[Emerging Infectious Diseases * Volume 4 * Number 1 * January - March 1998]
Update
International Editors Update
------------------------------------------------------------------------
In this new section, we welcome commentary and updates from our newly formed Board of
International Editors. (See inside front cover for a list of names.)
Emerging Infectious Diseases-Brazil
Hooman Momen
Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil
[Picture of Hooman Momen]
Dr. Momen is a senior research scientist; head of the Department of
Biochemistry and Molecular Biology, Oswaldo Cruz Institute, FIOCRUZ; and
Editor of Memorias do Instituto Oswaldo Cruz. His research interests
include biochemical systematics of pathogenic microorganisms. Dr. Momen is
temporary adviser to both WHO and PAHO at several technical consultations
and scientific working groups.
Brazil's large size (more than 8.5 million km[sup 2] and 150 million population)
and inadequate public health infrastructure pose a considerable challenge
in assessing the status of emerging infectious diseases. Under the current
system, most infectious diseases are not notifiable. In diseases for which
notification is required, underreporting is common and varies widely by
region and disease, and notification is often delayed, which causes the
data to be revised frequently. Moreover, in hospital and clinical settings,
the etiologic agent of an infectious disease is often not identified. For
example, more than a million hospital admissions are recorded per year by
the public health system under parasitic and infectious diseases (the
category excludes AIDS and respiratory illnesses); of these diseases, more
than 70% are diagnosed as ill-defined intestinal infections and a further
10% as food poisoning and septicemia, both without identification of the
etiologic agents. For these reasons, the numbers reported here (1996 data,
unless otherwise stated) may not reflect the true numbers of cases, and
only diseases whose prevalence has changed markedly in recent years will be
included in the review.
Parasitic Diseases
Malaria
Among parasitic diseases, malaria causes the most illness, approximately
half a million cases annually, nearly all from the northern (Amazon)
region. The disease has been controlled in the remainder of the country for
years. Where systematic and sustained efforts have been made in the Amazon,
control has also been successful. Due to the different epidemiologic
situations in the Amazon, no single strategy is effective. Worrying
developments in this region include the establishment of the disease in the
periphery of the principal cities and the increase in drug- resistant
parasites.
American Trypanosomiasis
American trypanosomiasis (Chagas disease) is the most lethal parasitic
disease in Brazil, causing more than 5,000 deaths per year. Several million
people are chronically infected carriers of this disease, for which there
is still no effective treatment. A very effective control program against
the principal insect vector, Triatoma infestans, and improved control of
the blood supply have reduced the incidence of new cases to a very low
level. Factors that continue to make the disease a re-emerging threat
include secondary vectors, forest clearance, and congenital transmission.
Leishmaniasis
Leishmaniasis (cutaneous and visceral) has increased in incidence in recent
years. This increase is modified by apparent cyclic variations of
undetermined cause (cyclic duration for the visceral form has been
estimated at 10 years). The disease has increased not only in the usual
areas of forest and recent clearance, but also in areas of traditional
colonization as well as in the urban areas of the northeast. One factor
involved in the spread of this disease, uncontrolled urban growth due
principally to rural migration, has led to pockets (normally on the
periphery of the cities) of extreme poverty that favor the spread of many
diseases.
Viral Diseases
Dengue
Among the notifiable viral diseases, dengue causes the most illness. Its
mosquito vector, Aedes aegypti, was reintroduced into Brazil in the 1970s;
large outbreaks followed in the next decade. Since the vector's
reintroduction, more than 700,000 cases have been reported (180,000 cases
in 1996). The presence of dengue-1 and -2 indicates that cases of
hemorrhagic dengue are also occurring. Ae. aegypti is now present in all
states, and a plan has been formulated by the federal ministry of health to
eradicate the vector, although plan implementation has met with operational
difficulties. The widespread distribution of Ae. aegypti, also the vector
of yellow fever, in areas where this disease was considered endemic poses a
serious risk for the reemergence of the epidemic form of yellow fever.
Vaccination and sanitation campaigns earlier in the century considerably
reduced the incidence of the disease (fewer than 100 cases per year-the
last urban cases were reported in 1942).
Measles
Measles-a viral disease that was controlled and nearly eradicated-reemerged
with a vengeance in 1997. The outbreak began in the state of So Paulo,
where only 15 cases of measles were confirmed in 1996, and has spread to
other states. As of October 1997, 61,000 cases (48,000 in So Paulo) have
been reported, and 17,000 (13,000 in So Paulo) have been confirmed. Most
cases have occurred in young adults under 30 years of age. The vaccination
campaign has been reformulated in response to the outbreak, and a national
campaign has been launched. Given Brazil's history of success in organizing
such campaigns, it is likely that the outbreak will be rapidly contained
and that the effort for measles eradication will be resumed.
Other Viral Diseases
Other viral diseases include AIDS, with more than 17,000 new cases
registered in the last year. The number of deaths due to AIDS was 30% lower
in the first half of 1997 than in the first half of 1996. This decrease is
attributed to the introduction of new antiviral drug combinations. The
spread of the AIDS epidemic, particularly into rural areas, has resulted in
coinfections with different infectious agents, producing a variety of novel
pathologies. Several subtypes of HIV, as well as novel recombinations, have
been reported.
Enteric transmission of hepatitis continues; hepatitis A and D comprise
most of the cases, although the peak incidence is now in young adults
rather than in young children. The incidence of hepatitis B is declining as
a result of vaccination, while that of hepatitis C is increasing. A high
prevalence of hepatitis D exists in some regions of the Amazon, where in
conjunction with hepatitis B, it is believed to be the principal cause of
Labrea black fever.
Influenza is a major concern. Imported vaccine is expensive and not
generally available, and the proportion of elderly people (and the threat
for a large-scale epidemic) is increasing.
The vast forests that still cover large areas of Brazil are home to many
known and unknown viruses. The Evandro Chagas Institute in Belem alone has
isolated more than 183 new arboviruses. The increasing exploitation of
sylvatic resources put humans in direct contact with these viruses, but
because of inadequate or nonexistent medical facilities (entire
municipalities still lack a single medical doctor), fevers and even deaths
often are not diagnosed or are misattributed. Among new viruses causing
fatal infections recently discovered in Brazil are Rocio virus, which
causes encephalitis, and Sabi virus, which causes hemorrhagic fever.
Several cases of hantavirus infection have also been reported, but on these
and other occasions, the lack of a biosafety level 4 laboratory in the
country impeded further work.
Bacterial Diseases
Enteric bacterial infections are an important cause of illness in Brazil.
National figures on prevalence are not available, except for cholera.
However, an alarming increase has been reported in antibiotic-resistant
strains.
Cholera
The cholera epidemic in Brazil started with the reemergence of the disease
in Latin America in 1991, reached a peak of more than 60,000 confirmed
cases and 670 deaths in 1993, and then declined to 1,000 cases in 1996. In
1997, the disease resurged with more than 5,000 reported cases and 2,600
confirmed cases. The reawakening of epidemiologic research in cholera
caused by its resurgence led to the discovery of a new biotype of Vibrio
cholerae in the Amazon region. This biotype is of the O1 serotype; it has
distinct multilocus enzyme electrophoresis and RAPD profiles from other
pathogenic O1 V. cholerae. About 50 isolates have been made from cases of
diarrhea in the upper Amazon (Solimoes) River. The microbe apparently lacks
the principal known virulence factors (e.g., the toxin gene cassette and
the major colonization factor, TCP); however, some isolates present a
cytotoxic effect for Y-1 cells.
