dorothe Sandvang for providing veterinary samples and Escherichia coli controls, respectively.

Ms. Lucey, senior biomedical scientist, Molecular Diagnostics Unit, Cork's Institute of Technology, and Department of Medical Microbiology, Cork University Hospital, is completing a Masters thesis under the direction of Samus Fanning. Her research interests include the molecular epidemiology of Campylobacter spp. and the
genetic mechanisms underlying antimicrobial resistance in these organisms. She is a recipient of the Abbott Research Prize 1996 awarded to Medical Laboratory Scientists.

Address for correspondence: Samus Fanning, Molecular Diagnostics Unit, Cork Institute of Technology, Bishopstown, Cork, Ireland; fax: 353-21-545-343; e-mail: sfanning@cit.ie.

References

  1. Stern NJ. Reservoirs for Camplobacter jejuni and approaches for intervention in poultry. In: Nachamkin I, Blaser MJ, Tompkins LS, editors. Campylobacter jejuni: current status and future trends. Washington: American Society for Microbiology; 1992. p. 49-60.
  2. Skirrow MB. Diseases due to Campylobacter, Helicobacter and related bacteria. J Comp Pathol 1994;111;113-49.
  3. Nachamkin I, Allos BM, Ho T. Campylobacter species and
Guillain-Barr syndrome. Clin Microbiol Rev 1998;11:555-67.
  4. Recchia GD, Hall RM. Gene cassettes: a new class of mobile element. Microbiology 1995;141:3015-27.
  5. Hall RM, Collis CM. Mobile gene cassettes and integrons: capture and spread of genes by site-specific recombination. Molecular Microbiol 1995;15:593-600.
  6. Moore JE, Elisha BG. Workshop summary: session D. Campylobacter and Helicobacter: antibiotic resistance. In: Lastovica AJ, Newell DG, Lastovica EE, editors. Campylobacter, Helicobacter & related organisms, Institute of Child Health, University of Cape Town; 1998. p. 133-5.
  7. Ruiz J, GoZi P, Marco F, Gallardo F, Mirelis B, De Anta TJ, et al. Increased resistance to quinolones in Campylobacter jejuni: a genetic analysis of gyrA gene mutations in quinolone-resistant clinical isolates. Microbiol Immunol 1998;42:223-6.
  8. Sjgren E, Kaijser B, Werner M. Antimicrobial susceptibilities of Campylobacter jejuni and Campylobacter coli isolated in Sweden: a 10-year follow-up report. Antimicrob Agents Chemother 1992;36:2847-9.
  9. Reina J, Ros MJ, Serra A. Susceptibilities to ten antimicrobial agents of 1,120 Campylobacter strains isolated from 1987 to 1993 from faeces of paediatric patients. Antimicrob Agents Chemother 1994;39:2910-20.
 10. National Committee for Clinical Laboratory Standards.
  Performance standards for antimicrobial disc susceptibility tests. Vol. 1, p: 141-56 (1981). Approved standard. National Committee for Clinical Laboratory Standards, Villanova, Pa. 
 11. Mazurier S, Van de Giessen A, Heuvelman K, Wernars K. RAPD analysis of Campylobacter isolates: DNA fingerprinting without the need to purify DNA. Lett Appl Microbiol 1992;14:260-2. 
 12. Levesque C, Pich L, Larose C, Roy P. PCR mapping of integrons reveals several novel combinations of resistance genes. Antimicrob Agents Chemother 1995;39:185-91.
 13. Sandvang D, Aarestrup FM, Jensen LB. Characterisation of integrons and antibiotic resistance genes in Danish
multiresistant Salmonella enterica typhimurium DT104. FEMS
Microbiol Lett 1998;160:37-41.
 14. Daly M, Buckley J, Power E, O'Hare C, Cormican M, Cryan B, et al. Molecular characterization of Irish Salmonella enterica serotype Typhimurium: detection of class 1 integrons and assessment of genetic relationships by DNA fingerprinting. Appl Env Microbiol. In press, 2000.
 15. Gibreel A, Skld O. High-level resistance to trimethoprim in clinical isolates of Campylobacter jejuni by acquisition of foreign genes (dfr1 and dfr9) expressing drug-insensitive dihydrofolate reductases. Antimicrob Agents Chemother 1998;42:3059-64.
 16. Briggs C, Fratamico PM. Molecular characterization of an antibiotic resistance gene cluster of Salmonella typhimurium DT104. Antimicrob Agents Chemother 1999;43:846-9. 
 17. Altschul SF, Madden TL, Schffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997;25:3389-402.
 18. Wang Y, Taylor DE. Natural transformation in Campylobacter species. J Bacteriol 1990;172:949-55.
 19. Jackson CJ, Fox AJ, Jones DM, Wareing DRA, Hutchinson DN. Associations between heat-stable (O) and heat-labile (HL) serogroup antigens of Campylobacter jejuni: evidence for interstrain relationships within three O/HL serovars. J Clin Microbiol 1998;36:2223-8.
 20. On SLW, Nielsen EM, Engberg J, Madsen M. Validity of
SmaI-defined genotypes of Campylobacter jejuni examined by
SalI, KpnI, and BamHI polymorphisms: evidence of identical
clones infecting humans, poultry, and cattle. Epidemiol Infect 1998;120:231-7.
 21. Richardson PT, Park SF. Integration of heterologous plasmid DNA into multiple sites on the genome of Campylobacter coli following natural transformation. J Bacteriol 1997;179:1809-12.
 22. Kokotovic B, On SLW. High-resolution genomic fingerprinting of Campylobacter jejuni and Campylobacter coli by analysis of amplified fragment length polymorphisms. FEMS Microbiol Lett 1999;173:77-84.
 23. Sallen B, Rajoharison A, Desvarenne S, Mabilat C. Molecular epidemiology of integron-associated antibiotic resistance genes in clinical isolates of Enterobacteriaceae. Microb Drug Resist 1995;1:195-202.

Dispatches

Burkholderia pseudomallei Traced to Water Treatment
Plant in Australia

Timothy J.J. Inglis,* Stuart C. Garrow, Mandy
Henderson,* Adele Clair,* Judy Sampson,* Lyn O'Reilly,*
and Bob Cameron
*Western Australian Centre for Pathology and Medical
Research, Nedlands, Australia; Kimberley Public Health
Unit, Derby, Australia

--------------------------------------------------------

 Burkholderia pseudomallei was isolated from
 environmental specimens 1 year after an
 outbreak of acute melioidosis in a remote
 coastal community in northwestern
 Australia. B. pseudomallei was isolated
 from a water storage tank and from spray
 formed in a pH-raising aerator unit.
 Pulsed-field gel electrophoresis confirmed
 the aerator and storage tank isolates were
 identical to the outbreak strain, WKo97.

Melioidosis is endemic in northern Australia. Since the
first description of the disease from far north
Queensland in 1962 (1), melioidosis has spread west and
south. A small focus of endemic infection was
identified in western Australia in 1967 (2). Cases are
reported throughout the year but peak during the rainy
season (3). An unusually large number of cases was
diagnosed in the Northern Territory during the
near-record rainfall of 1990-91 (4). A cluster of acute
melioidosis cases in a remote, coastal community
(population 300) in western Australia during the dry
season in late 1997 was, therefore, unexpected (5).
Five cases of acute septicemic or pneumonic melioidosis
were diagnosed during a 6-week period. All five
patients lived in the same remote community and had
recognized risk factors for acute melioidosis (e.g.,
diabetes or renal failure). Burkholderia pseudomallei
was isolated from all patients; three died. Late-onset
septicemia was diagnosed in a resident of this
community 6 months later. This infection was preceded
by a febrile episode during the presumed exposure
period and soft tissue infection 1 month later.

All six isolates were indistinguishable by pulsed-field
gel electrophoresis (PFGE). Environmental sampling
during the outbreak investigation identified the
potable water supply as a possible source of B.
pseudomallei. Epidemiologic investigations implicated
acid bore water, chlorination failure, recent
replacement of water pipes, and climate.

Twelve months later, after maintenance work on the
water supply was completed, microbiologic samples were
collected again. Isolation of B. pseudomallei prompted
a more detailed environmental investigation with
emphasis on the water treatment plant and bore water.
The water supply to the community comes from a
subterranean source through a group of capped and
sealed bore holes. These are fed by an underground
common bore main into the water treatment compound,
where the bore water is passed through an aerator
tower, then stored in two ground-level tanks before
chlorination. After passing through a gas chlorinator,
water is fed through another underground main pipe to
the community, is pumped up into a high-level tank, and
flows into the underground domestic reticulation
system. Outlets in family dwellings and community
buildings are plumbed faucets, showerheads, and water
closets. Potable water is supplied to the community at
a rate of approximately 500 kL per day.

Preliminary Bacteriology

We collected a 5-L water specimen from the previously
culture-positive yard tap and another 5-L specimen from
the sample access point before the gas chlorinator
unit, after flame-sterilizing the outlet and letting
water run for a prolonged period. A clump of wild grass
above the bore line entering the compound, near the
place where an excavator punctured the pipeline, was
dug up with its roots intact. Water specimens were
filtered, and the filter membrane was cultured in
Ashdown broth and subcultured onto Ashdown agar. A 5-g
sample of root soil was suspended in sterile saline and
processed as for water specimens. Suspect colonies were
identified by standard laboratory methods and confirmed
by polymerase chain reaction (PCR) amplification of B.
pseudomallei DNA sequences (6). B. pseudomallei was
isolated from all three tested specimens, although the
species was recovered only from the tap water after
enrichment with trypticase soy broth and prolonged
incubation (2 weeks). Sediment from the bottom of the
water tank and a 5-L specimen of tank contents were
cultured. Both were positive for B. pseudomallei.

Bacteriology of the Water Treatment Plant

Sampling of the water supply system progressively further upstream drew our attention to an aerator in the treatment plant that during the initial outbreak investigation had been temporarily removed for renovation. The aerator is installed to raise water pH (acid due to dissolved carbon dioxide) by spraying groundwater through a series of open 1 m x 1 m wire mesh trays located in a frame on an elevated
platform. A series of water samples were collected in the community, immediately after chlorination, before chlorination, from the ground tank, the aerator, bore lines, and all four capped bores. Aerator spray was collected from the top and lower down the tower. Biofilm was scraped from the sides of the aerator trays. Water and scum from
the aerator sump were also sampled. The pH was measured in the spray generated at each level of the aerator. Roots and soil were collected from a shrub (Acacia coleii) growing around the stop valve at the entry to the treatment plant complex, in case roots had penetrated the pipeline and soil had entered the water supply. All water specimens were filtered and spread onto Ashdown and trypticase
soy broth media. Isolates were identified as described above. B. pseudomallei was isolated from the spray at the top and bottom of the aerator in abundant growth on direct culture plates, without any prior enrichment (Table), and from soil around the acacia roots. Despite the use of selective media, the growth of various environmental bacterial and fungal species was so abundant in the
lower aerator trays and sump that the presence of B. pseudomallei could not be excluded, although all cultures were negative. All samples from the bore lines and bore field upstream of the aerator were negative for B. pseudomallei. Chlorine levels were maintained above 1 ppm.

Table. Results of 1999 environmental samples from outbreak vicinity
-------------------------------------------------------
				Burkholderia
Sample  			pseudomallei	WKo97
-------------------------------------------------------
All bores				 -
Bore line root soil    		+ 		-
Acacia root soil  		+ 		-
Aerator
Upper spray  			+		+
Lower spray  			+		+
Tray biofilm 			-
Sump water				-
Sump biofilm 			-
Storage tank
Surface water			-
Deep water				+		+
Sediment  				+		+
Prechlorinator    		-
Postchlorinator   		-
High-level tank   		-
Domestic tap water		-
Backyard tap 			+		-
Storage tank repeat
    Deep water    		-
--------------------------------------------------------

Molecular Epidemiology

Environmental and clinical isolates of B. 
pseudomallei were typed by PFGE of an XbaI
restriction digest of bacterial chromosomal DNA
(Figure). After completion of these studies,
environmental isolates from parts of the    
potable water supply, nearby soil, and two  
distant locations (250 and 1,000 km away) were 
run simultaneously with human and animal

Figure. Results of pulsed-field electrophoresis
(PFGE) of clinical and environmental
isolates of Burkholderia pseudomallei from
western Australia.... (Figures not available 
in ascii)

clinical isolates. As two gels were required, clinical isolates were run on one gel with a gel
duplicate of the initial tap water isolate, and  
environmental isolates were run with a 
duplicated digest from the fifth case in the cluster. Soil isolates were distinct from the outbreak strain, but the aerator spray and ground tank isolates were identical to the  outbreak strain, WKo97 (Figure; Table).

Biocontainment Measures   

Confirmation that a specific device in the water treatment plant had become heavily contaminated with B. pseudomallei prompted biocontainment questions. Arrangements were made to isolate the aerator from the water inflow, dismantle it, and remove its parts for incineration. Before being dismantled, the aerator was drained and soaked in concentrated hypochlorite, then wrapped in a polythene
sheet. Workers wore submicron particle filter masks, protective overalls, and heavy-duty gloves. The ground-level tank was drained, treated with concentrated hypochlorite solution, and cleaned before refilling. Samples collected from the ground-level tank 2 months after these measures had been completed contained no detectable B. pseudomallei.

Conclusions

In our investigation of the persistence of B. pseudomallei in the potable water supply, we traced the source of contamination upstream to the water treatment plant and identified the aerator as a possible source. If it had been connected during the initial outbreak investigation and had been sampled then, the aerator might have been identified as a potential source 1 year earlier. Moreover, failure to fully digest the prechlorination isolate in this and a
second reference laboratory led to the erroneous belief that the prechlorination point result was unrelated to the case-cluster. We cannot prove beyond doubt that the aerator was the primary source of contamination without contemporaneous bacteriologic evidence. It is unlikely the aerator became contaminated later as a result of retrograde flow, especially from a point downstream to the chlorinator unit. There are thus several possible sources of contamination: inflowing water from a leak in the bore pipeline,
rupture of the pipeline during renovation work, dust or vegetation blown through the mesh walls of the aerator by wind, or direct contact with contaminated soil during refurbishment of the dismantled aerator. The 1997 outbreak may have occurred because a shower of bacteria was released from the aerator into the potable water supply during renovation work on the aerator at a time when
chlorine levels were at or near zero. The aerator unit had only a modest effect on pH but clearly provided optimal conditions for multiplication of aquatic bacteria with an aerobic metabolism (7). Whether or not the aerator became contaminated more recently than the outbreak, its ability to act as a persistent source or amplifier of the outbreak strain of B. pseudomallei justified its removal.

Aerators are used to correct acidic, ferric, or unusually warm potable groundwater supplies in many remote communities in northern Australia and represent a potential melioidosis risk. Deliberate or natural aeration of a contaminated water supply could amplify B. pseudomallei growth. In northeastern Thailand, where melioidosis is endemic and the groundwater is unusually acidic (8), natural or artificial aeration of the potable water supply should be considered as an additional contributory factor.

Measures taken after the initial western Australian outbreak
investigation appear to have prevented further cases, despite the presence of multiple B. pseudomallei types in soil. Initial water engineering measures have cleared detectable B. pseudomallei from the water treatment plant.

Acknowledgments

Permission to publish this report was granted by the community council. We thank Nicky Buller and the Agriculture Department of Western Australia for supplying the animal and related environmental strains of B. pseudomallei and David Dance and Thomas Riley for reviewing the manuscript.

Dr. Inglis is a medical microbiologist at PathCentre, the Western Australian Centre for Pathology and Medical Research. His duties include public health microbiology and hospital infection control. Other than melioidosis, current research interests include the laboratory-based surveillance of infectious disease and the pathogenesis of ventilator-associated pneumonia.