Mycotic Diseases
Fungal infections, including histoplasmosis, paracoccidioidomycosis, and
cryptococcosis, occur; however, their cause is often unknown and even if a
diagnosis is made, the diseases are not reported.
Public Health Infrastructure
The emerging infectious disease picture in Brazil will not change markedly
without a sustained and determined effort to improve the country's public
health infrastructure. The existing, generally passive epidemiologic
surveillance system produces information that arrives too late to be
effective; however, a number of measures, if implemented immediately, can
mitigate the impact of any future epidemic: a containment laboratory
(biosafety level 4) that can handle known and unknown microbes of high
virulence; at least one infirmary, properly designed and fully equipped, to
treat highly contagious and virulent diseases. The current lack of this
facility poses a great danger to the population should an outbreak of such
a disease occur.
The financial, technical, and human resources needed to activate these
measures already exist. In infectious diseases, Brazil has a long tradition
of fruitful international collaboration that can be tapped for additional
support. The success of national vaccination and sanitation campaigns in
the eradication and control of some infectious diseases at the beginning of
this century and in more recent times demonstrates that much can be
accomplished. Brazil can become a successful model for other developing
countries.
Acknowledgments
I thank the Fundao Nacional de Saude for providing data on different
diseases; Drs. H. Schatzmayr, J.R. Coura, and P. Mason for interesting
discussions and criticisms of the manuscript; and Drs. Ana Gaspar, Clara
Yoshida, Ana Carolina Vicente, and Marilda Siqueira for useful information.
Address for correspondence: Hooman Momen, Instituto Oswaldo Cruz, FIOCRUZ,
Av. Brazil 4365, Rio de Janeiro 21045-900, Brazil; fax: 55-21-590-3545;
e-mail: hmomen@gene.dbbm.fiocruz.br.
------------------------------------------------------------------------
Perspectives
Risk for Transfusion-Transmitted Infectious Diseases in Central and South
America
Gabriel A. Schmunis, Fabio Zicker, Francisco Pinheiro, and David
Brandling-Bennett
Pan American Health Organization, Washington, D.C., USA
------------------------------------------------------------------------
      We report the potential risk for an infectious disease through
      tainted transfusion in 10 countries of South and Central
      America in 1993 and in two countries of South America in 1994,
      as well as the cost of reagents as partial estimation of
      screening costs. Of the 12 countries included in the study,
      nine screened all donors for HIV; three screened all donors for
      hepatitis B virus (HBV); two screened all donors for
      _Trypanosoma cruzi_; none screened all donors for hepatitis C
      virus (HCV); and six screened some donors for syphilis.
      Estimates of the risk of acquiring HIV through blood
      transfusion were much lower than for acquiring HBV, HCV, or _T.
      cruzi_ because of significantly higher screening and lower
      prevalence rates for HIV. An index of infectious disease spread
      through blood transfusion was calculated for each country. The
      highest value was obtained for Bolivia (233 infections per
      10,000 transfusions); in five other countries, it was 68 to 103
      infections per 10,000. The risks were lower in Honduras (nine
      per 10,000), Ecuador (16 per 10,000), and Paraguay (19 per
      10,000). While the real number of potentially infected units or
      infected persons is probably lower than our estimates because
      of false positives and already infected recipients, the data
      reinforce the need for an information system to assess the
      level of screening for infectious diseases in the blood supply.
      Since this information was collected, Chile, Colombia, Costa
      Rica, and Venezuela have made HCV screening mandatory;
      serologic testing for HCV has increased in those countries, as
      well as in El Salvador and Honduras. _T. cruzi_ screening is
      now mandatory in Colombia, and the percentage of screened
      donors increased not only in Colombia, but also in Ecuador, El
      Salvador, and Paraguay. Laws to regulate blood transfusion
      practices have been enacted in Bolivia, Guatemala, and Peru.
      However, donor screening still needs to improve for one or more
      diseases in most countries.
Preventing the transmission of infectious diseases through blood
transfusion in developing countries is difficult given that the resources
needed are not always available, even when policies and strategies are in
place (1). Avoiding paid donors, selecting blood donors through
questionnaires, and limiting the number of transfusions can prevent the
transmission of infections. Testing for specific antibodies is the final
measure for eliminating unsafe blood.
The risk for transfusion-transmitted infectious diseases can be estimated
on the basis of screening level for each infectious agent and the
prevalence rate of the infection in the donor population. Estimates may
also take into account the sensitivity, specificity, and window period of
the testing assays. We report here an estimate of such a risk in 12 Central
and South American countries and the cost of reagents required for the
screening of these infectious diseases as a proxy of resources needed to
reduce the risk.
Source of Information
This report analyzes data from 1993 on screening of blood donors from five
countries (Costa Rica, El Salvador, Guatemala, Honduras, and Nicaragua) of
Central America. Data were also analyzed for 1993 from five countries
(Bolivia, Chile, Colombia, Peru, and Venezuela) and for 1994 from two
countries (Ecuador and Paraguay) of South America. Information was obtained
from Ministry of Health reports during technical meetings in which the
situation of each country was reviewed (2-5) or from an official report
(6).
In addition, data are presented on the least expensive reagents for
detecting antibodies for HIV, hepatitis C virus (HCV), _Trypanosoma cruzi
_, and _Treponema pallidum_, and for detecting hepatitis B virus (HBV)
antigen (HBsAg) (2,3). All data are national except for Peru, where the
information was for the city of Lima only (3). Population data were from
the Pan American Health Organization's publication Health Conditions in the
Americas (7). Estimates are based on reported results of donor screening
activities (2-6).
Assumptions
For the best possible scenario, the following assumptions were made: 1)
Because the laboratory procedures and brands of reagents used in the 12
countries may differ in sensitivity and specificity, comparisons between
them are not straightforward. In addition, results of the screening are
influenced by the existence of an organized system of quality control and
proficiency testing for the serology and for the evaluation of the
diagnostic kits, which most countries lacked from 1993 to 1994. Most
countries reported the use of different brands of second, third, and fourth
generation immunologic assays for screenings of HCV, HIV, and HBV,
respectively. Therefore, we assumed that the specificity of the tests for
viral diagnosis was 100%, but the sensitivity was 90.00% for HCV, 99.99%
for HIV, and 99.90% for HBV. These specificity and sensitivity estimates
fit well with those reported for second, third, and fourth generations of
assays for HCV, HIV, and HBV, respectively, as mentioned in the package
insert by two of the manufacturers of reagents used in the countries.
Average window periods for those assays were 20 to 25 days (8,9), 82 to 84
days (9,10), and 51 days (9) for HIV, HBV, and HCV, respectively. In the
case of _T. cruzi_ serology, we selected the upper range of reported
sensitivity and specificity (90% and 95%, respectively) (11,12). For _T.
cruzi_, the probability that a person may become a donor during the window
period is low because infection is usually acquired in childhood and in
rural areas. 2) We assumed that prevalence of infection in unscreened
donors was the same as the national average prevalence for each infectious
disease. 3) Chile (6) and Peru (3) were the only countries that reported a
fractionation index, 1.85 and 1.5, respectively. As no other country
provided data on the fractionation index or data allowing one to be
calculated, to put the countries in the same category, it was assumed that
every blood donation corresponded to a single transfusion to one recipient.
Screening Coverage and Prevalence Rates
Table 1 shows coverage of screening and prevalence rates of seropositive
tests for specific infectious agents among blood donors reported by each of
the 12 countries. For HIV, 100% of the donors were screened in all
countries, except Bolivia (36.20%), Ecuador (89.50%), and Colombia
(98.80%). Prevalence rates for HIV varied from 3.90 per 1,000 in Honduras
to 0.04 per 1,000 in Nicaragua. For HBV, only Costa Rica, Peru, and
Venezuela screened 100% of donors. The highest values of HBV prevalence
estimated were 14.40 per 1,000 for Venezuela and 13.00 per 1,000 for
Paraguay. Bolivia, Costa Rica, and Paraguay did not screen for HCV at all,
and all other countries screened fewer than 58% of donors; prevalence rates
varied from 0.50 to 9.40 per 1,000. Screening for syphilis was not complete
in Bolivia, Chile, Colombia, Ecuador, Nicaragua, and Paraguay; prevalence
rates were 5.00 to 28.00 per 1,000. For _T. cruzi_ infection, only
Venezuela and Honduras screened 100% of donors; prevalence rates were 2.00
per 1,000 in Ecuador to 147.90 per 1,000 in Bolivia. In 1993, Peru and
Costa Rica had not yet introduced screening for _T. cruzi_.
Table 1. Coverage of screening (sup a) of blood donors and seroprevalence
rates (per 1,000) of infectious diseases, by country (sup b)
---------------------------------------------------------------------------
                HIV      HBV (sup c)     HCV       Syphilis   _T. cruzi_
                   Prev.
           Cov.    (sup        Prev.       Prev.      Prev.
Country    (sup     e)   Cov.  (/10  Cov.  (/10  Cov.  (/10  Cov.   Prev.
           d)      (/10   (%)  (sup   (%)  (sup  (%)   (sup   (%)   (/10
           (%)     (sup         3))        3))          3))       (sup 3))
                    3))
---------------------------------------------------------------------------
Bolivia     36.2   0.10  14.5  2.00   0   ? (sup 37.9  18.10 29.4  147.90
                                            f)
Chile        100   3.40  98.7  2.00  34.0  6.40  95.2  11.40 76.7   12.00
Colombia    98.8   2.00  98.3  7.00  24.7  9.00  87.3 13.00   1.4  12.00
Costa Rica   100   0.34   100  4.50   0      ?    100  5.00   0      ?
Ecuador     89.5   1.00  88.2  3.80  32.9  1.40  86.7  11.50 51.0   2.00
El
Salvador    100    1.30  96.0  8.00  31.4  2.50   100  19.00 42.5  14.70
Guatemala    100   3.00  79.8  7.00  37.2  8.00  100  19.00  75.0  14.00
Honduras     100   3.90  83.5  2.70  27.8  0.50   100  7.00   100  12.40
Nicaragua    100   0.04  53.1  4.00  53.1  4.40  88.4 16.00  58.4   2.40
Paraguay     100   0.70  92.9  13.00    0    ?   66.9 28.00  86.8  45.00
Peru         100   2.80  100   8.60  57.4  4.40  100   9.60   0    23.60(sup g)
Venezuela   100    2.10   100  14.40 31.0  9.40   100 10.70   100  13.20
---------------------------------------------------------------------------
(sup a)Coverage of screening = (number of screened donors  total number
of donors) x 100.
(sup b)Data as reported by the countries from 1993, except for Ecuador and
Paraguay, which were for 1994.
(sup c)HBsAg only.
(sup d)Coverage.
(sup e)Prevalence.
(sup f)Screening not performed and prevalence not known.
(sup g)Data from a survey of 2,237 samples.
---------------------------------------------------------------------------
Estimating Potential Infectivity of the Blood Supply
The probability of receiving an infected transfusion unit _P(R)_ in each
country was estimated by multiplying the prevalence of a specific infection
by 1-level of screening (Table 1). For those estimates, the sensitivity and
specificity of the different tests were taken into account. As the overall
assumed sensitivity of HIV screening was 99.99%, the adjustment of
prevalence rates makes no material difference to the precision of the
figures in Table 1. The probability of getting a transfusion-transmitted
infection _P(I)_ was calculated as the result of the probability of
receiving an infected transfusion _P(R)_ multiplied by the infectivity
risk. For countries reporting 100% of screening coverage for a specific
disease, a residual _P(R)_ was estimated as prevalence x 1-screening
sensitivity (Table 2). Infectivity risk (defined as the likelihood of being
infected when receiving an infected transfusion unit) was assumed to be 90%
for HIV (13), 75% for HBV (14), 90% for HCV (15), and 20% for _T. cruzi_
(16) (Table 2). Estimates for transfusion-acquired syphilis are not
presented because the infectivity risk depends on length of refrigeration
(17).
Table 2. Probability of receiving an infected transfusion _P(R)_(sup a) and
probability of getting a transfusion-transmitted infection _P(I)_ (sup b),
by country(sup c)
---------------------------------------------------------------------------
           HIV (x10(sup   HBV  (x10(sup   HCV (x10(sup      _T. cruzi_
                4))           4))             4))          (x10(sup 4))
Country    _P(R)_ _P(I)_  _P(R)_  _P(I)_  _P(R)_ _P(I)_   _P(R)_    _P(I)_
---------------------------------------------------------------------------
Bolivia     0.64  0.57    17.27   12.95  NSP(sup   NSP    1096.38  219.28
                                            d)
Chile       0.00   0.00    0.26   0.20    46.46  41.82    29.36     5.87
Colombia    0.24   0.22   1.20     0.90   74.55   67.09   124.24    24.85
Costa                    0.45(sup
Rica        0.00   0.00    x)      0.34    NSP    NSP      NSP       NSP
Ecuador     1.05   0.95    4.52   3.39    10.33   9.38     10.29    2.06
El
Salvador    0.00   0.00    3.23    2.42   18.87  16.97     88.75    17.75
Guatemala   0.00   0.00   14.28   10.71   55.26   49.74    36.75    7.35
Honduras    0.00   0.00    4.49    3.37    3.97   3.57   13.02(sup   2.60
                                                            x)
Nicaragua   0.00   0.00   18.95   14.21   22.70   20.43    10.48     2.10
Paraguay    0.00   0.00    9.32    6.99    NSP     NSP     62.37    12.47
Peru        0.00   0.00  0.87(sup  0.65   20.62   18.56   247.80    49.56
                            x)
Venezuela   0.00   0.00  1.45(sup  1.09   71.35   64.21 13.86 (sup  2.77
                            x)                              x)
---------------------------------------------------------------------------
(sup a)_P(R)_ = probability of receiving an infected transfusion =
prevalence of infection x 1- level of screening; (sup x)for countries in
which reported screening level was 100%, a residual _P(R)_ was estimated
as prevalence x 1- screening sensitivity rate x 10,000.