Address for correspondence: Timothy J.J. Inglis, Division of
Microbiology and Infectious Diseases, PathCentre, Locked Bag 2009, Nedlands, WA 6009, Australia; fax: 619-9381-7139; e-mail: tim.inglis@health.wa.gov.au

References

  1. Rimington RA. Melioidosis in north Queensland. Med J Australia 1962;1:50-4.
  2. Ketterer PJ, Bamford VW. A case of melioidosis in lambs in southwestern Australia. Austr Vet J 1967;43:79-80.
  3. Ashdown LR, Guard RW. The prevalence of human melioidosis in northern Queensland. Am J Trop Med Hyg 1984;33:474-8.
  4. Merianos A, Patel M, Lane MJ, Noonan CN, Sharrock D, Mock P et al. The 1990-1991 outbreak of melioidosis in the northern territory of Australia: epidemiology and environmental studies. Southeast Asian J Trop Med Pub Health 1993;24:425-35.
  5. Inglis TJJ, Garrow SC, Adams C, Henderson M, Mayo M, Currie BJ. Acute melioidosis outbreak in Western Australia. Epidemiol Infect 1999;123:437-44.
  6. Kunakorn M, Markham B. Clinically practical seminested PCR for Burkholderia pseudomallei quantitated by enzyme immunoassay with and without solution hybridization. J Clin Microbiol 1995;33:2131-5.
  7. Redfearn MS, Palleroni NJ, Stanier RY. A comparative study of Pseudomonas pseudomallei and Bacillus mallei. J Gen Microbiol 1966;43:293-313.
  8. Kanai K, Kondo E, Dejsirilert S, Naigowit P. Growth and
survival of Pseudomonas pseudomallei in acidic environment with possible relation to the ecology and epidemiology of
melioidosis. In: Puthucheary SD, Malik YA, editors.
Melioidosis: prevailing problems and future directions.
Malaysian Society of Infectious Diseases and Chemotherapy,
Kuala Lumpur, Malaysia; 1994. p 26-38.

Dispatches

Molecular Typing of Multidrug-Resistant Salmonella
Blockley Outbreak Isolates from Greece

Panayotis T. Tassios,* Christos Chadjichristodoulou,
Maria Lambiri, Athina Kansouzidou-Kanakoudi, Zannina
Sarandopoulou,* Jenny Kourea-Kremastinou, Leonidas S.
Tzouvelekis,* Nicholas J. Legakis*
*University of Athens, Athens, Greece; National Center
for Surveillance and Intervention, Athens, Greece;
National School of Public Health, Athens, Greece; and
Salmonella Reference Center for Macedonia and Thrace,
Thessaloniki, Greece

--------------------------------------------------------

 During 1998, a marked increase (35 cases)
 in human gastroenteritis due to Salmonella
 Blockley, a serotype rarely isolated from
 humans in the Western Hemisphere, was noted
 in Greece. The two dominant
 multidrug-resistance phenotypes (23 of the
 29 isolates studied) were associated with
 two distinct DNA fingerprints, obtained by
 pulsed-field gel electrophoresis of genomic
 DNA.

Salmonella Blockley is rarely isolated in the Western
Hemisphere. According to Enter-net, the international
network for surveillance of Salmonella and
verocytotoxin-producing Escherichia coli infections, S.
Blockley represented 0.6% of all Salmonella serotypes
isolated in Europe during the first quarter of 1998, a
full 100-fold lower than the dominant serotype, S.
Enteritidis (67.1%) (1). However, S. Blockley is among
the five most frequently isolated serotypes from both
avian and human sources in Japan (2,3), Malaysia (4,5),
and Thailand (6). A single foodborne outbreak in the
United States (7) and sporadic human infections in
Europe associated with travel to the Far East (8),
animal infection (9) or carriage (10,11), and
environmental isolates have also been reported (12,13).

Regardless of the frequency of S. Blockley isolation,
its rates of resistance to antibiotics have been high.
Among Spanish salmonellae isolated from natural water
reservoirs, S. Blockley and S. Typhimurium had the
highest rates of multidrug resistance (12). Comparing
1980-1989 with 1990-1994, researchers from Tokyo noted
an increase in the number of S. Blockley isolates
resistant to one or more antibiotics, from 92.0% to
98.2% for imported cases and from 57.4% to 88.7% for
domestic cases (3,14). In Thailand, isolates from human
or other sources also had high rates of resistance to
streptomycin, tetracycline, kanamycin, and
chloramphenicol and lower rates to ampicillin and
trimethoprim/sulfamethoxazole (15).

Nevertheless, few attempts at typing S. Blockley
isolates with molecular methods have been described,
and these have been limited to the characterization of
plasmid content (2,16).

During the second and third quarters of 1998, Enter-net
reported higher numbers of S. Blockley isolates than
during the same period of the previous year in several
European countries (1). The epidemiologic
investigations conducted in Germany, England and Wales,
and Greece did not confirm a source for this increase
(17-19).

In this study, we characterized the Greek outbreak
isolates further, both with respect to their antibiotic
resistance phenotypes and DNA fingerprints obtained by
pulsed-field gel electrophoresis of genomic DNA.

The Study

The study sample consisted of 28 of 35 S. Blockley
strains isolated from May to December 1998 (19), one
strain from February 1999, and four epidemiologically
unrelated control strains: one from 1996 and three from
1997. All isolates were from human cases of enteritis.
Identification was performed by the API 20E system
(BioMerieux S.A., Marcy l'Etoile, France) and
serotyping with commercially obtained antisera
(BioMerieux) (20).

Susceptibility to kanamycin, streptomycin, ampicillin,
amoxicillin/clavulanic acid, cefepime, tetracycline,
chloramphenicol, trimethoprim/sulfamethoxazole,
gentamicin, nalidixic acid, and ciprofloxacin was
tested by a disk diffusion assay according to National
Committee for Clinical Laboratory Standards guidelines
(21). Genomic DNA was prepared and digested with XbaI
(New England Biolabs) (22). Chi-square tests or Fisher
exact tests were used to calculate two-tailed
probabilities.

S. Blockley accounted for seven of the 13,199
salmonella isolates identified in Greece from 1976 to
1997. However, 35 gastroenteritis cases due to this
serotype were reported from May to December 1998 (19).
Twenty-nine S. Blockley strains isolated from fecal
specimens of patients with gastroenteritis during May
1998 to February 1999, along with four epidemiologically unrelated clinical isolates from 1996 and 1997, were therefore studied for susceptibility to antibiotics. The 1998 outbreak isolates were scattered throughout Greece; S. Blockley was isolated later, starting in August 1998, in northern Greece.

All isolates were susceptible to 
trimethoprim/sulfamethoxazole, ampicillin,
amoxicillin/clavulanic acid, gentamicin, and
ciprofloxacin (Table). High resistance rates were
observed to tetracycline (100%), streptomycin and
kanamycin (90%), chloramphenicol (83%), and nalidixic
acid (52%). Six resistance phenotypes could be
distinguished (Table) with the two major phenotypes of
outbreak isolates being resistant to kanamycin,
streptomycin, tetracycline, and chloramphenicol (ATC)
or kanamycin, streptomycin, tetracycline,
chloramphenicol, and nalidixic acid (ATCN). Most (76%)
strains isolated after August 24, 1998, were nalidixic
acid-resistant (resistance phenotypes ATCN, TCN, ATN),
unlike strains isolated up to August 17, 1998 (17%)
(1.29 <RR = 3.03 <7.11, p = 0.005).

Table. Resistant phenotypes for isolates of
Salmonella Blockley in Greece (Table not 
available in ascii)

When pulsed-field gel electrophoresis was used
to obtain DNA fingerprints for these isolates
(Figure 1), all belonged to the same type, A,
although eight subtypes, A1-A8, could be
distinguished on the basis of one to three DNA
fragment differences (Figures 1, 2). Two of the
four isolates from previous years belonged to
unique subtypes A6 and A7; the other two
belonged to subtypes A2 and A8, shared by
outbreak isolates (Table). In contrast, 93% of
the 1998 outbreak strains yielded PFGE patterns
common to two or more isolates. Indeed, most
outbreak isolates were grouped in subtypes A2
and A4, consisting of 11 and 12 isolates,
respectively (Table).

Figure 1. Sample pulsed-field gel electrophoresis
(PFGE) gel of representative Salmonella Blockley
isolates, indicating common and unique DNA
fingerprints.... (Figures not available in ascii)

Figure 2. Dendrogram of similarity among the observed
pulsed-field gel electrophoresis patterns.
(Figures not available in ascii)

Pulsed-field gel electrophoresis subtypes were
associated with resistance phenotypes. Most
resistance phenotype ATC isolates belonged to
subtype A2 (3.03. <RR = 11.86 <46.5, p =
0.0000098), while most resistance phenotype
ATCN isolates belonged to A4 (2.76 <RR = 7.11
<18.30, p = 0.0000416). In addition, most
isolates in the two major subtypes appearing
before August 17, 1998, belonged to the A2
group, while most isolates appearing after
August 24, 1998, belonged to the A4 group (1.47
<RR = 9.82 <65.45, p = 0.0006). Finally, unlike
A4, the earlier A2 subtype was not isolated in
northern Greece.

Conclusions

Our results indicate that PFGE is useful in
distinguishing epidemiologically related S.
Blockley isolates since two of the four
nonoutbreak isolates displayed unique PFGE
patterns, A7 and A8, while PFGE patterns A2 and
A4 grouped most of the 29 outbreak isolates (11 and 12,
respectively).

These two chromosomal fingerprints, differing by two DNA fragments, were associated with two distinct resistance phenotypes. The resistance phenotype of A4 isolates, ATCN, was identical to the earlier resistance phenotype of A2 isolates, ATC, except for the resistance to nalidixic acid. Nevertheless, these two PFGE/antibiotic resistance types, A2/ATC and A4/ATCN, displayed a clear distribution both in time and space.

The data may, therefore, indicate two main sources for the outbreak. Alternatively, and perhaps more likely, these two closely related types may together constitute the outbreak clone, evolved with time to acquire resistance to nalidixic acid. Resistance may well have originated in the food source, since several antibiotic classes are
used as feed supplements in animal rearing and aquaculture in Greece: sulfonamides (trimethoprim/sulfathiazine), tetracyclines (oxytetracycline), and quinolones (oxolinic acid). However, as in other European countries (17,18), the epidemiologic investigation did not locate a common source to account for the wide geographic spread of cases (19). Although travel was not mentioned in the Greek
patients' questionnaire responses, the possibility that the source was an imported food cannot be ruled out. The association with smoked eel of Italian origin in the German outbreak has not been microbiologically confirmed (17). The only other previous European report of a human outbreak attributed to S. Blockley, probably from vegetables contaminated by this organism, which was prevalent in
irrigation water in the Spanish region of Granada, is anecdotal (13). A documented S. Blockley enteritis epidemic in a U.S. hospital in 1966 was attributed to contaminated ice cream; however, this was also not microbiologically confirmed (7).

While this serotype may remain important in Europe, its high rates of resistance to kanamycin, streptomycin, tetracycline, and chloramphenicol, which were in agreement with studies from the Far East (3) and Spain (12), are cause for concern. Unlike the Far Eastern strains, no resistance to -lactam antibiotics or cotrimoxazole was observed in our study. The two dominant resistant phenotypes of S. Blockley from natural polluted waters in Spain were sulfonamides, streptomycin, and tetracycline; and neomycin, streptomycin, kanamycin, tetracycline, and chloramphenicol (12), as
in the Greek strains, except for the absence of resistance to nalidixic acid.

In agreement with differences in animal reservoirs and transmission routes and therefore the mechanism of resistance acquisition among different Salmonella serotypes, the main patterns of resistance observed in S. Blockley were distinct from those predominating in the two major serotypes from isolates of both human and animal food origin in Greece. In S. Enteritidis, the most frequent resistance
phenotype was resistance to ampicillin (24), while in S.
Typhimurium, the most frequent resistant phenotype was resistance to sulfonamides and streptomycin (A. Markogiannakis, P.T. Tassios, N.J. Legakis, unpub. obs.). Furthermore, the considerably high rate of resistance to nalidixic acid is equally unprecedented in both the
Far Eastern and Spanish S. Blockley isolates and in other salmonella serotypes from Greece. Since resistance to nalidixic acid can be a precursor of resistance to fluoroquinolones, one of the two drug classes of choice for invasive salmonella disease, this feature of
these S. Blockley strains is particularly disturbing. S. Blockley, previously a prevalent serotype in the Far East but rare elsewhere, nevertheless posed a public health problem in several European countries. The source of the European outbreaks, however, remains unclear. Given the increased international commerce in food, a collaborative study would be useful in identifying potential similarities between the recent European strains and established
strains from the Far East.

Acknowledgments

We thank Veneta Lukova and Soula Christou for assistance with antibiotic susceptibility testing.

This work was funded in part by the Ministry of Health and Welfare.

Dr. Legakis is professor and head of the department of Microbiology of the Medical School of the National University of Athens. His research interests include molecular typing, mechanisms of antibiotic resistance of bacterial pathogens, and molecular diagnosis of infections.

Address for correspondence: Nicholas J. Legakis, Department of Microbiology, Medical School, University of Athens, M. Asias 75, 115 27 Athens, Greece; fax: 301-7709-180; e-mail: nlegakis@cc.uoa.gr

References

  1. Fisher IST, on behalf of Enter-net participants. Enter-net Quarterly Salmonella Report - 98/3 (1998 Jul-Sep). Available from: URL:  http://www2.phls.co.uk/reports/latest.htm
  2. Limawongpranee S, Hayashidani H, Okatani AT, Ono K, Hirota C, Kaneko K, et al. Prevalence and persistence of Salmonella in broiler chicken flocks. J Vet Med Sci 1999;61:255-9.
  3. Matsushita S, Yamada S, Sekiguchi K, Kusunokui J, Ohta K, Kudoh Y. Serovar-distribution and drug-resistance of Salmonella strains isolated from domestic and imported cases in 1990-1994 in Tokyo. Kansenshogaku Zasshi 1996;70:42-50.
  4. Yasin RM, Jegathesan MM, Tiew CC. Salmonella serotypes isolated in Malaysia over the ten-year period 1983-1992. Asia Pac J Public Health 1996-97;9:1-5.
  5. Rusul G, Khair J, Radu S, Cheah CT, Yassin RM. Prevalence of Salmonella in broilers at retail outlets, processing plants and farms in Malaysia. Int J Food Microbiol 1996;33:183-94.
  6. Sasipreeyajan J, Jerngklinchan J, Koowatananukul C, Saitanu K. Prevalence of salmonellae in broiler, layer and breeder flocks in Thailand. Trop Anim Health Prod 1996;28:174-80.
  7. Morse LJ, Rubenstein AD. A food-borne institutional outbreak of enteritis due to Salmonella blockley. JAMA 1967;202:939-40.
  8. Albert S, Weber B, Schafer V, Rosenthal P, Simonsohn M, Doerr HW. Six enteropathogens from a case of acute gastroenteritis. Infection 1990;18:381-2.
  9. van Duijkeren E, Sloet van Oldruitenborgh-Oosterbaan MM, Houwers DJ, van Leeuwen WJ, Kalsbeek HC. Equine salmonellosis in a Dutch veterinary teaching hospital. Vet Rec 1994;135:248-50.
 10. Mallaret MR, Turquand O, Blatier JF, Croize J, Gledel J, Micoud M, et al. Human salmonellosis and turtles in France. Rev Epidemiol Sante Publique 1990;38:71-5.
 11. Levre E, Valentini P, Brunetti M, Sacchelli F. Stationary and migratory avifauna as reservoirs of Salmonella, Yersinia and Campylobacter. Ann Ig 1989;1:729-40.
 12. Morinigo MA, Cornax R, Castro D, Jimenez-Notaro M, Romero P, Borrego JJ. Antibiotic resistance of Salmonella strains isolated from natural polluted waters. Journal of Applied Bacteriology 1990;68:297-302.
 13. Garcia-Villanova Ruiz B, Cueto Espinar A, Bolanos Carmona MJ. A comparative study of strains of salmonella isolated from irrigation waters, vegetables and human infections. Epidemiol Infect 1987;98:271-6.
 14. Matsushita S, Yamada S, Inaba M, Kusunoki J, Kudoh Y, Ohashi M. Serovar distribution and drug resistance of Salmonella isolated from imported and domestic cases in 1980-1989 in Tokyo. Kansenshogaku Zasshi 1992;66:327-39.
 15. Bangtrakulobth A, Suthienkul O, Kitjakara A, Pornrungwong S, Siripanichgon K. First isolation of Salmonella blockley in Thailand. Southeast Asian J Trop Med Public Health 1994;25:688-92.
 16. Atanassova V, Matthes S, Muhlbauer E, Helmuth R, Schroeter A, Ellendorff F. Plasmid profiles of different Salmonella serovars from poultry flocks in Germany. Berl Munch Tierarztl Wochenschr 1993;106:404-7.
 17. Hamouda O. Outbreak of Salmonella blockley infections in Germanypreliminary investigation results.  Eurosurveillance Weekly 1998;2:980924. Available at URL:
http://www.eurosurv.org/1998/980924.htm
 18. Benons L. Salmonella blockley infections in England and Wales, 1998. Eurosurveillance Weekly 1998;2:980924. Available at URL: http://www.eurosurv.org/1998/980924.htm
 19. Vassilogiannakopoulos A, Tassios P, Lampiri M. Salmonella blockley infection in Greece. Eurosurveillance Weekly 1999;3:990408. Available at URL:
http://www.eurosurv.org/1999/990408.htm
 20. Kauffman F. Serological diagnosis of Salmonella species. Copenhagen (Denmark): Munksgaard; 1972.
 21. National Committee for Clinical Laboratory Standards. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved Standard M7-A2. Wayne (PA): The Committee; 1997.
 22. Tassios PT, Vatopoulos AC, Mainas E, Gennimata D, Papadakis J, Tsiftsoglou A, et al. Molecular analysis of
ampicillin-resistant sporadic Salmonella typhi and Salmonella paratyphi B clinical isolates. Clin Microbiol Infect 1997;3:317-23.
 23. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995;33:2233-9.
 24. Tassios PT, Markogiannakis A, Vatopoulos AC, Katsanikou E, Velonakis EN, Kourea-Kremastinou K, et al. Molecular
epidemiology of antibiotic resistance of Salmonella enteritidis during a 7-year period in Greece. J Clin Microbiol 1997;35:1316-21.

Dispatches

Risk Factors for Carriage of Neisseria meningitidis
during an Outbreak in Wales

Patricia E. Fitzpatrick,* Roland L. Salmon,* Paul R.
Hunter, Richard J Roberts, and Stephen R. Palmer*
*PHLS Communicable Disease Surveillance Centre (Wales),
Cardiff, United Kingdom; Chester Public Health
Laboratory, Chester, United Kingdom; and North Wales
Health Authority, Mold, United Kingdom

---------------------------------------------------------

 In a school outbreak of meningococcal
 disease in Wales, we compared risk factors
 for the carriage of Neisseria meningitidis
 B15 P1.16 with carriage of any meningococci.
 Students had throat swabs and completed a
 questionnaire. Sixty (7.9%) carried
 meningococci; risk for carriage was higher
 in those >14 years of age.

Outbreaks of meningococcal disease, although rare, may
have become more common in the United Kingdom,
particularly among teenagers and young adults (1-3). In
an investigation of a school-based outbreak in north
Wales, extensive nasopharyngeal swabbing and subtyping
allowed comparison of risk factors for carriage of the
epidemic strain of Neisseria meningitidis B15 P1.16 and
carriage of other meningococci.

On consecutive days in May 1996, two cases of
meningococcal disease were reported in a single year
group (year 11, ages 15 to 16 years) in a large (760
students) secondary school. One case was confirmed as
due to N. meningitidis group B, type 15 P1.16. The
second involved characteristic clinical symptoms,
although blood culture and polymerase chain reaction
(PCR) of serum were negative. In addition, five cases of
meningococcal disease from the surrounding areas (total
population 8,000) had been reported in the preceding 11
months. Three of these cases were in students of the
school. One had been confirmed as N. meningitidis B15
P1.16 and one as serogroup C. The observed incidence of
notified disease in England and Wales for 1995 was 3.7
per 100,000 and of culture-confirmed disease 2.9 per
100,000 total population (4). We conducted an
investigation to determine the prevalence of N.
meningitidis B15 P1.16 carriage in the school and
examine the associated risk factors.

The Study

Throat swabs from students and staff were spread onto 5%
Columbia blood agar containing polymyxin 25,000 units/L
and vancomycin 3 mg/L. Primary incubation was conducted
at 37C for 48 hours in 10% CO2. Plates showing
preliminary growth were sent to the Meningococcal
Reference Unit at Manchester Public Health Laboratory
for further examination and serotyping. Rifampicin was
given to students in year 11, which included all those
subsequently found to be carriers of the epidemic
strain.

Epidemiology

All students from whom a throat swab was taken were
asked to complete a questionnaire about personal and
household details, lifestyle and social behavior
(including travel), and health. A household density
ratio was calculated from the ratio of number of
household members to the number of rooms in the house.
Socioeconomic background was determined by occupation of
the head of the household, according to the Office of
Population Census and Surveys classification of
occupations (5). As stress has been proposed as a risk
factor for meningococcal disease (6), we asked about
stressful events in the month before the diagnosis of
the index cases (e.g., a death in the family, household
move, or bad news).

Univariate analysis of risk factors for meningococcal
carriage was performed by using Epi-Info (7); the
chi-square test was used for statistical significance.
Multivariate analysis of carriage of N. meningitidis was
performed with SAS (SAS Institute Inc., Cary, NC);
variables from univariate analysis were entered into a
forward stepwise logistic regression, and conditional
odds ratios and 95% confidence intervals were calculated
for the resulting significant variables.

Swabs were taken from 744 (97.8%) of 760 pupils at the
school. No pupil had received prior antimicrobial
chemoprophylaxis. N. meningitidis was cultured from
throat swabs of 60 (7.9%) students; 33 (55%) were in
year 11. Of 17 group B isolates, 12 were type B15P1.16
(Table 1). Of 626 students (83.4%) who completed
questionnaires, N. meningitidis was isolated from 53
students (8.5%) (Table 1).

Table 1. Subtyping of Neisseria
meningitidis isolates in a Welsh
secondary school (Table not available 
in ascii)
Table 2. Personal, family, and household factors and meningococcal carriage (Table not available in ascii)

Students who reported that they had smoked cigarettes or lived with a smoker were more likely to carry meningococci (Table 3). Students in the same two classerooms and the same year as the index patients were more likely to carry N. meningitidis. Attendance at an informal party held by year 11 students 10 days before onset of illness was associated with carriage (Table 3). This informal gathering had no
list of invitees; therefore, the number of those who attended but did not have throat swabs taken is not known.

Being in a sports team or regular attendance at youth clubs, Sunday schools, cubs, scouts, brownies, or guides was not associated with carriage, nor was recent travel.

Students who had been in regular contact with one of the patients were more likely to be carriers (Table 4). On multivariate analysis, having more than two smokers in the household, being in the same year in school as the index patients, and having received bad news in the preceding 3 months remained associated with meningococcal carriage (Table 5).

Table 3. Risk factors for meningococcal carriage in a Welsh secondary school (Table not available in ascii)
Table 4. Medical factors associated with memingococcal carriage in a Welsh secondary school (Table not available
in ascii)
Table 5. Factors remaining significant
for meningitidis carriage in final model
----------------------------------------
             Odds ratio Odds ratio
              (95% CI)   (95% CI)
Variable     unadjusted  adjusteda
------------------------------------------
Year 11b           8.20  8.61
            (4.34-15.56)(4.66-15.91)
>2 smokers in      2.96  2.99
household    (1.24-6.89) (1.25-7.15)
Received bad news  2.46  2.67
             (1.22-4.91) (1.32-5.40)
------------------------------------------
aadjusted for other significant variables.
bsame school year as index case.

For carriage of the epidemic strain, N. meningitidis B15 P1.16, four factors were associated: being in year 11, being older than 14, having attended the end-of-year party, and being male. Because carriage of the epidemic strain was confined to year 11 students (who were >14 years of age), multivariate analysis was not performed. Within year 11, no single factor was associated with carriage of N. meningitidis B15 P1.16.

Conclusions

In February 1995, we investigated risk factors for the carriage of any meningococci among the contacts of three ill students at another Welsh secondary school (8). These index patients were students in different school years, and 2 (1.7%) of 119 contacts carried the epidemic strain (type B2b P1.10). In the current outbreak, we identified 11 (1.5%) of 744 students with the epidemic strain (type B15 P1.16). In contrast, outbreaks of disease associated with serogroup C disease are typically accompanied by lower rates of
carriage in populations at risk. However, outbreaks of group C disease involving higher carriage rates are occasionally described. In an outbreak at an agricultural college in England, carriage of the epidemic strain of serogroup C organisms among students and staff was 6.2% (9). In the current outbreak, the fact that all carriers of the epidemic strain were in the same school year as the two index patients enabled us to examine risk factors for carriage
of the epidemic strain. Risk factors for the carriage of any
meningococci may differ. For example, in an outbreak of six cases among first-year students at Southampton University (United Kingdom) in 1997, 0.9% of students surveyed carried serogroup C strains; however, no first year students were carriers (10). 

Better knowledge of risk factors for carriage of epidemic
meningococci may help identify close contacts who are candidates for antimicrobial therapy to eliminate nasal carriage and prevent spread of disease (11). Carriers of an epidemic strain with the potential to infect others may be missed, and a number of people may receive unnecessary antibiotics, which has implications for the spread of
antibiotic resistance.

Carriage of any meningococci was associated in the univariate analysis with the well-described risk factors of increasing age and smoking (active and passive) (12-14). Smoking may predispose to colonization by inhibiting bronchial ciliary action (12). We also found increasing age and active smoking to be associated with carriage in our previous study (8). In the current study, receipt of
bad news was associated. No obvious biological mechanism exists to explain what may be a chance finding, although recent stress has been described as a risk factor for meningococcal disease (6). Being in the same year as the index patients and attending the end-of-year party were risk factors for carriage of any strain of meningococci
in this outbreak, mainly because these were the only two risk factors associated with carriage of the epidemic strain. These two factors reflect the kinds of social contacts among teenagers and young adults that may permit spread of meningococci. In a review of 22 school-based clusters between 1989 and 1993, the patients in nine
of the clusters had contact through extracurricular activities; these activities in four clusters were parties or dances (15). Patronage of a particular bar was implicated in the university outbreak in Illinois (16), and a particular discotheque in an outbreak among eight adults (five of whom were teenagers) in Corrientes, Argentina (17). Such social settings differ from the residential settings, of outbreaks among military recruits and prisoners, where overcrowding and proximity of beds may permit transmission (18).

Attendance at the party may have been the critical factor in
carriage of the epidemic strain among year 11 students. However, another hypothesis may account for our observations. Young adults who socialize frequently at discotheques and parties may, particularly if they smoke, be at higher risk for carriage of meningococci of all types. This increased long-term risk for disease may have a protective effect against a virulent outbreak strain
(19). In contrast, those who participate infrequently in social events such as the end-of-year party may be at higher risk. This hypothesis may explain some of the risk for first-year university students. In addition to longitudinal studies, combining the results of surveys during outbreaks may help provide a more scientific basis for the management of future outbreaks.

Dr. Fitzpatrick is a lecturer in the department of Public Health Medicine and Epidemiology, University College, Dublin, Ireland. Her areas of expertise and research interests include epidemiology of prostate cancer and infectious and chronic diseases and health services research.

Acknowledgment

The authors thank the Meningococcal Reference Unit, Manchester PHL, for further examination and serogrouping of isolates.

Address for correspondence: Roland L. Salmon, PHLS Communicable Disease Surveillance Centre (Wales), Abton House, Wedal Rd, Roath, Cardiff, CF4 3QX, United Kingdom; fax: 44-1222-521-987; e-mail: roland.salmon@phls.wales.nhs.uk

References

  1. Ramsay M, Kaczmarski E, Rush M, Mallard R, Farrington P, White J. Changing pattern of case ascertainment in meningococcal disease in England and Wales. Communicable Disease Report Review 1997;7:R49-54.
  2. PHLS Meningococcal Working Group and Public Health Medicine Environment Group. Control of meningococcal disease: guidance for consultants in communicable disease control. Communicable Disease Report Review 1995;5:R189-95.
  3. Stuart JM, Monk PN, Lewis DA, Constantine C, Kaczmarski EB, Cartwright KAV, et al. Management of clusters of meningococcal disease. Communicable Disease Report Review 1997;7:R3-5.
  4. Kaczmarski EB. Meningococcal disease in England and Wales. Communicable Disease Report Review 1997;7:R55-9.
  5. Office of Population Census and Surveys. Occupation and social class. London: The Office; 1990.
  6. Haneberg B, Tonjum T, Rodahl K, Gedde-Dahl T. Factors preceding the onset of meningoccoal disease, with special emphasis on passive smoking, stressful events, physical fitness and general symptoms of ill-health. Symposium "Causes and control of meningococcal disease," Oslo 1982.
  7. Dean AD, Dean JA, Burton JH, Dicker RC. Epi-Info, version 5. Centers for Disease Control, Atlanta 1990.
  8. Davies AL, O'Flanagan D, Salmon RL, Coleman TJ. Risk factors for Neisseria meningitidis carriage in a school during a community outbreak of meningococcal infection. Epidemiol Infect 1996;117:259-66.
  9. Riordan T. A college outbreak of group C meningococcal
infection: how widely should investigation and prophylaxis
extend? Communicable Disease Report Review 1997;7:R5-9.
 10. Gilmore A, Jones G, Barker M, Soltanpoor N, Stuart JM.
Meningococcal disease at the University of Southampton:
outbreak investigation. Epidemiol Infect 1999;123:185-92.
 11. Kristiansen B-E, Tveten Y, Jenkins A. Which contacts of
patients with meningococcal disease carry the pathogenic strain of Neisseria meningitidis? A population based study. BMJ 1998;317:621-5.
 12. Stuart JM, Cartwright KAV, Robinson PM, Noah ND. Effect of smoking on meningococcal carriage. Lancet 1989;ii:723-5.
 13. Stanwell Smith R, Stuart J, Hughes A, Robinson P, Griffin M, Cartwright K. Smoking, the environment and meningococcal disease: a case control study. Epidemiol Infect 1994;112:315-28.
 14. Cartwright KAV, Stuart JM, Jones DM, Noah ND. The Stonehouse survey: carriage of meningococci and Neisseria lactamica. Epidemiol Infect 1987;99:591-601.
 15. Zangwill KM, Schuchat A, Riedo FX, Pinner RW, Koo DT, Reeves MW, et al. School-based clusters of meningococcal disease in the United States; descriptive epidemiology and case-control analysis. JAMA 1997;277:389-95.
 16. Imrey PB, Jackson LA, Ludwinski PH, England AC, Fella GA, Fox BC, et al. Outbreak of serogroup C meningococcal disease associated with campus bar patronage. Am J Epidemiol
1996;143:624-30.
 17. Cookson ST, Corrales JL, Lotero JO, Regueira M, Binsztein N, Reeves MW, et al. Disco fever: epidemic meningococcal disease in Northeastern Argentina associated with disco patronage. J Infect Dis 1998;178:266-9.
 18. Tappero JW, Reporter R, Wenger JD, Ward BA, Reeves MW, Missbach TS, et al. Meningococcal disease in Los Angeles County, California, and among men in the county jails. N Engl J Med 1996;335:833-9.
 19. Kristiansen BE, Knapskog AB. Secondary prevention of
meningococcal disease. BMJ 1996;312:591-2.