(sup b)_P(I)_= probability of getting a transfusion-transmitted infection
= _P(R)_ (sup x) infectivity index (infectivity indexes used were HIV=90%;
HBV=75%; HCV=90%; _T.cruzi_=20%). For calculations of _P(R)_ and _P(I)_
the prevalence was corrected taking into account the sensitivity of the
screening.
(sup c)Data from 1993, except for Ecuador and Paraguay, which were for
1994.
(sup d)No screening performed, so _P(R)_ and _P(I)_ not known.
---------------------------------------------------------------------------
Considering the low prevalence rates and the incompleteness of HIV
screening, only Bolivia, Colombia, and Ecuador could have missed detecting
an HIV-infected transfusion unit; the probability of getting an infection
in these countries was estimated at 0.57, 0.22, and 0.95 per 10,000
transfusions, respectively. For HBV and HCV this risk is higher. Up to
14.21 HBV infections (Nicaragua) and 67.09 HCV infections (Colombia) per
10,000 transfusions may have occurred. The highest risk for
transfusion-transmitted infection was estimated for _T. cruzi:_ 219.28
per 10,000 and 49.56 per 10,000 for Bolivia and Peru, respectively, and
approximately 2 to 24 per 10,000 for the other seven countries (Table 2).
Table 3 shows estimates of the absolute number of infections that may have
been induced by transfusion, calculated as [no. of donors x _P(I)_], for
each country. Because Chile (6) and Peru (3) reported fractionation of
blood units by 1.85 and 1.5, respectively, the estimated number of infected
units transfused in those countries was multiplied by these factors. For
the remaining countries, it was assumed that each donated unit was given to
only one recipient. An index of infectious disease spread through blood
transfusion was calculated by dividing the estimated total number of
transfusion-related infections (for any one of the infectious agents
considered) by the total number of donors. This index indicates the health
risks associated with blood transfusion and can be used as an outcome
indicator to assess the cost-effectiveness of screening programs.
Table 3. Estimates of transfusion-transmitted infectious diseases, by
country (sup a)
---------------------------------------------------------------------------
                                                  Infection
                                                  spreading
                    Absolute no. of transfusion-  index(sup
                       transmitted infectious     c)          Ratio of
           No. of         diseases (sup b)        /10 (sup    infections:
Country    donors  -------------------------------4)          donations
                                    _T.
                   HIV HBV   HCV    cruzi_  Total
---------------------------------------------------------------------------
                             NA
Bolivia    37,9
48  2   49    (sup   832     883   233         1:43
                             e)
Chile
(sup d)    217,312 0   8     1681   236     1925  88          1:113
Colombia   352,316 8   32    2364   875     3279  93          1:107
Costa                  2
Rica       50,692  0   (sup  NA     NA      NA    NA          NA
                       f)
Ecuador    98,473  9   33    92     20      154   16          1:639
El
Salvador   48,048  0   12    82     85      179   37          1:268
Guatemala  45,426  0   49    226    33      308   68          1:147
Honduras   27,885  0   9     10     7 (sup  26    9           1:1072
                                    f)
Nicaragua  46,001  0   65    94     10      169   37          1:272
Paraguay   32,893  0   23    NA     41      64    19          1:514
Peru (sup              4
g)         52,909  0   (sup  147    393     544   103         1:97
                       f)
                       22
Venezuela  204,316 0   (sup  1312   57 (sup 1391  68          1:147
                       f)           f)
---------------------------------------------------------------------------
(sup a)Data from 1993 except for Ecuador and Paraguay, which were for
1994.
(sup b)Number of cases transmitted by blood transfusion = [number of
donors x _P(I)_ ]. For calculations of number of infections, the
prevalence was corrected taking into account the sensitivity of the
screening.
(sup c)Infection spreading index = (total number of infections transmitted
 number of donors) x 10,000.
(sup d)Fractionation index = 1.85.
(sup e)Data not available.
(sup f)Residual infectivity considering that sensitivity of diagnostic
tests is not 100%.
(sup g)Fractionation index = 1.5.
---------------------------------------------------------------------------
The highest value for the infection spreading index was obtained for
Bolivia, where 233 transfusion-related infections may have occurred per
10,000 donations. This was a result of a very high prevalence rate of
antibodies to _T. cruzi_ and a lower level of screening. For most other
countries considered, the index was 68 to 103 infections per 10,000
donations. Due to low seroprevalence rates and good screening levels in
some cases, the risk for transfusion-related infections was relatively low
in Honduras (nine per 10,000), Ecuador (16 per 10,000), and Paraguay (19
per 10,000).
Table 3 also shows the ratio of number of infections per donation by
country. One infection (HIV, HBV, HCV, or _T. cruzi_) might have been
transmitted in every 43 (Bolivia) to 1,072 (Honduras) donated units.
Screening Costs
The unitary cost for serologic screening, estimated solely from
expenditures on the least expensive laboratory reagents in each country and
considering the prevalence rates reported by the countries was US$0.9 to
US$2.4 for an HIV enzyme-linked immunosorbent assay (ELISA), US$0.5 to
US$3.5 for HBV screening (enzyme immunoassay, radioimmunoassay, or passive
reverse hemagglutination), US$3.5 to US$10.0 for HCV ELISA, US$0.25 to
US$1.0 for a _T. cruzi_ test (ELISA, radioimmunoassay, or indirect
hemagglutination) (Table 4), and US$0.09 to US$0.60 for syphilis serology
(RPR or VDRL). Using other tests might have increased the costs
significantly for some of the infections. For example, the rapid
agglutination test for HIV is usually more expensive than ELISA.
Table 4. Estimated unitary cost of preventing transfusion-transmitted
infections, by country, 1993 (sup a)
---------------------------------------------------------------------------
                                        Cost (US$)
          ----------------------------------------------------------------------
                 HIV           HBV (sup b)          HCV           _T. cruzi_
          ----------------------------------- ----------------------------------
                  Preventing       Preventing       Preventing       Preventing
                     one              one               one              one
          Single   infected Single  infected  Single infected  Single infected
Country     test     unit    test     unit     test    unit     test    unit
--------------------------------------------------------------------------------
                              1.2              3.5               NA
Chile       2.3      676     (sup     599     (sup d    547     (sup     NA
                              c)                )                e)
Costa                         0.5
Rica        1.1     3,280    (sup     111       NA      NA       NA      NA
                              c)
Ecuador                       1.0                               0.35
(sup f)     1.7     1,708    (sup     263      10.0    7,136    (sup     175
                              c)                                 g)
El          2.0     1,550     1.9     238      4.5     1,802    1.0      68
Salvador                     (sup                               (sup
                              c)                                g,h)
                                                                1.0
                              1.7                               0.9
Guatemala   1.8      601     (sup     243      3.5      438     (sup     65
                              c)                                 g)
Honduras    0.9      232      0.9     334      3.5     6,971    0.45     36
                             (sup     186                       (sup
                              c)                                 h)
                              0.5
                             (sup
                              h)
                              0.5                               0.5
Nicaragua   1.0     23,000   (sup     125      3.5      797     (sup     209
                              h)                                 h)
Peru        2.4      858      3.5     407      8.2     1,862    0.25     11
(Lima)                       (sup     279                       (sup
                              c)                                 g)
                              2.4
                             (sup
                              i)
Venezuela   1.3      619      1.3      90      4.5      479     0.5      38
                             (sup                               0.3      23
                              c)                                (sup
                                                                 g)
--------------------------------------------------------------------------------
(sup a)Cost of preventing (=detecting) one infected unit was calculated as
[(number of donors x test cost)  (total number of
positive donors detected)]. All costs refer to enzyme-linked immunosorbent
assay, unless otherwise indicated.