Dispatches

Salmonellosis in the Republic of Georgia: Using
Molecular Typing to Identify the Outbreak-Causing
Strain

Alexander Sulakvelidze,* Merab Kekelidze, Durmishkhan
Turabelidze,* Shota Tsanava, Lia Tevsadze, Lamara
Devdariani, Romesh Gautom, Robert Myers, J. Glenn
Morris, Jr.,* and Paata Imnadze
*University of Maryland School of Medicine, Baltimore,
Maryland, USA; National Center for Disease Control,
Tbilisi, Republic of Georgia; Public Health
Laboratories, Department of Health, Seattle,
Washington, USA; and Maryland Department of Health and
Mental Hygiene Laboratories, Baltimore, Maryland, USA

--------------------------------------------------------

 In May 1998, three large outbreaks of
 salmonellosis, affecting 91 persons, were
 identified in the Republic of Georgia.
 Eighteen Salmonella Typhimurium strains
 were characterized by arbitrary primed
 polymerase chain reaction and pulsed-field
 gel electrophoresis; the results suggested
 that all cases were part of a single
 outbreak caused by a distinct clonal
 strain.

Salmonella species, which cause a variety of clinical
manifestations from mild gastroenteritis to septicemia,
are one of the leading causes of foodborne illness
worldwide (1-2). Approximately 50% of cases of human
disease caused by salmonellae are produced by S.
Enteritidis and S. Typhimurium (3). S. Typhimurium is
of particular concern because of the recent emergence
of a highly antibiotic-resistant strain (resistant to
ampicillin, chloramphenicol, streptomycin,
sulfonamides, and tetracycline) designated as
definitive type (DT) 104 (4). In approximately 75% of
human Salmonella cases, the bacterium is acquired from
meat, poultry, or eggs (2,3).

The Study

In May 1998, three apparently distinct outbreaks of
salmonellosis were identified in the Republic of
Georgia. Symptoms in all three included acute diarrhea
(100% of cases), severe stomach cramps (35%), nausea
and vomiting (22%), and fever (97%). The first outbreak
affected children attending a birthday party in Kojori,
a suburb near Tbilisi, Georgia. Ten of 14 children
attending the party came down with acute diarrhea and
were hospitalized within 3 days. S. Typhimurium was
isolated from stool samples of four patients.

The second outbreak, 4 days later, affected guests at a
wedding reception in the village of Asureti (40
kilometers east of Tbilisi). Fifty of approximately 100
guests had diarrhea, and 18 were hospitalized.
Salmonellosis was confirmed by the isolation of S.
Typhimurium from seven of the hospitalized patients.

The third outbreak occurred in Tbilisi approximately 14
days after the first outbreak. Thirty-one of 50 guests
at a birthday party were hospitalized for acute
diarrhea within 3 days. Salmonellosis was
culture-confirmed in 14 cases (i.e., S. Typhimurium was
isolated from 14 of the patients). All persons with
diarrhea in all three outbreaks took various doses of
antibiotics (ciprofloxacin or ceftriaxone) when they
first became ill.

All 59 hospitalized persons were interviewed to
determine a possible common source of infection. In
addition, 32 persons who had diarrhea but were not
hospitalized and 71 healthy guests were interviewed.
Eggs were implicated in the first outbreak; the eggs
were used to prepare uncooked icing for a homemade
cake. In the second outbreak, eating chicken served
during the wedding banquet was associated with illness.
In the third outbreak, chicken from a farmers' market
in Tbilisi was implicated. No food samples were
available for bacteriologic analysis.

Stool samples from the 59 hospitalized patients were
examined for Salmonella, Shigella, and Yersinia by
standard techniques. Salmonella were isolated from
stool samples of 25 (42%) of the patients; all 25
isolates were identified as S. Typhimurium strains by
the API20E test system, and 18 of them were randomly
chosen for subsequent analysis. No other pathogen was
isolated.

Arbitrary primed polymerase chain reaction (AP-PCR) was performed by using an RAPD kit (Amersham Pharmacia Biotech, Piscataway, NJ). All typing was done with primer #6 of the kit (5'- CCC GTC AGC A - 3'), which gave distinctive, reproducible patterns with three or more major bands. Bacterial DNA for AP-PCR was obtained, and amplification was performed (5). After the amplification cycles, the samples were incubated at 72C for 5 minutes and analyzed by
electrophoresis in 2% agarose gel in TAE buffer.

The rapid pulse-field gel electrophoresis Figure 1
(PFGE) procedure developed for typing Escherichia coli O157H7 strains was used for PFGE typing of the outbreak strains (6). The strains were analyzed, in separate experiments, by digesting DNA with Xba I, Avr II, and Spe I   enlarged restriction enzymes, and Xba I-digested S.
Newport strain amO1144 was used as the

Figure 1.  Pulse-field gel electrophoresis
patterns of Xba I-digested Salmonella
Typhimurium strains.... (Figures not available
in ascii)

Figure 2. Pulse-field gel electrophoresis patters of AVR II-
digested DNA of Salmonella Typhimurium strains... (Figures
not available in ascii)

reference strain in all experiments (Figure 1). A distinct pattern was observed in all 18 strains when they were analyzed by AP-PCR (data  DNA of not shown), which suggests close genetic relatedness. This finding was confirmed by PFGE  typing, i.e., Xba I-, Avr II-; Spe I-macrorestriction patterns generated by PFGE showed that all strains tested had an identical PFGE pattern. The pattern was distinct from
those obtained for 18 other S. Typhimurium 
strains isolated in the Republic of Georgia at
about the same time, which were grouped into
five PFGE types distinct from that of the 
outbreak-causing strain.  The national molecular subtyping network, PulseNet, includes a database of the patterns of E. coli O157 and Salmonella group B and D strains isolated in the United States. We screened local databases of two public health laboratories in Washington and Maryland that
participate in PulseNet, and we used the 
Internet to compare the PFGE pattern of the
Georgian outbreak strain with Salmonella
patterns obtained in these two laboratories. At
least three strains among >300 S. Typhimurium
isolates (grouped into approximately 50 PFGE types) in the two databases were almost identical to the Georgian isolate. However, the PFGE patterns of the strains differed slightly when their DNA was digested with Avr II before PFGE. The patterns obtained by AP-PCR and PFGE (Xba I- and Spe I-digests) were almost indistinguishable; however, when DNA of the same strains was analyzed by PFGE after digestion with Avr II, differences were detected. For example, the PFGE pattern of strain 00354 (a Seattle isolate) had a large fragment not present in the Georgian outbreak strain and had lost one small fragment (Figure 2, lane 5). In addition, the PFGE pattern of strain 01587 (also from Seattle) lacked one fragment present in the Georgian outbreak strain and had
two other small fragments (Figure 2, lane 6). These differences may result from a single genetic event in the bacterial DNA and are associated with a spontaneous point mutation resulting in either creation or loss of a restriction site. Therefore, the Seattle and Maryland isolates were classified as closely related (7,8) to the
Georgian outbreak strain. In Washington, the strain was associated with severe diarrhea like that of the Georgian patients, which suggests that the strain was strongly virulent. (Clinical data for the Maryland isolate were not available.) Detailed evaluation of the pathogenic potential of the outbreak strain (and the closely related
U.S. isolates) will require determination of the possible links between unusually severe cases of salmonellosis and isolation of S. Typhimurium strains having closely related PFGE patterns, and testing the strain for virulence in laboratory animals.

The outbreak-causing S. Typhimurium strain was resistant to
ampicillin, chloramphenicol, streptomycin, and tetracycline, but was susceptible to cephalosporins and sulfonamides. Two of the above closely related strains (00354 and 9294-99) had an antibiotic-susceptibility pattern similar to that of the Georgian outbreak strain. The only difference in the pattern for the third closely related strain (01587) was that it was susceptible to chloramphenicol.

Conclusions

Epidemiologically, the outbreaks had no obvious connection. Patients were hospitalized in two different hospitals in two different cities and were not associated with more than one outbreak. In addition, no common source of infection could be identified. Thus, the three outbreaks were initially thought to be separate. However, the observation that they occurred within a short period (2 weeks between the first and third outbreaks), were caused by the same serotype of Salmonella, were associated with severe diarrhea, and
were limited geographically to a 80-kilometer radius raised the possibility that the outbreaks may have been related. Therefore, we used molecular typing techniques to characterize the Salmonella strains isolated from the patients in each outbreak. Our observations suggest that what appeared to be three distinct outbreaks of salmonellosis were, in reality, parts of one large
outbreak caused by a distinct clonal strain of S. Typhimurium. The common source of infection and the transmission route for the outbreak-causing Salmonella strain are not known.

The clinical diagnosis of salmonellosis was confirmed by isolating S. Typhimurium from 25 (42%) of the hospitalized patients. All the patients had treated themselves with ciprofloxacin and ceftriaxone (readily available without prescription in Georgia) before hospitalization, which may have contributed to our inability to isolate Salmonella (or other potential pathogens) from most of them. Fifty-nine (65%) of 91 persons diagnosed as having salmonellosis had
diarrhea severe enough to require hospitalization. The
hospitalization rate of these patients was approximately seven times higher than the usual hospitalization rate (<10%) for Salmonella cases in Georgia and approximately three times higher than the average hospitalization rate reported for Salmonella patients in the United States (9). Although some persons with diarrhea may have requested hospitalization after hearing that other ill guests were
hospitalized, the clinical reports indicate that the high
hospitalization rate reflects a severe manifestation of the disease. Since the AP-PCR and PFGE typing results suggested that a single clone was the causative agent in all cases, the question of its possibly increased virulence arose. It has recently been reported (J.G. Morris, et al., unpub. data) that some Salmonella serotypes/strains cause more severe illness. The recent emergence of S. Typhimurium DT104 strains having increased virulence (4), in addition to being multidrug resistant, also highlights the possibility of supervirulent strains emerging worldwide.

S. Typhimurium, in contrast to S. Enteritidis, which is a highly clonal organism (5), is a fairly diverse serotype (10). Therefore, detection of closely related S. Typhimurium strains in geographically distinct loci (the Republic of Georgia, and the East Coast and West Coast of the United States) may signal worldwide spread or emergence of closely related clonal groups of Salmonella having increased virulence. This possibility may be confirmed by worldwide (or nationwide) standardization of molecular typing
protocols and further strengthening of data-sharing capabilities between laboratories involved in the molecular typing of pathogenic microorganisms. An example demonstrating the potential value of such a database is our finding, during screening of the two participating
PulseNet laboratories, that two seemingly unrelated strains (Seattle isolate 00354 and Maryland strain 9294-99) were clonal. As described above, this observation was confirmed by both antibiotic-susceptibility testing and side-by-side molecular typing of the strains in question. An  epidemiologic link between the two isolates is being investigated.

Acknowledgments

The authors thank Arnold Kreger for his helpful discussions and editorial comments and Lia Tsintsadze, Judith Johnson, and Sadaf Qaiyumi for their help in determining the antibiotic susceptibility and resistance of selected Salmonella strains used in this study. 

The study was funded in part by the International Training and Research in Emerging Infectious Diseases grant from the Fogarty International Center, National Institutes of Health.

Dr. Sulakvelidze is assistant professor of Medicine at the
University of Maryland School of Medicine. His research interest include molecular epidemiology and the pathogenesis of diseases caused by bacterial enteric pathogens and toxins. He is also involved in studies evaluating the utility of bacteriophages in the prophylaxis and treatment of diseases caused by various multidrug-resistant bacteria.

Address for correspondence: Alexander Sulakvelidze, Division of Hospital Epidemiology, University of Maryland School of Medicine, MSTF Bldg., Room 9-34, 10 S. Pine Street, Baltimore, Maryland 21201, USA; fax: 410-706-4581; e-mail: asulakve@medicine.umaryland.edu.

References

  1. Goldberg MB, Rubin RH. The spectrum of Salmonella infection. Infect Dis Clin N Am 1988;2:571-98.
  2. Hook EW. Salmonella species (including typhoid fever). In: Mandell GL, Douglas RG, Bennett JE, editors. Principles and practice of infectious diseases. New York: Churchill
Livingstone; 1990.
  3. Centers for Disease Control and Prevention. Salmonella
surveillance, annual summary, 1993-1995. Atlanta (GA): The
Centers; 1996.
  4. Centers for Disease Control and Prevention. Multidrug resistant Salmonella serotype typhimuriumUnited States. MMWR Morb Mortal Wkly Rep 1997;46:308-10.
  5. Lin AW, Usera MA, Barrett TJ, Goldsby RA. Application of random amplified polymorphic DNA analysis to differentiate strains of Salmonella enteritidis. J Clin Microbiol 1996;34:870-6.
  6. Gautom RK. Rapid pulsed-field gel electrophoresis protocol for typing of Escherichia coli O157:H7 and other gram-negative organisms in one day. J Clin Microbiol 1997;11:2997-80.
  7. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 1995;33:2233-9.
  8. Olive MD, Bean P. Principles and applications of methods for DNA-based typing of microbial organisms. J Clin Microbiol 1999;6:1661-9.
  9. Mead PS, Slutsker L, Dietz V, McCaig LF, Bresee JS, Shapiro C, et al. Food-related illness and death in the United States. Emerg Infect Dis 1999;5:607-25.
 10. Nastasi A, Mammina C, Villafrate MR. Epidemiology of Salmonella typhimurium: ribosomal DNA analysis of strains from human and animal sources. Epidemiol Infect 1993;3:553-65.

Dispatches

First Report of Q Fever in Oman

Euan M. Scrimgeour,* William J. Johnston,* Said H.S. Al
Dhahry,* Hussein S. El-Khatim, Valliath John, and
Mohamed Musa
*Sultan Qaboos University, Al-Khod, Sultanate of Oman;
Sultan Qaboos University Hospital, Al-Khod, Sultanate
of Oman; Royal Hospital, Muscat, Sultanate of Oman

--------------------------------------------------------

 Although serologic evidence suggests the
 presence of Q fever in humans and animals
 in Saudi Arabia and the United Arab
 Emirates, acute Q fever has not been
 reported on the Arabian Peninsula. We
 report the first two cases of acute Q fever
 in Oman.

Acute Q fever has not been reported on the Arabian
Peninsula; however, serologic studies have documented
Coxiella burnetii in eastern Saudi Arabia. In Riyadh,
in the late 1960s, 8.4% of residents >5 years of age
were seropositive (1). Veterinary studies in Saudi
Arabia have shown that Q fever occurs in livestock (2)
and in Arabian oryx (Oryx leucoryx) at the National
Wildlife Research Centre at Taif (3). In Abu Dhabi,
United Arab Emirates (UAE), the disease was reported in
racing camels (4).

Acute Q fever is usually a nonspecific febrile illness,
often with atypical pneumonia or transient hepatitis.
Seroconversion without symptoms is common, especially
in children (5). As a rule, it is self-limiting and
resolves without treatment, but some untreated cases
may progress to chronic Q fever (endocarditis,
granulomatous hepatitis, osteomyelitis, interstitial
pulmonary fibrosis) (6). Pericarditis is rare (7),
mostly reported from France (6,8) and Spain (9), and
sporadically worldwide (10).

In acute Q fever, diagnosis is supported by a fourfold
increase in antibody titers to C. burnetii in acute-
and convalescent-phase serum samples by
complement-fixation test (CFT) or indirect
immunofluorescence assay (IF). The IF is more sensitive
and specific than CFT in chronic Q fever. In the
National Reference Center in France, the diagnosis of
acute infection is supported by IgG phase II > 1:200,
IgM phase II > 1:50; and of chronic disease, IgG phase
I > 1:800 (11). Diagnosis can also be made by
identifying C. burnetii in tissue culture or its
antigens by polymerase chain reaction (PCR) in biopsy
specimens. We report the first two human cases of Q
fever in Oman.