(sup b)HBsAg only
(sup c)Enzyme immunoassay.
(sup d)Estimated cost based on cost of test in other countries.
(sup e)Data not available.
(sup f)Donors and prevalence for 1994, costs for 1993.
(sup g)Indirect hemagglutination.
(sup h)Radioimmunoassay.
(sup i)Passive reverse hemagglutination.
--------------------------------------------------------------------------------
The cost of preventing the transfusion of one infected unit was estimated
as [(no. of donors x cost of each test)/ total number of positive donors]
as reported by each country. This value represents the cost of detecting
one unit positive for any one of the infections studied in each country by
using one diagnostic test for each infectious disease. For example, using
two tests, one for antibody detection and one for antigen detection of HIV,
increases costs. Detection of _T. cruzi_ was the least expensive
(US$11-$209 per positive unit), followed by HBV (US$90-US$599 per unit),
HCV (US$438-$7,136 per unit), and HIV (US$232-$23,000 per unit) (Table 4).
The wide variation of cost primarily reflects differences in the prevalence
of each infection and in the cost of each test in the countries.
The costs per capita to carry out a complete screening of blood donors in
each country was US$0.008 to US$0.04 for HIV, US$0.008 to US$0.02 for HBV,
US$0.01 to US$0.08 for HCV, US$0.0008 to US$0.003 for syphilis, and
US$0.0025 to US$0.009 for _T. cruzi._
Limitations of the Data
Difficulties and limitations of the use of public health data for policy
decisions, even in industralized countries, are well recognized. Figures
presented here were generated to establish an approximation of the problem
by providing an overview of the risk of receiving tainted blood in
different countries in Central and South America. One potential cause of
underestimation of viral infections transmitted by blood is the residual
risk because of the window period, even when 100% of donors are screened by
serology (8,9). However, this residual risk would be difficult to ascertain
in most countries of Central and South America. Investigation of clinically
identified cases after a transfusion, follow-up of recipients for
seroconversion, and special laboratory studies detecting seronegative
donors for missed infections are laborious and expensive and could seldom
be undertaken in those countries. Another possibility would be studies that
combine estimates of incidence rates of infection among repeated and
first-time donors (who seroconvert) with estimates for the duration of the
preseroconversion period for a specific infectious agent (22). Excellent
results were obtained by this method in the United States, where more than
80% of donations come from repeat donors. Those studies involved hundreds
of thousands of donors and millions of donations. To have incidence data on
repeat donors, it is necessary to have a significant number of voluntary
donors who will repeat donations. Therefore, it is unlikely that studies of
that sort could be carried out in the countries mentioned here. First, the
population of the countries is much smaller; therefore, the number of
donations is smaller. For example, in all Central America the number of
donations is approximately 210,000 per year. Second, the number of repeat
donations from voluntary donors is small. Voluntary donors accounted for
30% and 40% of all donations in Colombia and Costa Rica and 4% to 10% of
all donors in Chile, Bolivia, Peru, and Venezuela (2-6). The number of
voluntary donors was also small in the remaining countries. In all
countries of Central and South America, most donations come from directed
donors, relatives or friends of patients. In addition, there is no national
registry of donors to allow for follow-up. Using incidence rates for
first-time donors instead of repeat donors is not a solution because
official incidence rates for HIV or other viruses were not available at the
time of this study.
The risk for transfusion-related infection could also be overestimated.
Recipients may already be infected. This is especially likely for _T. cruzi
_ infection in Bolivia, where the seroprevalence in the general population
could be higher than 20% (18,19). Another source of overestimation is that
only some of the cases detected by screening would be confirmed. In several
countries, a confirmatory test is mandatory for HIV, syphilis, and HCV.
However, as the primary function of blood banks is donor screening,
seropositive donors for any of the diseases mentioned here are usually
referred to specialized services or reference laboratories for confirmation
of the results of the screening, and if results are confirmed, for
treatment and counseling. Results of this confirmatory serology are not
often sent back to the blood bank, even when privacy concerns allow for it.
Chile was the only country that reported results of confirmatory tests for
HIV: results indicated that only 9% of those found positive by the
screening were confirmed positive (6). With _T. cruzi_, as there is no
confirmatory test, it is assumed that a true positive is a unit that is
positive on more than one test. By these criteria, a recent study in Brazil
suggested that only one out of five donors positive for _T. cruzi_ could
be considered a true positive (23). Those facts, however, do not reduce the
public health relevance of the problem presented here, although the real
numbers of potentially infected units/infected persons may be still lower
than our estimates.
Establishment of a screening process in every country will depend on
balancing the benefits and costs. Although costs for preventing transfusion
of one tainted unit or preventing one infection seem high for some
etiologic agents, they are not so. Even in the case of Nicaragua, the
country with the lowest HIV prevalence, the cost to prevent the transfusion
of one potentially HIV-infected unit (by testing all donors with ELISA) was
estimated at US$23,000, while treatment costs (drugs only) for an AIDS
patient would be approximately US$12,000 per year.
In general, the risk for an infectious disease through tainted transfusion
is not as high as that reported from some countries of Africa (24). Since
1993, donor screening has improved in several countries. Chile, Colombia,
Costa Rica, and Venezuela, for example, have made screening for HCV
mandatory, and coverage for serology for that infection has increased in
those countries, as well as in El Salvador and Honduras. _T. cruzi_
screening is now mandatory in Colombia, and the percentage of screened
donors not only increased in Colombia but also in Ecuador, El Salvador, and
Paraguay. Laws to regulate blood transfusion practices have been enacted in
Bolivia, Guatemala, and Peru. The figures presented, however, underline the
need for improvement and stress the importance of an information system
that allows assessing the level of screening for infectious diseases in the
blood supply. Universal screening of donors for HCV is still a priority in
most countries, and increased donor screening for _T. cruzi_ is a priority
for Bolivia and possibly for Peru.
Continuous collection of the type of information shown here, which has only
been partially available (1), provides a baseline against which future
achievements can be measured and is essential for obtaining the support
needed to maintain or expand the screening of blood donors.
Address for correspondence: Gabriel A. Schmunis, Pan American Health
Organization-PAHO, 525 23th Street NW, Washington, D.C. 20037, USA; fax:
202-974-3688; e-mail: schmunig@paho.org.
References
  1. Linares J, Vinelli E, editors. Taller Latinoamericano de servicios de
     transfusin sangunea y ptimo uso de los recursos. Cruz Roja
     Finlandesa; 1994. p. 167.
  2. Organizacin Panamericana de la Salud. Taller para el control de
     calidad de sangre en transfusiones: serologa para la deteccin de
     Chagas, hepatitis B y C, sfilis y HIV/SIDA. Document
     OPS/HPC/HCT/94.42.
  3. Organizacin Panamericana de la Salud. Pases andinos. Taller sobre
     control de calidad de sangre en transfusiones: serologa para la
     deteccin de hepatitis B y C, sfilis, tripanosomiasis americana y
     VIH/SIDA. Document OPS/HCP/HCT/95-61.