Case 1

A 45-year-old Omani woman was
hospitalized on December 13, 1997,
with a 4-week history of fever and
productive cough. The patient's
temperature was 39C, she was
jaundiced, and she had signs of
right lower lobe pneumonia, which
was confirmed by X-ray (Figure 1).
Hemoglobin was 9.5 g/dL, white
blood cells 6.6 x 109/L
(neutrophils 76%, lymphocytes
14.2%), platelets 317 x 109/L, and erythrocyte
sedimentation rate 101 mm/hour. An electrocardiogram
showed nonspecific abnormalities, but an echocardiogram
revealed a small pericardial effusion. Liver function
tests showed normal serum bilirubin, serum aspartate
aminotransferase 111 /L (normal range 14-42 /L),
serum alkaline phosphatase 256 /L (normal range 3-210
/L), and serum albumen 26 g/L. Community-acquired
pneumonia, with pericarditis and hepatitis, was
diagnosed, and the patient was treated with intravenous
erythromycin, 500 mg 4 times a day.

Since the patient lived on a farm with cattle and
goats, Q fever was considered, although C. burnetii
infection had never been reported in Oman. Sera were
sent to England for Q-fever antibody tests, which were
not available locally. The patient became afebrile,
signs of pneumonia resolved, and she was discharged on
December 31, 1997 (before serologic results were
received).

On January 12, 1998, the patient had signs of
constrictive pericarditis and small bilateral pleural
effusions. The results of CFT for Q fever were total
antibody titers to phase-II antigen > 1:80 (threshold >
1:10). Phase-I antigen and immunoglobulin (Ig) M tests
were not performed, but the results indicated recent
Q-fever infection, so doxycycline (100 mg twice a day)
was prescribed. Repeat serologic tests (January 12,
1998) revealed total CFT antibody titers to phase-I and
phase-II IgG antigens > 1:40 (threshold > 1:10); and
IgM and IgA < 1:10 (threshold > 1:10). These results
were interpreted as evidence that C. burnetii infection
was the probable cause of chronic pericarditis.

Figure 1. Q-fever pneumonia. (Figures not available in 
ascii)

Echocardiography revealed a grossly thickened
pericardium with abnormal septal motion
encroaching on the right ventricular cavity in
diastole, and the CT scan confirmed a diffusely
thickened pericardium (Figure 2). Cardiac
catheterization revealed normal left
ventricular systolic function, but the central
venous pressure was 26 cm. On April 5, at
pericardiectomy, the pericardium was 1 cm thick
and densely adherent to the heart.
Histopathologic examination revealed
granulomatous pericarditis without evidence of
tuberculosis (Figure 3). The patient recovered
well after surgery.

Figure 2. CT scan of heart, showing
thickened pericardium. (Figures not available in ascii)

Figure 3. Histologic results of pericardial biopsy, which
show granulomatous pericarditis. (Figures not available 
in ascii)

Case 2

A 50-year-old Omani man from a rural area was
hospitalized on June 2, 1999, with a 3-week
history of fever of unknown cause, not
responding to cefuroxime. Hematologic
examination revealed hemoglobin 13 g/dl, white
blood cells 6.7 x 109/L (neutrophils 53%,
lymphocytes 37%, monocytes 9%), platelets 195 x
109/L, and erythrocyte sedimentation rate 2
mm/hour. Signs of bilateral basal pneumonia
developed, and Q-fever serologic tests were
positive: total antibody titers to phase-II
antigen by IF > 1:1280 (threshold > 1:80), and
IgM titer > 1:320 (threshold > 1:20). After
treatment with doxycycline, the patient's
symptoms resolved. Repeated serologic results
were total IF antibody titer 1:2560 and IgM titer 1:160, evidence of the convalescent phase of infection.

Conclusions

In most countries on the Arabian Peninsula, including Oman, most people live in rural areas and are exposed to livestock. In countries where it is endemic, Q fever usually occurs sporadically and may be underdiagnosed and underreported (6,8). In Zimbabwe, where acute Q fever had never been reported, a recent community survey revealed C. burnetii antibodies in 37% of humans, 39% of cattle, and 10% of goats (12).

In Case 1, pericardial involvement in the acute stage of Q fever likely progressed to chronic constrictive pericarditis because diagnosis and appropriate treatment were delayed. For acute Q fever, the treatment of choice is doxycycline or orfloxacin, alone or in combination with rifampicin, for 21 days. For treatment of chronic Q fever (e.g., with endocarditis), doxycycline and a quinolone for a minimum of 3 months have been advocated (5-7). 

Q fever may be widely endemic but undiagnosed in livestock and domestic animals in Oman. Similarly, the evidence suggests that acute Q fever is prevalent, but undiagnosed (or unreported) in other Arabian Peninsula countries, including Saudi Arabia. Diagnostic serologic tests are not yet available in Oman but will be introduced soon, which will permit us to determine prevalence and distribution
of C. burnetii in humans and in livestock.

Acknowledgments

The authors thank Didier Raoult, Centre for Rickettsial Studies, University of Marseilles, for PCR testing of the biopsy specimen. The Marcel Merieux Laboratory, Lyon, France, conducted the immunofluorescence tests, and Elizabeth Richens provided sera from Case 1 for immunologic studies.

Dr. Scrimgeour is an associate professor in Infectious and Tropical Diseases, and heads the Communicable Diseases unit at the  Sultan Qaboos University. His research interests include epidemiology of infectious and tropical diseases and parasitic disease of the central nervous system.

Address for correspondence: Euan M. Scrimgeour, Department of Medicine, Sultan Qaboos University, P.O. Box 35, Al-Khod, 123, Sultanate of Oman; fax: 968-513-419; e-mail: scrim@squ.edu.om.

References

  1. Lippe M, Seebastiani A, El-Mutabakani H. Investigation of serum antibodies to rheovirus, adenovirus, and Coxiella burnetii in a group of inhabitants of Riyadh, Saudi Arabia. Arch Ital Sc Med Trop 1968;49:129-36.
  2. Gelpi AP. Q fever in Saudi Arabia. Am J Trop Med Hyg
1966;15:784-98. 
  3. Greth A, Calvez D, Vassart M, Lefevre PC. Serological survey for bovine bacterial pathogens in captive Arabian oryx (Oryx leuocoryx Pallas, 1776). Revue Scientifique et Technique Office International des Epizooties (France). 1992:11:1163-8.
  4. Afzal M, Sakkir M. Survey of antibodies to various infectious disease agents in racing camels in Abu Dhabi, UAE. Rev Sci Tech 1994;13:787-92.
  5. Dupuis G, Peter O, Pedroni D. Aspects cliniques observes lors d'une epidemie de 415 cas de fievre Q. Schweiz Med Wschr 1985;115:814-8.
  6. Marrie TJ. Coxiella burnetii (Q fever). In: Mandell GL, Bennett JE, Dolin R, editors). Principles and practice of infectious diseases. New York: Churchill Livingstone Inc.; 1995. p. 1727-35.
  7. Pinto JR. Pleuropericardial lesions in Q fever. BMJ
1973;71;2:1542.
  8. Brouqui P, Tissot-Dupont H, Drancourt M, Berland Y, Etienne J, Leport C, et al. Chronic Q fever: ninety-two cases from France including 27 cases without endocarditis. Arch Intern Med 1993;153:642-8.
  9. Diaz Morant V, Mateo Sanchez JI, Lara Fernandez A, Cabello Rueda F. Pleuropericarditis caused by Q fever [in Spanish] An Med Interna 1995;12:568-9.
 10. Beamon MH, Hung J. Pericarditis associated with tick-borne Q fever. Aus NZ J Med 1989;19:254-6.
 11. Tissot-Dupont H, Thirion X, Raoult D. Q fever serology cut-off determination for microimmunofluorescence. Clin Diagn Lab Immunol 1994;1:189-96.
 12. Kelly PJ, Matthewman LA, Mason PR, Raoult D. Q fever in
Zimbabwe. S Afr Med J 1993;83:21-5.

Another Dimension

The Philosophic Origins of Science and the Evolution of
the Two Cultures1

Ntinos C. Myrianthopoulos

"A small but growing number of American philosophers
have opened private practices as `philosopher
practitioners' offering a therapy based on the idea
that solutions to many personal, moral, and ethical
problems can be found not in psychotherapy or Prozac
but deep within the 2,500-year-old body of
philosophical discourse."

--------------------------------------------------------

This quotation from the New York Times of March 8,
1998, may have been startling to some and amusing to
others. The New Yorker used it as a preamble to a
humorous article but not to anyone, myself included,
who has enjoyed the pleasure of delving into the
history of philosophy and who appreciates its relevance
to the scientific process. What a splendid opportunity,
then, to explore the philosophic origins of science and
its long and fruitful legacy.

The quest for knowledge is an old preoccupation with
roots in prehistory, starting with Adam and Eve, who
did not go about it scientifically and you know what
happened to them. Even so, ancient humans continued the
quest to understand and study the nature around them:
the trees and the animals, the bearing of children, the
heavenly bodies; that is, the natural phenomena that
today we refer to as the natural sciences.

Among the old civilizations, the Babylonians and
Egyptians contributed considerably to these sciences,
practiced primitive medicine and surgery, and collected
facts about natural history and biology. It was,
however, left to the Greeks to enlarge the scope of
these collections and formulate from the facts a
unified concept of nature and the laws that govern it.

The oldest Greek thinkers were natural philosophers,
and it was much later that ethical issues and other
problems found a place in Greek thought. Practically
all philosophers were teachers; many had their own
schools, had to teach several subjects (rhetoric,
ethics, poetics, astronomy, physics, biology), and
wrote treatises on these subjects, which were surely
sometimes used as textbooks. A few of these
philosophers were also poets and wrote their own books
in verse; Empedocles, for example, wrote two treatises,
"On Nature" and "Purifications," in dactylic hexameter.
I shall mention only one of these early philosophers,
Democritos (approximately 450 BC), for two reasons. The
first is that he was the very antithesis of the usual
image of a brooding philosopher; he was known by
several nicknames, one being "the laughing philosopher"
because he was good humored and jolly all the time
(gelousivs - note: text in Greek characters, please refer
to html version for accuracy). In fact, one of his noteworthy treatises is entitled "On Cheerfulness." The other reason is that to most of us Democritos is known mainly as the father of the atomic theory, which is not quite the case. It was his teacher, Leukippos, who first conceived and formulated the atomic theory, which Democritos
immediately espoused and refined. He wrote on almost
every field known at the time: physics, psychology,
logic, astronomy, the senses, the mind, music, poetics.
Aristotle thought highly of Democritos and refers to
his work frequently, particularly in his various
biological writings.

Aristotle was certainly the greatest of this genre of
philosophers and is justly considered one of the
greatest thinkers of all time. He was born in 384 B.C.
in Stageira, Macedonia, where his father was the
physician to the royal court. At age 17, he was sent to
Athens to study with Plato in the Academy, where later
he also taught. He spent 3 years in Asia Minor in the
court of his former student Hermeias, who gave him his
niece in marriage. He was then appointed by King Philip
of Macedon to be the tutor of his impetuous and
brilliant teenaged son Alexander, and after 3 years, he
moved back to Athens. Now a well-to-do man under the
protection of Alexander, he founded his own school, the
Lyceum (named after the nearby temple of Apollo
Lycaeus). The Lyceum served as the prototype of a
learned educational institution throughout the world.
Here Aristotle taught while taking walks with his
students and collaborators and wrote an incredible
amount on various and different subjects, including
logic, physics, ethics, art poetry, politics,
economics, psychology, and biology. He retired to
Chalkis, Euboea, where he died at age 62.

To the biologist, Aristotle's work on the "Generation
of the Animals" is of special interest because it is
the first systematic treatise on animal reproduction
and embryology, taxonomy, and evolution. The
"Generation of the Animals" is the culmination of
Aristotle's zoological works that comprise 10 volumes
and include "On the History of Animals," "On the Parts
of Animals," and "On the Soul." I shall only touch upon
those areas in which he made lasting contributions in
the development of the biological sciences that have
come down to us.

Aristotle repeatedly pointed out that his predecessors'
work and conclusions were often marred by insufficient
observation. He himself, after a remarkable analysis of
the reproduction of bees, states that he cannot arrive
at certain conclusions because "the facts have not yet
been sufficiently ascertained. And if at any future
time they are ascertained, then credence must be given
to the direct evidence rather than to the theories; and
to the theories also, provided that the results which
they show agree with what is observed." This, indeed,
is the principle upon which his work is based. It is
also the definition of the scientific method, which was
later broadened in scope, especially by Bacon, and by
and large constitutes the basis of the scientific
method we practice today. Note the subtle yet critical
point: Aristotle does not say "the results prove the
theory," but "the results agree with the observations."
Today, we take this reasoning for granted, that science
proceeds and progresses not by proving hypotheses, but
by disproving them. If the observations do not agree
with a hypothesis, we shelve it; if it does agree with
a high enough level of certainty and consistent
repetition of the results, we accept it, but we can
never prove it.

Up to the time of Aristotle, there had been no serious
attempts at classification of animals. Thus, his
classification was based almost entirely on his own
observations. For animals not found in Greece, he
referred to credible observations by others, e.g.,
Herodotos. In this area also, Aristotle made very
important contributions by characterizing and
differentiating among a number of systematic
categories. In his own words, "Animals may be
characterized according to their way of living, their
actions, their habits, and their bodily parts." The
most important criterion is certainly the parts of the
animals, both external and internal: organs of
movement, respiration, sense, blood circulation. By
combining various qualities, he defined and
characterized the groups. Aristotle's two major
categories are blooded animals (he refers to red blood
only) and bloodless animals.

Under blooded animals: humans, viviparous quadrupeds,
oviparous quadrupeds, and footless animals (reptiles,
amphibians), birds, and fishes.

Under bloodless animals: malacostraca (soft-shelled,
crustacea); malakia (soft, without shell, cephalopods);
entoma (insected animals, insects); and ostrakoderma
(shell-skinned, testacea). These categories and
nomenclature are still used today.

Aristotle also classified animals according to their
mode of reproduction, but the most important part of
his classification is the final two categories, the
genus (genos - note: text in Greek; refer to html version
for accuracy) and the species (eidos - note: text in Greek; refer to html version for accuracy), the latter
referring to the individual animal form: horse, dog,
lion. This is a farsighted classification, and though
it cannot be compared with the Linnaean with its
manifold categories, it is certainly a pioneering
achievement.

In his work on the reproduction of animals, Aristotle
differentiates sexual and asexual reproduction. In
sexual reproduction, male and female contribute
equally, and in his thorough investigation of the
development of animals from egg and embryo, Aristotle
points out the phenomenon that we know today as
"ontogeny recapitulates phylogeny." He disagreed
sharply with the opinion of earlier philosophers that
the seed is derived from all parts of the body and thus
gives rise to similar individuals. On the contrary, he
asserts that the seed goes to all parts of the body to
form an individual, an explanation shown to be correct
2,000 years later. In addition, during embryonic
development, there is a specific movement or substance
in each part of the body which brings about its
development as a specific part of the embryo. Today, we
call such substances organizers. Aristotle also
recognized congenital malformations as imperfect
developmental events in the embryo due to various
causes, one being some irregularity in the seminal
fluid. He correctly understood the functions of the
placenta and the umbilical cord and was an ardent
supporter of epigenesis. He made his observations in
several animals, and it can be said that he introduced
the comparative method to embryology.

Aristotle has been called the first evolutionist. His
theory of evolution lies not only in the sphere of
discovery, but also in his system of thought, embracing
all phenomena of life. Here we find enunciated for the
first time a truly complete theory of evolution,
subject to natural laws and progressing from the lower
to the higher forms of being. Although partly based on
metaphysical speculation, the theory has proven fertile
ground for future biologists.

Aristotle constantly compares nature and the products
of nature with art and the products of art. Like
nature, the artist or craftsman works to produce a
finished product. Like the artist, nature uses
instruments charged with specific modulations to bring
these products to fulfillment. The most typical of
these products of nature are, of course, living
creatures. Nature aims always to produce a finality in
the sense of a completely formed individual and that is
the Final Cause in each case. "There is," Aristotle
says, "more beauty and purpose found in the works of
nature than in those of art." And who can disagree?

Although Aristotle was not the last of the era in which
the study of nature was in the province of philosophy,
by the time of his death, there were already signs of
specialization, that is, philosophers began to be
concerned mainly with ethics and metaphysics, leaving
the other subjects to those more informed about them.
This trend continued through the Hellenistic and Roman
times. Later, with the increase of knowledge and ease
in its dissemination, the establishment of libraries,
and invention of the printing press, the graduates of
schools of higher education came to be recognized
either as scientists, biological or physical, or as
artists, poets, writers, painters, or musicians and
received different credentials, in our time Bachelor of
Arts or Bachelor of Science. In spite of the widening
schism, the philosophic origin of the sciences and the
arts is acknowledged and maintained today in the award
of the highest academic degree, that of Doctor of
Philosophy.

In his controversial Rede lecture presented in
Cambridge in 1959, C.P. Snow (1905-1980), the British
intellectual, first used the phrase "The Two Cultures"
to describe the world of the sciences and the world of
the arts, which had become culturally isolated. Sir
Charles Percy Snow was himself a distinguished
physicist, who during World War II made significant
contributions to the British and Allied war effort and
for his services was elevated to the peerage. He was
also an excellent novelist. His magnum opus, "Strangers
and Brothers," comprises eleven volumes written over 30
years, in which he recounts a saga of lives, events,
and the passage of time, both for individuals and for
English society as a whole. As Snow himself described
his existence, "There have been plenty of days when I
have spent the working hours with scientists and then
gone off at night with some literary colleagues. I mean
that literally. It was through living with these groups
and much more, I think, through moving regularly from
one to the other and back again that I got occupied
with the problem of what, long before I put it on
paper, I christened to myself as `the two cultures.'
For constantly I felt I was moving among two
groupscomparable in intelligence, identical in race,
not grossly different in social origin, earning about
the same incomes, who have almost ceased to communicate
at all, who in intellectual, moral and psychological
climate had so little in common, that instead of going
from Burlington House or South Kensington to Chelsea,
one might have crossed an ocean."

Snow, of course, was addressing a situation prevalent
in England and Europe in general in the late 1950s, but
at that time, conditions on this side of the Atlantic
may have been a little better. Nevertheless, the two
cultures still exist and combine infrequently in rare
and exceptional individuals.

Every era has had such exceptional individuals. The
Renaissance produced a sprinkling of them, the towering
and awesome figure of that era being Leonardo da Vinci
(1452-1519), the Italian painter par excellence, and
sculptor, but also architect, engineer, musician,
inventor, anatomist, physiologist, geologist, botanist,
and everything that you can imagine, and many things
you cannot. Indeed, a thumbing through his voluminous
diaries, originally written in "looking-glass writing,"
will find him discussing, in addition to the subjects
that I have already referred to, philosophy, geography,
flight physics, mathematics, warfare, sailing, and
scores of prophesies on scores of subjects. While in
the long service of Ludovic Sforza in Milan, in
addition to art, he performed various functions,
including tutoring; designing churches, scientific
instruments, and war machines; and organizing
spectacular pageants. His biological writings
constitute about 20% of his total output. They
encompass discourses on anatomy, physiology, medicine,
optics, and acoustics and are illustrated with
exquisite drawings, which he did from his own
observations and dissections. He dissected more than 30
bodies of men and women of all ages and made drawings
and casts of anatomical structures, most of them at the
hospital of Santa Maria Nuova in Florence and at the
University of Pavia, where his friend Antonio della
Torre held the chair of anatomy. It is impossible to
discuss this work at length, but I would like to quote
his ruminating on the nervous system: "The frog
instantly dies when the spinal cord is pierced.
Previous to this it lived without head, without heart,
or any bowels or intestines or skin. And here,
therefore, it would seem lies the foundation of
movement and life."

Leonardo's counterpart in the 20th century is
undoubtedly Albert Schweitzer (1875-1965), the Alsatian
physician, philosopher, musician, theologian, and great
humanitarian. Those of our generation who recognize his
name know him as the doctor who founded a hospital in
Equatorial Africa. Actually, he came to medicine rather
late, and by that time, he was an accomplished musician
and organist and had written a monumental biography of
J.S. Bach and an analysis of his work, to be followed
later with his edition of Bach's organ music, all of
which had made him the outstanding authority on Bach.
He also attained the rank of professor of theology and
director of the Strasbourg Seminary. But he was
resolved to become a doctor in French Equatorial
Africa, and at age 30 he began his medical studies,
meanwhile continuing his other activities. In his
mid-30s, he went to Lambarene in Gabon, where he
established a primitive hospital, which eventually grew
into an extensive medical facility, including a
leprosarium. Except for a long period during World War
I when he was a prisoner of war, he remained there most
of the time to minister to the poor and the sick, with
the help of a small, dedicated staff. He traveled
frequently to the continent to raise money for the
hospital, and once to the United States, in 1949, to
address the Goethe Festival in Colorado. On these
trips, he was honored in many countries for his work as
a scientist, his artistry as an organist and musical
scholar, and his contributions as a philosopher. In a
three-volume series entitled Philosophy of
Civilization, Schweitzer developed his own philosophy,
which is summed up in the term Reverence for Life, a
universal code of ethics that requires respect for the
lives of all other beings. His theological writings
include "The Quest of the Historical Jesus," in which
he expresses a dissatisfaction with the way that the
gospels treat the facts of the life of Jesus, and "The
Mysticism of Paul the Apostle." He described his early
years at Lambarene in "On the Edge of the Primeval
Forest," and in the mid-30s, he wrote his
autobiography, "Out of My Life and Thought." Schweitzer
was awarded the Nobel Prize for Peace in 1952.

Norman Cousins, the late editor of the now sadly
defunct Saturday Review, visited Schweitzer and, in a
book about this visit, describes this scene of life in
Lambarene, the customary singing of a hymn after
dinner: "There was a piano in the dining room, old,
dilapidated, unable to hold its tune because of the
heat and the moisture. When I saw Dr. Schweitzer sit
down at the piano and prop up the hymnbook, I winced.
Here was one of history's greatest interpreters of
Bach, a man who could fill any concert hall in the
world. The best grand piano made would not be too good
for him. But he was now about to play a dilapidated
upright virtually beyond repair. And he went at it
easily and with the dignity that never leaves him."

The Aristotles, the da Vincis, the Goethes, the
Jeffersons, the Schweitzers are, indeed, rare
individuals, true descendants of those early
philosophers of the ancient world. Their works and
their writings had, and continue to have, profound
influence on life and civilization worldwide and are
kthma es ae, possessions forever. We admire them,
stand in awe of them, but they are in a sense remote to
us, far beyond us. It is the work, the writings and
contributions of individuals in whom the elements of
the two cultures are combined in a more modest scale
that are understandable and accessible to us, and they
are the ones that influence us in our occupations and
interests and enrich our personal lives.

Some of these gifted people have attained a measure of
fame, and we can read about them in any encyclopedia or
good dictionary; most of them remain unknown, unless we
come upon them and their work by accident or
acquaintance. One of these, unknown even among
biologists, is the British naturalist Langdon W. Smith,
who did some excellent biological research and also
wrote exquisite poetry. Smith was born in Scotland in
1877 and came to the United States when he was 14.
Practically nothing is known about his education,
except that in his early 20s he was engaged by the
Museum of Natural History in New York to do research
and was often invited by scientific societies to
lecture. He also wrote articles on scientific subjects
for newspapers. He wrote a particularly beautiful poem
about evolution titled "A Tadpole and a Fish." A friend
of his found this poem, which Smith had carelessly laid
aside, and recognized it as something exceptional. He
prevailed upon Smith to submit the poem to some of the
best papers for an opinion. The first to examine the
poem was the editor of the New York Herald, who gave
Smith a check for $500, a considerable sum in those
times, for the right to publish it.

Smith became ill and returned to England, where he died
some months later of tuberculosis. The poem, which was
later published under the title "Evolution" in 1909 and
was included in anthologies published by the
Haldeman-Julius company of Girard, Kansas, in 1922 and
1924, makes a felicitous conclusion to this essay: like
the essay, but in a grand context, it takes us back to
our roots:

	A Tadpole and a Fish
		Langdon Smith

      I

     When you were a tadpole and I was a fish,
     In the Paleozoic time,
     And side by side in the ebbing tide
     We sprawled through the ooze and slime,
     Or skittered with many a caudal flip
     Through the depths of the Cambrian fen,
     My heart was rife with the joy of life
     For I loved you even then.

     II

     Mindless we lived and mindless we loved,
     And mindless at last we died;
     And deep in a rift of the Caradoc drift
     We slumbered side by side.
     The world turned on in the lathe of time,
     The hot land heaved amain,
     Till we caught our breath from the womb of death,
     And crept into light again.

     III

     We were Amphibians, scaled and tailed
     And drab as a dead man's hand;
     We coiled at ease 'neath the dripping trees,
     Or trailed through the mud and the sand,
     Croaking and blind with our three-clawed feet
     Writing a language dumb,
     With never a spark in the empty dark
     To hint at a life to come.

     IV

     Yet happy we lived, and happy we loved,
     And happy we died once more;
     Our forms were rolled in the clinging mold
     Of a Neocomian shore.
     The eons came, and the eons fled,
     And the sleep that wrapped us fast
     Was riven away in a newer day,
     And the night of death was past.

     V

     Then light and swift through the jungle trees
     We swung in our airy flights,
     Or breathed in the balms of the fronded palms,
     In the hush of the moonless nights.
     And oh! What beautiful years were these,
     When our hearts clung each to each;
     When life was filled, and our senses thrilled
     In the first faint dawn of speech.

     VI

     Thus life by life, and love by love,
     We passed through the cycles strange,
     And breath by breath, and death by death,
     We followed the chain of change.
     Till there came a time in the law of life
     When over the nursing sod
     The shadows broke, and the soul awoke
     In a strange, dim dream of God.

     VII

     I was thewed like an Auroch bull,
     And tusked like the great Cave Bear;
     And you, my sweet, from head to feet,
     Were gowned in your glorious hair.
     Deep in the gloom of a fireless cave,
     When the night fell o'er the plain,
     And the moon hung red o'er the river bed,
     We mumbled the bones of the slain.

     VIII

     I flaked a flint to a cutting edge,
     And shaped it with brutish craft;
     I broke a shank from a woodland dank,
     And fitted it, head and haft.
     Then I hid me close to the ready tarn,
     Where the Mammoth came to drink;
     Through brawn and bone I drave the stone,
     And slew him upon the brink.

     IX

     Loud I howled through the moonlight wastes,
     Loud answered our kith and kin;
     From west and east to the crimson feast
     The clan came trooping in.
     O'er joint and gristle and padded hoof,
     We fought and clawed and tore,
     And cheek by jowl, with many a growl,
     We talked the marvel o'er.

     X

     I carved that fight on a reindeer bone,
     With rude and hairy hand,
     I pictured his fall on the cavern wall
     That men might understand.
     For we lived by blood, and the right of might,
     Ere human laws were drawn,
     And the Age of Sin did not begin
     Till our brutal tusks were gone.

     XI

     And that was a million years ago,
     In a time that no man knows;
     Yet here tonight in the mellow light,
     We sit in Delmonico's;
     Your eyes are as deep as the Devon springs,
     Your hair is as dark as jet,
     Your years are few, your life is new,
     Your soul untried; and yet

     XII

     Our trail is on the Kimmeridge clay,
     And the scarp of the Purbeck flags,
     We have left our bones in the Bagshot stones,
     And deep in the Coraline crags;
     Our love is old, our lives are old,
     And death shall come amain;
     Should it come today, what man may say
     We shall not live again?

     XIII

     God wrought our souls from the Tremadoc beds
     And furnished them wings to fly;
     He sowed our spawn in the world's dim dawn,
     And I know that it shall not die,
     Though cities have sprung above the graves
     Where the crook-boned men made war,
     And the ox-wain creaks, o'er the buried caves
     Where the mummied mammoths are.

     XIV

     Then as we linger at luncheon here,
     O'er many a dainty dish,
     Let us drink anew to the time when you
     Were a Tadpole and I was a Fish.

Dr. Myrianthopoulos is a scientist emeritus, the National Institutes of Health, where he conducted research on the genetics of nervous system disorders. He was born in Cyprus, then a British colony. His studies at the University of Athens were interrupted by World War II, during which he served as an officer with the British forces in the Middle East. He completed his doctoral studies at the University
of Minnesota. He and his wife Marjorie have three children, each with two X chromosomes.

1Aristotelian lecture given at the 9th International Clinical Genetics Seminar, July 4, 1998, Limassol, Cyprus. Parts of this lecture were published in "Cosmos, 1999," the Journal of the Cosmos Club of Washington, D.C.

Letters

Isolation of a Dengue Type 1 Virus from a Soldier in
West Africa (Cte d'Ivoire)

To the Editor: In Africa, recent reports of epidemic or
endemic dengue activity usually come from the eastern
part of the continent (1), and the serotype most
frequently identified is dengue 2.

We report the isolation of a dengue 1 virus strain from
the blood of a young soldier living in Abidjan, the
capital of Cte d'Ivoire. This 20-year-old man arrived
from France on October 19, 1998. On December 28, 1998,
he consulted a physician of his regiment because of
headache, diarrhea, and fever (40C). The results of
the laboratory investigations were as follows:
platelets (193 109 /L), leukocytes (2.2109 /L); no
malaria was found. He was hospitalized for possible
arboviral infection, and treatment with paracetamol was
prescribed. On December 29, a blood sample was
collected; serum and buffy coat were frozen at -20C
for further examination at the Institute of Tropical
Medicine of the Military Health Services. On day 3, the
patient's temperature dropped to 38C, then rose to
39.5C on day 5. All symptoms were resolved on day 6.

On February 15, 1999, the frozen blood was defrosted
and the lysed buffy coat was immediately cocultured
with C6/36 cells. On day 6, a blind passage was made on
the same cells and on Vero. On day 12, no cytopathic
effect was observed, but a dengue 1 virus was
identified by indirect fluorescent antibody assay (2)
with type-specific monoclonal antibodies. This
diagnosis was confirmed by reverse
transcription-polymerase chain reaction, with a
technique slightly modified from Lanciotti (3).

On the first blood specimen, the serologic
immunoglobulin (Ig)M assays (M antibody capture
enzyme-linked immunosorbent assay [ELISA]) with our
antigen screening panel were negative (dengue, West
Nile, Chikungunya, Rift Valley fever). The IgG assay
(ELISA) against dengue antigen was also negative.

The patient returned to France in February 1999. A
second blood sample was collected and tested on April
7, 1999, 3 months after the illness. The serologic
assays were positive against the dengue antigens at the
following dilutions of the serum: IgM: dengue 1:
1/120,000; dengue 2: 1/40,000; dengue 3: 1/12,000;
dengue 4: 1/25,000. IgG: dengue 1: 1/75,000; dengue 2:
1/90,000; dengue 3: 1/60,000; dengue 4: 1/120,000. This
seroconversion allowed us to confirm infection of this
patient by a dengue virus.

The lack of similar reported cases or epidemics among
the local and expatriate populations of Abidjan may
indicate poor transmission, recent introduction of the
strain, or low virulence of the virus, as previously
hypothesized for dengue in West Africa (4). However,
the serologic status of the human population needs to
be further investigated. Characterization of the
isolated viral strain would be of interest for dengue
epidemiology. Complete sequencing of the viral RNA is
in progress in our laboratory.