  4. Organizacin Panamericana de la Salud. Simposio internacional sobre
     control de calidad en bancos de sangre del Cono Sur y de Brasil.
     Informe final OPS/HCP/HCT/95.55.
  5. Organizacin Panamericana de la Salud. Taller sobre control de calidad
     en serologa de bancos de sangre. OPS/HCP/HCT/96/79.
  6. Ministerio de Salud, Chile. Diagnstico de la situacin de los bancos
     de sangre y medicina transfusionl en Chile 1993. Santiago, Chile: La
     Ministerio; 1995. Ser Inf Tc No.14.
  7. Pan American Health Organization. Health conditions in the Americas.
     Washington (DC): The Organization; 1994. PAHO Sci Pub No. 549.
  8. Lackritz EM, Satten GA, Aberle-Grasse J, Dodd RY, Raimondi VP, Janssen
     RS, et al. Estimated risk of transmission of the human
     immunodeficiency virus by screened blood in the United States. N Eng J
     Med 1995;333:1721-5.
  9. Schreiber GB, Busch MP, Kleinman SH, Korelitz JJ. The risk of
     transfusion-transmitted viral infections. N Eng J Med
     1996;334:1685-90.
 10. Van de Poel CL, Cuypers HT, Reesink HW, Hepatitis C virus six years
     on. Lancet 1994;344:1475-9.
 11. Andrade ANS, Martelli CMT, Luquetti AO, Oliveira OS, Almeida e Silva
     S, Zicker F. Triagem sorologica ra o Trypanosoma cruzi entre doadores
     de sangue do Brasil central. Bol Oficina Sanit Panam 1992;113:19-27.
 12. Lorca MH, Child RB, Garcia AC, Silva MG, Osorio JS, Atias M.
     Evaluacin de reactivos comerciales empleados en el diagnstico de la
     enfermedad de Chagas en bancos de sangre de Chile. I Seleccin de
     reactivos. Rev Med Chile. 1992;120:420-6.
 13. Donegan E, Stuart M, Niland JC, Sacks HS, Azen SP, Dietrich SL, et al.
     Infection with human immunodeficiency virus type 1 (HIV-1) among
     recipients of antibody-positive blood donations. Ann Intern Med
     1990;113:733-9.
 14. Gocke DJ. A prospective study of post-transfusional hepatitis. JAMA
     1972;219:1165-70.
 15. Aach RD, Stevens CE, Hollinger FB, Mosley JW, Peterson DA, Taylor PE,
     et al. Hepatitis C virus infection in post-transfusion hepatitis. An
     analysis with first- and second-generation assays. N Engl J Med
     1991;325:1325-9.
 16. World Health Organization. The control of Chagas disease. Geneva: WHO
     Tech Rep Ser No.811:32;990.
 17. National Institutes of Health. Infectious disease testing for blood
     transfusion. NIH Consens Statement 1995;13:13-4.
 18. Schmunis GA. _Trypanosoma cruzi_, the etiologic agent of Chagas
     disease: status in the blood supply in endemic and non endemic
     countries. Transfusion 1991;31:547-55.
 19. Schmunis GA. American trypanosomiasis as a public health problem.
     Chagas disease and the nervous system. PAHO Sci Pub 994;547:3-29.
 20. Pedersen C, Lindhardt BO, Jensen BL, Lavritzen E, Gerstoft J,
     Dickmeiss E, et al. Clinical course of primary HIV infection:
     consequences for subsequent course of infection. BMJ 1989;299:154-7.
 21. Alter M. Residual risk of transfusion associated hepatitis. In:
     Program and Abstracts of the National Institutes of Health Development
     Conference on Infectious Diseases Testing for Blood Transfusions. 1995
     Jan 9-11; Bethesda (MD): National Institutes of Health;1995. p. 23-7.
 22. Busch MP. Incidence of infectious disease markers in blood donors,
     implications for residual risk of viral transmission by transfusion.
     In: Program and Abstracts of the National Institutes of Health
     Development Conference; 1995. Jan 9-11; Bethesda (MD): National
     Institutes of Health; 1995. p. 29-30.
 23. Hamerschlak N, Pasternak J, Amato Neto V, Carvalho MB, Guerra CS,
     Coscina AL, et al. Chagas' disease, an algorithm for donor screening
     and positive donor counseling. Rev Soc Bras Med Trop 1997;30:205-9.
 24. McFarland W, Mvere D, Shandera W, Reingold A. Epidemiology and
     prevention of transfusion-associated human immunodeficiency virus
     transmission in sub-Saharan Africa. Vox Sang 1997;72:85-92.
------------------------------------------------------------------------
Perspectives
Calicivirus Emergence from Ocean Reservoirs:  Zoonotic and
Interspecies Movements
Alvin W. Smith,* Douglas E. Skilling,* Neil Cherry, Jay H. Mead, and
David O. Matson
*Oregon State University, Corvallis, Oregon, USA; Lincoln University, New
Zealand; Red Cross, Portland, Oregon, USA; and Children's Hospital of the
King's Daughters, Eastern Virginia Medical School, Norfolk, Virginia, USA
---------------------------------------------------------------------------
      Caliciviral infections in humans, among the most common causes
      of viral-induced vomiting and diarrhea, are caused by the
      Norwalk group of small round structured viruses, the Sapporo
      caliciviruses, and the hepatitis E agent. Human caliciviruses
      have been resistant to in vitro cultivation, and direct study
      of their origins and reservoirs outside infected humans or
      water and foods (such as shellfish contaminated with human
      sewage) has been difficult. Modes of transmission, other than
      direct fecal-oral routes, are not well understood. In contrast,
      animal viruses found in ocean reservoirs, which make up a
      second calicivirus group, can be cultivated in vitro. These
      viruses can emerge and infect terrestrial hosts, including
      humans. This article reviews the history of animal
      caliciviruses, their eventual recognition as zoonotic agents,
      and their potential usefulness as a predictive model for
      noncultivatable human and other animal caliciviruses (e.g.,
      those seen in association with rabbit hemorrhagic disease).
In vitro cultivation of caliciviruses indicates that these pathogens have
been emerging periodically from ocean sources for 65 years (1). The
best-documented example of ocean caliciviruses causing disease in
terrestrial species is the animal disease vesicular exanthema of swine
(VES) (1). Feline calicivirus (the only member of the group with a
seemingly ubiquitous and continuous terrestrial presence) also appears to
have ocean reservoirs (2). The source of caliciviruses causing
gastroenteritis in humans is frequently shellfish, which do not always come
from beds contaminated with human waste (3,4). The origins of hepatitis E
are often obscure, but water is one suspected source (5). The most recent
emerging calicivirus is associated with rabbit hemorrhagic disease (RHD),
and although an ocean association has not been reported, the agent readily
moves between continents and crosses ocean channels (6). Finally, the only
reported in vitro isolation and sequential propagation of a calicivirus
pathogenic for humans is a virus residing in the sea (7).