Human infection with dengue virus has been rarely
reported and studied in West Africa, and the
epidemiology of serotype 1 is poorly documented. During
the past 35 years, the Pasteur Institute of Dakar
confirmed three dengue 1 strains (two from humans in
Senegal; one from mosquitoes in Cte d'Ivoire), while
during the same period, more than 300 dengue 2 strains
were studied, most from mosquitoes (5). In the past 10
years, medical and entomologic surveys showed
circulation of dengue 2 virus in Senegal (one isolate
in the blood of a French soldier) (6). However, during
the 1970s, Nigerian virologists demonstrated
circulation of dengue 1 and 2 in their country: more
than 50% of the adults living in the savannah had
neutralizing antibodies (7). Of 148 blood samples of
febrile patients, three viral strains (yellow fever,
dengue 1, and Zika) were isolated, all from children
(8).

In Africa, outside the epidemics of yellow fever, it is
difficult to isolate arboviruses from adult humans.
Isolation is often more successful from children or
naive expatriates. Accordingly, soldiers participating
in international operations constitute a very exposed
population. During recent operations in Somalia
(1992-93), dengue fever was an important cause of
febrile illness among U.S. troops (9). Thirty-nine
dengue 2 and three dengue 3 strains were isolated from
96 collected sera.

Acknowledgments

We thank F. Tock and S. Guesdon for technical
assistance and D. Gubler and N. Karabatsos for the
monoclonal antibodies used for virus typing.

This work was supported by the Dlgation Gnrale 
L'Armement.

J.P. Durand,* L. Valle, J.J. de Pina, and H. Tolou*

Institut de Mdecine Tropical du Service de Sant des
Armes, Marseille Armes, France; Service Mdical du
43 BIMa, Abidjan, Rpublique de Cte d'Ivoire; and
Hpital Laveran, Marseille Armes, France

References

  1. Gubler DJ, Kuno G. Dengue and dengue hemorrhagic
fever. Center for Agriculture and Biosciences
International; 1997; p. 10-25.
  2. Henchal EA, Gentry M, McCown JM, Brandt WE. Dengue
virus specific and flavivirus group determinants
identified with monoclonal antibodies by indirect
immunofluorescence. Am J Trop Med Hyg
1982;31:830-6.
  3. Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ,
Vorndam AV. Rapid detection and typing of dengue
viruses from clinical samples by using reverse
transcriptase-polymerase chain reaction. J Clin
Microb 1992;30:545-51.
  4. Deubel V, Nogueira RM, Drouet MT. Direct
sequencing of genomic cDNA fragments amplified by
the polymerase chain reaction for molecular
epidemiology of dengue 2 virus. Arch Virol
1993;129:197-210.
  5. Centre Collaborateur OMS de Reference et de
Recherche pour les Arbovirus et les Fivres
Hmorragiques. Rapport Annuel 1995; Institut
Pasteur de Dakar.
  6. Monlun E, Zeller H, Le Guenno B, Traore-Lamizana
M, Hervy JP, Ferrara L, et al. Surveillance of the
circulation of arbovirus of medical interest in
the region of eastern Senegal. Bull Soc Pathol
Exot 1993;86:21-8.
  7. Fagmani A. Epidemiological investigations on
arbovirus infections at Igbo-Ora, Nigeria.
Tropical and Geographical Medicine 1977;29:187-91.
  8. Fagmani AH, Monath TP, Fabiyi A. Dengue virus
infection in Nigeria: a survey for antibodies in
monkeys and humans. Trans R Soc Trop Med Hyg
1977;71:60-5.
  9. Sharp TW, Wallace MR, Hayes CG, Sanchez JL,
DeFraites RF, Arthur RR, et al. Dengue fever in
U.S. troops during Operation Restore Hope,
Somalia, 1992-1993. Am J Trop Med Hyg
1995;53:89-94.

--------------------------------------------------------

Carbapenem-Hydrolyzing Metallo--Lactamase from a
Nosocomial Isolate of Pseudomonas aeruginosa in France

To the Editor: The carbapenems (meropenem and
imipenem), the -lactams with the broadest spectrum,
are stable to most -lactamases (1). Therefore, they
are often used as antibiotics of last resort for
treating nosocomial infections due to gram-negative
bacteria resistant to other -lactams. Resistance to
carbapenems and susceptibility to other -lactams in
Pseudomonas aeruginosa is common as a result of reduced
drug accumulation or increased expression of pump
efflux (1).

Several extended-spectrum -lactamases have been
reported in P. aeruginosa, but only two, IMP-1 and
VIM-1, possess an extended hydrolysis profile that
includes carbapenems (2-5). The chromosome-borne and
plasmid-mediated carbapenem-hydrolyzing -lactamase,
IMP-1, has been described in several gram-negative
rods, including P. aeruginosa, P. cepacia, Alcaligenes
xylosoxydans, and Enterobacteriaceae isolates in Japan
(4,6). Recently, a chromosome-borne
carbapenem-hydrolyzing -lactamase, VIM-1, was reported
from a clinical isolate of P. aeruginosa in Italy (5),
and uncharacterized carbapenem-hydrolyzing -lactamases
have been reported in the United Kingdom and Portugal
(7,8). The weakly related IMP-1 and VIM-1 (31.4% amino
acid identity) are both zinc-dependent
(metallo-enzymes) and confer resistance to all
-lactams except monobactams (3,5).

In 1996, a 39-year-old French woman was hospitalized in
Marseille for chronic myelogenous leukemia,
pancytopenia, and allogeneic bone marrow
transplantation. After a 15-day stay in the
transplantation unit, fever developed and imipenem and
amikacin were administered. Despite this treatment, the
patient died of septic shock syndrome 5 days later.
Three-day-old blood cultures grew a
carbapenem-resistant P. aeruginosa isolate. This P.
aeruginosa COL-1 isolate was resistant to most
-lactams, including piperacillin/tazobactam, imipenem,
meropenem, ceftazidime, cefepime (minimum inhibitory
concentrations [MICs] of 128, 32, 16, 64, 32 mg/L,
respectively), amikacin, tobramycin, gentamicin,
netilmicin, and ciprofloxacin; however, the isolate was
susceptible to aztreonam (MIC determination, genetic
techniques and -lactamase assays are described
elsewhere [9]). A sonicate of crude extract of P.
aeruginosa COL-1 culture showed strong imipenem and
meropenem hydrolysis activity (0.7 mU/mg and 1.9 mU/mg;
reference P. aeruginosa strain <0.05 mU/mg) by UV
spectrophotometry with 0.1 mM of substrate, after
incubation in 50 mM phosphate buffer at 30C. This
activity was lost when the enzyme extract was
preincubated with 10 mM of edetic acid and was
partially restored by addition of 1 mM ZnCl2,
indicating the presence of a metallo-carbapenem
hydrolyzing -lactamase. Isoelectric focusing revealed
two -lactamase bands of pI 5.6 and 9. Only the pI 5.6
-lactamase band was inhibited if the gel was overlaid
with edetic acid before nitrocefin was added as the
indicator substrate; the other pI 9 -lactamase likely
corresponded to a naturally occurring AmpC
cephalosporinase. This pI 5.6 value differed from the
pI values of the carbapenem-hydrolyzing -lactamase
previously reported in P. aeruginosa (3-5,7,8).
Polymerase chain reaction amplification experiments
were negative when internal primers were used for the
only sequenced carbapenem-hydrolyzing -lactamase genes
from P. aeruginosa encoding IMP-1 and VIM-1 and genomic
DNA of P. aeruginosa COL-1. Transfer of the carbapenem
resistance marker by conjugation to laboratory strains
of P. aeruginosa or Escherichia coli was unsuccessful
(9), but transformation by electroporation of a
putative plasmid extract from P. aeruginosa COL-1 in E.
coli, followed by selection onto amoxicillin-containing
agar plates (9), gave a ca. 45-kb plasmid that produced
the carbapenem-hydrolyzing -lactamase with a pI value
of 5.6. Thus, the carbapenem-hydrolyzing -lactamase
gene was plasmid-borne.

This case indicates the presence of a novel
carbapenem-hydrolyzing -lactamase in P. aeruginosa in
Europe, the first in France; its spread in
gram-negative rods, as reported for IMP-1 in Japan, is
of concern because, as seen in this case, routine
laboratory detection is difficult and therapeutic
options are extremely limited.

Laurent Poirel,* Louis Collet, and P. Nordmann*

*Hpital de Bictre, Le Kremlin-Bictre, France; and
Institut Paoli-Calmettes, Marseille, France

This work was supported by a grant from the Ministre
de l'Education Nationale, de la Recherche et de la
Technologie, Universit Paris XI, Facult de Mdecine
Paris Sud, UPRES-JE-2227, France.

References

  1. Livermore DM. Are all -lactams created equal?
Scand J Infect Dis 1996;Suppl 101S:33-43.
  2. Nordmann P, Guibert M. Extended-spectrum
-lactamases in Pseudomonas aeruginosa. J
Antimicrob Chemother 1998;42:128-31.
  3. Osano E, Arakawa Y, Wacharotayankun R, Ohta M,
Horii T, Ito H, et al. Molecular characterization
of an enterobacterial metallo -lactamase found in
a clinical isolate of Serratia marcescens that
shows imipenem resistance. Antimicrob Agents
Chemother 1994;38:71-8.
  4. Senda K, Arakawa Y, Nakashima K, Ito H, Ichiyama
S, Shimokata K, et al. Multifocal outbreaks of
metallo--lactamase-producing Pseudomonas
aeruginosa resistant to broad-spectrum -lactams,
including carbapenems. Antimicrob Agents Chemother
1996;40:349-53.
  5. Lauretti L, Riccio ML, Mazzariol A, Cornaglia G,
Amicosante G, Fontana R, et al. Cloning and
characterization of blaVIM, a new integron-borne
metallo--lactamase gene from a Pseudomonas
aeruginosa clinical isolate. Antimicrob Agents
Chemother 1999;43:1584-90.
  6. Senda K, Arakawa Y, Ichiyama S, Nakashima K, Ito
H, Shimokata K, et al. Characterization of metallo
-lactamase gene (blaIMP) in gram-negative rods
resistant to broad-spectrum -lactams. J Clin
Microbiol 1996;34:2909-13.
  7. Woodford N, Palepou MFI, Babini GS, Bates J,
Livermore DM. Carbapenemase-producing Pseudomonas
aeruginosa in UK. Lancet 1998;352:546-7.
  8. Cardoso O, Sousa JC, Leitao R, Peixe L.
Carbapenem-hydrolysing -lactamase from clinical
isolates of Pseudomonas aeruginosa in Portugal. J
Antimicrob Chemother 1999;44:135.
  9. Philippon LN, Naas T, Bouthors AT, Barakett V,
Nordmann P. OXA-18, a class D clavulanic
acid-inhibited extended-spectrum -lactamase from
Pseudomonas aeruginosa. Antimicrob Agents
Chemother 1997;41:2188-95.

---------------------------------------------------------

Population-Based Study of Invasive Kingella kingae Infections

To the Editor: For most of the 3 decades since the first description of Kingella kingae, this gram-negative bacillus was considered a rare cause of human disease (1). Since the late 1980s, however, reports of infections by the organism in young children have increased in the United States, Western Europe, and Israel (2-6). The rapid emergence of K. kingae as an important cause of pediatric disease does not necessarily imply that the organism is truly a new
pathogen. Better isolation techniques and awareness of the bacterium by microbiology laboratories may contribute to the apparent increase (4). Recent studies have demonstrated that primary isolation of K. kingae can be substantially improved by injection of synovial fluid and bone exudates into aerobic blood-culture bottles (4). Synovial fluid may inhibit the growth of K. kingae, and injection of the
clinical specimen into a 50-mL volume of broth reduces the
concentration of inhibitory factors, facilitating isolation of the organism (4).

In 1993, we reported results of routine use of blood-culture bottles for processing cultures of exudates (7) at the Soroka University Medical Center, Beer-Sheva, Israel. From 1988 to 1992, 25 children with invasive K. kingae infections, defined as isolation of the organism from blood or normally sterile body fluids, were identified
in southern Israel. From 1994 to 1998, 33 additional patients, including 32 children and a 21-year-old adult, were detected in the same area. Twenty-four (63.6%) of the 33 patients were male. Eight (24.2%) cases were diagnosed between January and June and 25 (75.8%) between July and December. Median age of children was 13 months (mean  SD: 15.0  7.6 months; range 6 to 37 months).

The fact that all children in southern Israel are born and receive inpatient medical services at the Soroka University Medical Center allowed us to calculate the incidence of invasive pediatric K. kingae infections in this population. During the 6-year period, the average annual number of births was 10,860. The annual incidence of invasive K. kingae infections during the same period was 11.9 per
100,000 in children < 48 months of age, 19.2 per 100,000 in children <24 months of age, and 20.0 per 100,000 in infants <12 months of age.

When medical attention was sought, patients had been ill for a median of 3 days. Symptoms of upper respiratory tract infection were recorded in 12 (36.4%) children, stomatitis in 8 (24.2%), and diarrhea in 4 (12.1%). Occult bacteremia (positive blood culture with no obvious focal infection) was diagnosed in 16 children. In 15 children, K. kingae had invaded the bones. Septic arthritis was diagnosed in 11 children, involving the ankle in 4; the knee or wrist in 2 patients each; and the hip, shoulder, or elbow in one
patient each. Osteomyelitis was diagnosed in two patients, affecting the femur in one and the tibia in the other. In two additional patients, both with fever and bacteremia, the location of the skeletal infection could not be determined. One limped and had tenderness over the femur, but X-rays and a Technecium99-labeled bone scan showed no abnormalities. The other had pain in the heel but no fluid could be aspirated. Bacteremic tracheobronchitis occurred in one child, and endocarditis of the mitral valve was
diagnosed in a 21-year-old woman who was receiving immunosuppressive therapy for systemic lupus erythematosus. All 33 patients were treated with -lactam drugs and recovered.

Injecting synovial fluid specimens into blood-culture bottles permitted the diagnosis of K. kingae in these patients and showed that this organism may be a common cause of invasive pediatric infections. The age distribution of the patients demonstrates that K. kingae is a pathogen of young children, especially those between the ages of 6 months and 2 years, among whom the incidence of invasive disease has remained stable since 1988. This age
distribution of K. kingae infections parallels that for respiratory carriage of the organism. In a surveillance study among 48 children ages 6 to 42 months attending a day-care center in Israel, K. kingae was isolated from 109 (17.5%) of 624 throat cultures, and 34 children (70.8%) carried the organism at least once during an 11-month period (8). However, the organism was not detected in healthy infants ages 2 to 4 months attending a well-baby care
clinic, which indicates some immunity to colonization and infection by K. kingae during the first months of life (8).

When the 1988 to 1993 surveillance data are added to those collected from 1994 to 1998, K. kingae infections show a significant seasonal pattern; 44 (75.9%) of 58 cases were diagnosed in the second half of the year (p = 0.007). This increase in K. kingae infections in winter has also been described in other respiratory pathogens. This finding, as well as the frequent detection of respiratory symptoms
in children with invasive K. kingae infections, suggests that seasonal viral infections may facilitate the spread of K. kingae from the throat, to the bloodstream and bones. In a prospective study, K. kingae bacteremia was documented in 4 (13.7%) of 29 young children with culture-proven herpetic gingivostomatitis, confirming the role played by viral infections in the pathogenesis of infections caused by the organism (9).

With few exceptions, isolates of K. kingae remain susceptible to antibiotic drugs (10). Our results demonstrate that the prognosis of invasive K. kingae infections is generally good and patients respond
promptly to appropriate antimicrobial therapy.

Pablo Yagupsky and Ron Dagan

Soroka Medical Center, Ben-Gurion University of the Negev,
Beer-Sheva, Israel 

References

  1. Graham DR, Band JD, Thornsberry C, Hollis DG, Weaver RE. Infections caused by Moraxella, Moraxella urethralis,
Moraxella-like groups M-5 and M-6, and Kingella kingae in the United States, 1953-1980. Rev Infect Dis 1990;12:423-31.
  2. deGroot R, Glover D, Clausen C, Smith AL, Wilson CB. Bone and joint infections caused by Kingella kingae: six cases and review of the literature. Rev Infect Dis 1988;10:998-1004.
  3. Goutzmanis JJ, Gonis G, Gilbert GL. Kingella kingae infection in children: ten cases and review of the literature. Pediatr Infect Dis 1991;10:677-83.
  4. Yagupsky P, Dagan R, Howard CB, Einhorn M, Kassis I, Simu A. High prevalence of Kingella kingae in joint fluid from children with septic arthritis revealed by the BACTEC blood culture system. J Clin Microbiol 1992;30:1278-81.
  5. Birgisson H, Steingrimsson O, Gudnasson T. Kingella kingae infections in paediatric patients; five cases of septic arthritis, osteomyelitis and bacteraemia. Scand J Infect Dis 1997;29:495-8.
  6. Lundy DW, Kehl DK. Increasing prevalence of Kingella kingae in osteoarticular infections in young children. J Pediatr Orthop 1998;18:262-7.
  7. Yagupsky P, Dagan R, Howard CB, Einhorn M, Kassis I, Simu A. Clinical features and epidemiology of invasive Kingella kingae infections in southern Israel. Pediatrics 1993;92:800-4.
  8. Yagupsky P, Dagan R, Prajgrod F, Merires M. Respiratory
carriage of Kingella kingae among healthy children. Pediatr
Infect Dis J 1995;14:673-8.
  9. Amir J, Yagupsky P. Invasive Kingella kingae infection
associated with stomatitis in children. Pediatr Infect Dis J
1998;17:757-8. 
 10. Jensen KT, Schonheyder H, Thomsen VF. In-vitro activity of -lactam and other antimicrobial agents against Kingella
kingae. J Antimicrob Chemother 1994;33:635-40.

----------------------------------------------------------

Involving Ornithologists in the Surveillance of Vancomycin-Resistant Enterococci

To the Editor: Because migratory birds cross national or
intercontinental borders, they are possible long-range vectors for human pathogens such as viruses, Borrelia burgdorferi sensu lato, and enteropathogenic bacteria with antibiotic resistance or virulence factors (1,2). Enterococci are ubiquitous in humans and animals and have a propensity for uptake and transfer of glycopeptide antibiotic resistance (3); therefore, the emergence of
glycopeptide-resistant enterococci (GRE) in humans is a public health concern. Low-level vancomycin resistance (genotype vanC-1-3) is intrinsic in enterococcal species (e.g., Enterococcus gallinarum, E. flavescens, and E. casseliflavus) that may normally occur in the intestinal flora of some birds. However, the finding of high levels
of GRE in wild birds suggests acquisition from an environmental source.

In March 1998, we obtained fecal samples while banding 318
northbound migrating gulls in Malm, southern Sweden. Using a selective culture procedure with enrichment broth (bile esculin azide broth, Acumedia, LabFab, Ljusne, Sweden) containing vancomycin (8 g/ml) and aztreonam (60 g/ml), we isolated vancomycin-resistant E. faecalis from a black-headed gull (Larus ridibundus). High-level glycopeptide resistance (>256 g/ml) was demonstrated by E-test (AB
Biodisc, Solna, Sweden), and a vanA genotype was found by polymerase chain reaction amplification (4). This survey protocol can also be used to detect medium to low levels of glycopeptide resistance. Using the same procedure in a study of 230 sub-Antarctic birds on Bird Island, South Georgia, in 1996, we found four GRE isolates with vanC1 genotype (MIC 3-8 g/ml).

Many species of gulls have moved into urban areas, where they commonly feed on human trash and deposit feces. The black-headed gull with GRE described above was banded as a fledgling in Malm in 1995. Birds of this population spend the winter mainly in Western Europe (5), where they forage at garbage dumps, sewage outlets, and agricultural areas. This bird may have acquired GRE in such an area.
VanA genotype E. faecium and E. faecalis have been found in poultry and pigs in the Netherlands and Denmark, where the vancomycin analog avoparcin has been used as a growth promoter (6). Manure from such farms may be a GRE source accessible to wild birds.

We have previously reported the introduction into Sweden of
multidrug-resistant Salmonella Typhimurium by migratory birds (7). The present report further emphasizes the possibility of migratory birds as long-range vectors of bacteria potentially associated with human disease. The risk to humans for GRE from migratory birds may seem insignificant compared with such risk from hospitalization or from eating meat products from GRE-colonized animals. However, if the frequency of birds carrying high-level GRE increases and if amplification in a secondary reservoir or spread through polluted water takes place, spread by migratory birds may become a problem. Bacteriologic surveys of birds may provide vital information for assessing the environmental dispersion of GRE from farms and hospitals. In combination with data about migration patterns and reports of banding recoveries from ornithologists, the potential
sources of GRE might be deduced.

This work was supported by the Center for Environmental Research, the Medical Faculty of Ume University, the Swedish Council for Agricultural Research, and the Swedish Society of Medicine.

M. Sellin,* H. Palmgren,* T. Broman,* S. Bergstrm,* and B. Olsen* 
*Ume University, Ume, Sweden; and Kalmar County Hospital, Kalmar, Sweden

References

  1. Coulson JC, Butterfield J, Thomas C. The herring gull Larus argentatus as a likely transmitting agent of Salmonella montevideo to sheep and cattle. Journal of Hygiene London 1983;91:437-43.
  2. Olsen B, Jaenson TGT, Bergstrm S. Prevalence of Borrelia burgdorferi sensu lato infected ticks on migrating birds. Appl Environ Microbiol 1995;61:3082-7.
  3. French GL. Enterococci and vancomycin resistance. Clin Infect Dis 1998;27:75-83.
  4. Dutka-Malen S, Evers S, Courvalin P. Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J Clin Microbiol 1995;33:24-7.
  5. Bengtsson K. Migratory routes and wintering areas of Swedish black-headed gull (Larus ridibundus) populations. Ornis Svecica 1996;6:17-38.
  6. Aarestrup FM, Ahrens P, Madsen M, Pallesen LV, Poulsen RL, Westh H. Glycopeptide susceptibility among Danish Enterococcus faecium and Enterococcus faecalis isolates of animal and human origin and PCR identification of genes within the VanA cluster.
Antimicrob Agents Chemother 1996;40:1938-40.
  7. Palmgren H, Sellin M, Bergstrm S, Olsen B. Enteropathogenic bacteria in migrating birds arriving in Sweden. Scand J Infect Dis 1997;29:565-8.

News and Notes

2nd International Conference on AIDS
in India, December 1999

The 2nd International Conference on AIDS in India, which was organized by the Department of Experimental Medicine, Tamil Nadu Dr. M.G.R. Medical University, was held in Chennai, India, on December 5-8, 1999. In addition to the national delegates, the conference was attended by 652 national and international experts.
	On December 6, the first AIDS Society of India was formed and inaugurated by Mark Wainberg, president of the International AIDS Society.
	HIV/AIDS prevalence is increasing in India, where as many as 8 million people may be infected. Available interventions are not adequate. The conference participants recommended the following  prevention measures:

The Chennai Statement

1.	Simplified, low-cost medical interventions (including antiviral drugs, which can substantially reduce mother-to-infant HIV transmission, should be implemented.
2.	Medicines against HIV and opportunistic infections should be made affordable and accessible, and discrimination against HIV-infected persons in the workplace, health-care industry, and other social contexts should be addressed.
3.	Medical professionals should receive special training in the treatment of HIV infection and disease. Formal training leading to postgraduate qualification in HIV medicine will improve the quality and equity of health care for infected persons in various clinical settings. Similarly, formal training toward postgraduate qualification in transfusion medicine will improve blood safety and the use of blood components, and thus the use of donated blood. The Medical Council of India and medical universities around the country should develop such postgraduate training.
4.	Folklore, traditions, and other elements of Indian culture should be systematically reviewed and adapted to teaching children and youth about responsible, safe behavior to prepare them for their roles as future parents. Medical systems in India should be rigorously and scientifically evaluated by multicenter studies, and improvements in treating HIV disease should be introduced.
5.	India must invest in research to develop vaccines and antiviral and antimicrobial drugs for the prevention and treatment of HIV disease and opportunistic infections.
6.	Cooperative efforts should be made within India to collaborate with international coalitions for the prevention, control, and treatment of HIV/AIDS.
7.	In all strategic planning, the voice of the people, particularly the voice of infected people, must be taken into account.

	We believe that the above recommendations will help control the AIDS epidemic, with all its socioeconomic implications, in India.

N.M. Samuel
AIDS Society of India

Workshop on Micronutrients and Infectious Diseases: Cellular and Molecular Immunomodulatory Mechanisms

	Epidemiologic and clinical data, as well as experimental studies, suggest a bidirectional interaction between micronutrients and infectious pathogens. While nutritional deficiencies can alter several aspects of the innate and acquired immune responses to pathogens, some infectious diseases alter the nutritional status of the host and the host's ability to absorb micronutrients. Nutritional deficiencies may also influence pathogen mutations directly, affecting virulence and clinical outcome. These interactions depend on a number of other variables, including the severity of micronutrient deficiency, the age and clinical condition of the host, and a variety of environmental factors.
	To address issues pertinent to understanding the role(s) of micronutrients in the control of infectious disease, the National Institute of Allergy and Infectious Diseases organized a 2-day meeting, cosponsored by the Office of Dietary Supplements, National Institutes of Health. Representatives attended from several other NIH institutes and centers, the U.S. Agency for International Development, the U.S. Department of Agriculture, the U.S. Food and Drug Administration, the Council for Responsible Nutrition, and the World Bank. The objectives of this meeting were to provide a forum for the exchange of ideas between basic scientists, nutritionists, infectious disease specialists, and clinical epidemiologists; to examine the mechanisms involved in the pathogenesis and clinical manifestations of infectious diseases, including the immunology, molecular biology, and potential direct interactions between micronutrients and pathogens; evaluate current strategies for intervention; and summarize research needs and new directions.
	The workshop provided information on the effects of micronutrients on innate and adaptive immunity, mucosal immunity, cytokine production, gene expression, and intracellular signaling pathways. In addition, a session focused on the implications of basic research findings for the treatment of disease. The workshop opened with an overview on how dietary characteristics modulate immune responses. Although the field is in its infancy, areas in particular need of research include the role of micronutrients in innate and mucosal immunity, as well as the early phases of development of immune cells. Instances of direct interaction between micronutrients and pathogens were also described, for example, how a benign strain of an infectious agent can become virulent when passaged through micronutrient-deficient mice. Examples of host-pathogen interactions, in which nutrition is an important modulator, provide new opportunities for study, especially as the host's genetic background and the role of specific genes are elucidated. One potential mechanism of immune suppression is the alteration of cytokine responses. Molecular studies using gene knock-out mice also provided new information about nutrient transport proteins and their relationship to normal immunity.
	At the conclusion of the workshop, a panel of experts, including basic scientists, infectious disease specialists, and clinical epidemiologists, submitted recommendations for future research: the development of a panel of assays that could be used for general screening of a nutrient's impact on immunocompetence; the need to define reproducible molecular and immunologic biomarkers that can be used in human and animal studies; the need to continue basic mechanistic studies on both the role of micronutrients as antioxidants or as regulatory molecules within the immune system and the effects of micronutrient status on the decline of immune function in the elderly; the need to encourage more efficient use of transgenic knock-outs and other appropriate animal models, as well as the use of microarray technology, by investigators in this area of research; the need for investigators to link basic molecular, mechanistic studies with field work in areas of endemic infectious diseases of greatest public health importance; the need for innovative approaches for combining nutritional supplementation and immunotherapy in new forms of intervention; and the need to recruit young investigators with state-of-the-art immunology skills and promote collaborative research ith nutrition or infectious disease professionals.
	As a result of this workshop, an open LISTSERV has been created for continued interaction among interested parties in the field. The Journal of Infectious Diseases plans to publish the proceedings of the workshop in a supplement in early 2000. For additional information, contact Christopher E. Taylor, telephone: 301-496-5305; e-mail: ct18m@nih.gov; or Elizabeth Higgs, telephone: 301-496-2544; e-mail: eh63a@nih.gov.

Third Annual Conference on Vaccine Research: Basic Science-Product Development-Clinical and Field Studies

Renaissance Hotel, Washington, D.C.
April 30-May 2, 2000
 
	The conference is sponsored by the National Foundation for Infectious Diseases, in collaboration with the Centers for Disease Control and Prevention; National Institute of Allergy and Infectious Diseases, National Institutes for Health; International Society for Vaccines; Albert B. Sabin Vaccine Institute at Georgetown University; World Health Organization; U.S. Department of Agriculture; and the Center for Biologics Evaluation and Research, Food and Drug Administration.
	For information, contact Kip Kantelo, National Foundation for Infectious Diseases, 4733 Bethesda Avenue, Suite 750, Bethesda, MD 20814-5228, USA; telephone: 1-301-656-0003 x19; fax: 1-301-907-0878; e-mail: kkantelo@nfid.org; URL: http://www.nfid.org/conferences.

Dangerous Pathogens 2000 Conference 

University of Plymouth
United Kingdom
September 4-7, 2000
	
	The Dangerous Pathogens 2000 Conference is organized by the Defence Evaluation and Research Agency (DERA), Porton Down; the National Institute of Allergy and Infectious Diseases, National Institutes of Health, United States; and the Society for Applied Microbiology, United Kingdom.  The conference will review the ongoing battle between humans and microorganisms, past, present, and future, and identify any gaps in our knowledge that need to be filled to ensure that humans come out on top.
	The conference will cover the following themes: The Past, including Plagues of Antiquity (plague, cholera, smallpox, syphilis, influenza), Disease and War, The Role of Sanitation and Clean Water, and The Impact of Science (Koch, Pasteur, Fleming); The Present, including Plagues of the Present (HIV, multidrug-resistant tuberculosis, malaria), Antibiotic-Resistant Organisms, Hospital-Acquired Infections, and Global Vaccine Programs; and The Future, including Emerging Pathogens, Genomics and Bioinformatics, and New Antimicrobial Agents.
	For more information about the conference, contact Les Baillie, Principal Scientist, DERA Porton, Porton Down, Salisbury SP4 OJQ, United Kingdom; telephone: 44-1980-613-881; fax: 44-1980-613-284; e-mail: lesbaillie@hotmail.com.

ICEID 2000-Call for Abstracts

	Abstract submission to ICEID 2000 is now possible through the Conference Web site, and we strongly encourage you to make use of this electronic method.  Not only will you be able to submit your abstract in a finished form (prepared using your own word processing program such as Word or WordPerfect, on PC or Macintosh platforms), but you can check back to learn acceptance status, presentation location, and time information.  In addition, it will save you both the costs of postage and the worry of a lost submission.
	ICEID 2000 is using the American Society for Microbiology's Web-based Abstract Submission System.  If you have used the system to submit to either the 1999 or 2000 ASM General Meetings or the 1999 ICAAC Meeting, you will be able to reenter the system as a returning user.
	For more information on ICEID abstract submission, see the ASM Web site: www.asmusa.org.  Follow the link to ICEID 2000.

	Emerging Infectious Diseases thanks the following reviewers for their support through thoughtful, thorough, and timely reviews from the journal's inception to the present. We apologize for any names we may have omitted inadvertently. 
(please see html version for list of reviewers)

Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA

URL: ftp://ftp.cdc.gov/pub/EID/vol6no1/ascii/vol6no1.txt

Please note that figures, equations, and most tables are not available in ASCII format.  Greek symbols are spelled out when possible. >/= is used for greater than or equal to.