The Caliciviridae are divided into five groups, tentatively designated
distinct genera, on the basis of sequence relatedness and genomic
organization (8). Four are known human pathogens-Sapporo, Norwalk-like
small round structured viruses, hepatitis E, and the marine (animal)
caliciviruses-while the fifth group, which includes RHD virus, is not yet
proven to be a human pathogen. The human Sapporo viruses are more closely
related to the marine caliciviruses than to the other human group causing
gastroenteritis, the Norwalk-like viruses. On the basis of homology and
genomic organization, RHD virus falls between these two groups. In
addition, the genomic organization of hepatitis E is most closely related
to that of the only other hepatotropic calicivirus currently described, RHD
virus (8).
Many marine calicivirus strains in the tentative genus of VES virus-like
caliciviruses have been passaged in vitro; their characterization has
facilitated understanding of calicivirus geographic distribution and host
versatility (1). Dozens of serotypes were described on the basis of serum
neutralization tests; this antigenic complexity complicated serodiagnosis
and hampered studies of effects on host species.
The illnesses associated with two recently discovered viruses classified as
Caliciviridae, hepatitis E virus and RHD virus, have altered the notion
that caliciviruses produce only transient clinical disease but not death
(1,9,10). Hepatitis E virus is fatal for 25% of the pregnant women in
developing countries who contract hepatitis E (11); RHD can kill 95% of
infected rabbits within 24 to 48 hours of exposure (6).
The oceans are reservoirs in which caliciviruses are exposed in a water
substrate to life forms from zooplankton to whales and in which they, like
other RNA viruses, can amplify to very high numbers with variants occurring
in every replicative cycle (12). Such a varied replicative setting has
served this parasite well. With the right tools, evidence of previous
infection with caliciviruses can often be shown in fish, avian, and many
mammalian species, including humans (1). It is not known why some
caliciviruses have become potential hemorrhagic agents associated with
purpura hemorrhagica in aborted piglets (13), neonatal hemorrhagic syndrome
in pinnipeds (A.W. Smith, D.E. Skilling, unpub. data), hemorrhagic disease
in fatal hepatitis E in humans (5), and RHD (6). However, it is known that
caliciviral diseases can be difficult or impossible to contain and
eradicate. Pathogenic caliciviruses can be expected to continue emerging
from the sea in unexpected forms at unexpected times in unexpected places.
Studying those that have emerged and are compliant to in vitro propagation
can provide insights into those that cannot be cell-culture adapted and
those yet to be discovered.
History and Its Lessons
The 66-year history of the caliciviruses with ocean reservoirs can be
divided into three periods: 1932 to 1972, the species-specific era (14);
1972 to 1982, the new era of virology, during which oceans were first found
to be reservoirs of viral disease infecting domestic animals (7,9); and
1976 to the present.
The first evidence of infection with caliciviruses of marine origin can be
traced to 1932. A large herd of swine in Orange County, California, was
being fed raw garbage collected from restaurants and institutions in the
Los Angeles area. When some animals became sick with vesicular lesions on
the feet and nose, regulatory veterinarians were notified because vesicular
diseases of livestock were reportable. The farm with sick swine and
adjacent farms were quarantined for foot-and-mouth disease, and more than
19,000 head of exposed cattle and swine were destroyed and buried in
quicklime (14). The outbreak was contained. One year later and 100 miles to
the south in San Diego, California, the second known outbreak occurred and
was contained (14). This time the disease was found not to be
foot-and-mouth disease, because the virus would not infect cattle, but
instead was described as a new disease of swine and was called VES (14). In
1934, a third outbreak occurred in San Francisco, California, and VES was
again contained (14). In 1935, the events repeated themselves, but from
1936 through mid-December 1939, the disease disappeared and then abruptly
reappeared, at times involving 40% of California swine herds. All of these
outbreaks in the 1930s and 1940s were shown by cross-infectivity studies to
be caused by many distinct but related VES virus strains.
The embargoes placed on raw California pork were successful in containing
VES within California until 1952. That year, a passenger train between San
Francisco and Chicago served California pork and discarded the raw pork
trimmings into the garbage in Cheyenne, Wyoming; the garbage was fed to
swine subsequently redistributed by auction sale yard. Within 14 months,
all major swine-growing areas in the United States (41 states) had reported
VES. For the first time, the federal government, rather than just the
California state government, activated eradication and quarantine measures
against VES, including enforcement of federal laws requiring garbage to be
cooked before it was fed to swine. By 1956, the last reported outbreak of
VES had been contained, and the disease was said to have been eradicated.
In 1959, VES was declared a foreign animal disease, even though it had
never been reported outside the United States (14).
Forty years later, the natural history of this calicivirus still contained
few details. Its origins were not known but were said to have been de novo
or from some unknown wild animal reservoir, which was extensively sought
but not found (14,15). Swine were the only naturally infected host species;
no evidence of human infection had been observed (14). Control of VES had
been a notable success story for regulatory veterinary medicine in the
United States; within 24 years of its discovery as an entirely new disease,
it was said to have been eradicated (14). Internationally accepted animal
disease diagnostic tests using swine, horse, and bovine infectivity
profiles for vesicular stomatitis virus, foot-and-mouth virus, and VES
virus were used routinely; VES virus did not infect cattle, whereas the
other viruses did (14,15).
The discovery of a new paradigm in "viral traffic" (16) began the second
period of calicivirus history. The movement of a member of the
Caliciviridae from ocean reservoirs to terrestrial hosts changed the
understanding of the natural history of a virus thought to be host specific
and eradicated (1,15,17).
The first virus isolate from a pinniped occurred in 1972. The agent, named
San Miguel sea lion virus type 1 (SMSV-1), was a calicivirus that caused
classic VES in swine (17). Thus began a series of isolating and
characterizing viruses in ocean species that were officially designated as
"viruses indistinguishable from VES virus" because they were additional VES
virus types. They could not be called VES virus (18) since VES had been
officially eradicated. Should the VES virus reappear, its status as a
foreign animal disease would mandate immediate implementation of
eradication measures; eradication was viewed as an impossibility because of
the wide range of reservoirs for VES virus, both migratory and ocean
species. The 13 marine caliciviruses serotypes (100 TCID50 vs. 20 units of
neutralizing antibody) isolated from swine before 1956 were called VES
viruses; those isolated after 1972 have been designated SMSV-1 through 17
or given more proper nomenclature, e.g., the pygmy chimpanzee isolate
(primate calicivirus Pan paniscus type 1) (1,19).
By 1982, 11 species of pinnipeds and cetaceans of the North Pacific Ocean
and Bering Sea (monk seals, California sea lions, northern sea lions,
northern elephant seals, northern fur seals, walrus, gray whales, sei
whales, sperm whales, bowhead whales, and Pacific bottlenosed dolphins)
were known to be susceptible to calicivirus infection, as was an ocean
fish, the opaleye perch (Girella nigricans) (20). Furthermore, in many
instances, the virus had crossed the intertidal zone to infect terrestrial
species (18). On the basis of these data and the established ocean ranges
of known calicivirus host species, the shores of Mexico, the United States,
Canada, Russia, Korea, Japan, China, and perhaps others bordering the North
Pacific Ocean had been regularly exposed to large numbers of marine
caliciviruses with unknown host ranges and tissue trophisms (21). By this
time, type-specific neutralizing antibodies to two of four serotypes tested
were reported in human patients in the United States (22). Cumulatively,
these findings lead to the conclusion that fish and perhaps other ocean
products provide a vehicle for transmission of these marine caliciviruses
to terrestrial animals.
The magnitude of potential exposure to marine caliciviruses from the sea is
substantial. For example, a 35-ton gray whale, shown by electron microscopy
to have more than 106 caliciviruses per gram of feces, can eat 5% or more
of its body weight per day and eliminate an equivalent quantity of feces
containing an estimated 1013 caliciviruses daily. Marine caliciviruses
remain viable more than 14 days in 15C seawater (20).
Although marine mammals were often infected, fish and fish products were
more likely to transport the virus from sea to land (23). In contrast to
the 1932 to 1936 introductions of VES from raw fish, the rapid and
uncontrolled spread of VES virus throughout California after 1939 and then
across the United States in 1952 was a pig-to-pig cycle through raw garbage
feed. However, new virus serotypes were also introduced through feeding raw
fish scraps to swine (1,15). That VES was not a species-specific disease
became accepted, but the possibility of human infection, although suspected
(22), was largely untested.
During the third historical period (1976 to present), which overlaps with
the second, viral traffic across the land/sea interface has been observed
repeatedly, as the following examples show. A calicivirus isolated from an
opaleye perch and designated SMSV-7 produced fulminating VES in exposed
swine and spread from pig to pig by contact transmission (23). A reptilian
calicivirus Crotalus-1 was isolated from three species of snakes and one
species of amphibian (24) and from three species of marine mammals whose
population distributions spanned the North Pacific from Mexico to the
Bering Sea (1). Mink fed a diet of ground-up calicivirus-infected seal meat
became infected with VES virus (25). Parasitic nematode larvae from
California sea lions in San Diego, California, were used to infect opaleye
fish with calicivirus (SMSV-5); when the fish were killed and fed to
Northern fur seals on the Pribilof Islands in the Bering Sea 30 days later,
the seals developed vesicular disease, and the virus was recovered from the
lesions (1). Shellfish resident to the tidal zone were exposed to marine
caliciviruses and held at less than 10C in a continuous flow of sterile
seawater. The caliciviruses were reisolated 60 days later in mammalian cell
lines (26). When tested with a cDNA calicivirus group-specific
hybridization probe from a marine calicivirus (SMSV-5), some shellfish beds
on U.S. coasts were positive for caliciviruses of unknown type (26). Feline
calicivirus was shown to cause disease not only in dogs, but also in seals
(on the basis of 17 of 20 adult sea lions having neutralizing antibody to
FCV-F9 with titers of 1:15 to 1:220). Only 11 of 20 of these sea lions had
neutralizing antibody to a sea lion isolate, SMSV-13 (titer 1:10). This
demonstrates a probable feline calicivirus ocean presence in California sea
lions (2). In swine, a so-called mystery pig disease (porcine respiratory
reproductive syndrome) was reproduced in pregnant sows in 1992 with a
three-plaque passage purified cytolytic calicivirus isolate from stillborn
piglets with mystery pig disease (13). A second calicivirus serotype
isolated from the same piglets was the same as that isolated from walruses
in 1976 (1). A white tern (Gygis alba rothschildi), a migratory sea bird
sampled in the mid-Pacific (French Frigate Shoals), had a blistering
disease caused by a calicivirus (27). In the first fully documented human
case of clinical disease caused by a marine calicivirus, SMSV-5 was
isolated from blisters on the hands and feet of a patient (7). A second,
less well-documented, case involved a field biologist who was handling sea
lions and developed severe facial blistering. An untypable calicivirus was
isolated in tissue culture (Vero cells) from throat washings (7).
Extent of Exposure
The extent of human disease is not known because test reagents are not
readily available and diagnosticians are not alerted to caliciviral causes
of human disease, except for diarrhea and occasionally hepatitis. However,
evidence of human exposure was shown when 150 serum specimens from normal
blood destined for donor use were tested. The samples were
antibody-negative for hepatitis B surface and core antigen, HIV-1 and -2,
HIV P-24 antigen, human T-cell lymphotropic virus Type 2, and hepatitis C
virus. Approximately 19% had antibodies reactive to a polyvalent antigen
made up of equal quantities of cesium chloride-banded SMSV-5, 13, and 17.
(Figure 1A). To demonstrate that these reactions were not cross-reactions
to the human Norwalk calicivirus antibody, serum samples from eight persons
with Norwalk virus-induced diarrhea were also tested. Both acute- and
convalescent-phase serum specimens were tested by enzyme-linked
immunosorbent assay with the same SMSV antigens and Norwalk capsid protein
(Figure 1B, C, and D). Although cross-reactivity was not detected, the
serum samples may still have been able to cross-react with the calicivirus
causing hepatitis E or Sapporo calicivirus, which were not tested. However,
sera typed to all 40 marine caliciviruses reacted negatively when tested
against Sapporo antigen (data not shown). These results suggest possible
human exposure and antigenic response to marine caliciviruses. If that is
not the case, such results present a confusing diagnostic picture of
calicivirus exposure and diagnosis of human disease.
                        [fig]
Figure 1. A) 150 blood donor sera tested against a polyvalent antigen
containing San Miguel sea lion viruses (SMSVs) 5, 13, and 17 purified by
CsCl; B) Eight acute- and eight convalescent-phase sera from a confirmed
outbreak of Norwalk gastroenteritis tested against the polyvalent SMSVs 5,
13, 17 antigen; C) The eight acute-phase sera from the same outbreak of
Norwalk gastroenteritis tested in B also tested against the baculovirus
expressed Norwalk virus capsid protein; D) The eight convalescent-phase
sera paired with the acute-phase sera (See C) tested as in C.
Organism Characteristics
The history of marine caliciviruses demonstrates that their biologic
properties have great plasticity. The VES virus-like caliciviruses can
replicate at temperatures of 15C to 39C, have diverse tissue trophisms,
and travel by land (terrestrial reptiles, amphibians, and mammals), sea
(pinnipeds, cetaceans, teleosts, and perhaps filter-feeding mollusks), and
air (pelagic birds, e.g., the white tern). They can persist in nonlytic
cycles in many reservoir hosts, and they have a wide diversity of
successful antigenic types (1) (more than 40 serotypes on the basis of
virus neutralization, e.g., no cross-protection between types). Their
cup-like surface morphology is characteristic (Figure 2). Finally, they are
zoonotic: this is a paradigm shift (7). No other virus has been shown to
have its origins and primary reservoirs in the sea yet emerge to cause
disease in humans.
To measure calicivirus adaptivity and preclude strong presumptions of host
specificity on the basis of calicivirus type or species of origin, the
following list of 16 hosts is given for a single virus serotype, SMSV-5:
known natural hosts-five genera of seals, cattle, three genera of whales,
donkeys, fox, and humans-and susceptible hostso-paleye fish, horses,
domestic swine, and primates (1). The lists are still growing. SMSV-5 can
also persist for 60 days in shellfish, but infectivity has not been
measured (26). Feline calicivirus (FCV-F9) has an apparent ocean presence
among California sea lions and is not host-specific (2). All members of the
family Felidae are susceptible to infection, not just domestic cats. In
addition, cheetahs are susceptible; the agent has naturally infected and
caused disease in dogs and experimentally infected coyotes (28,29). Reports
of human antibody against the feline virus suggest zoonotic potential for
the feline calicivirus (30).
                        [Fig]
Figure 2. Electron photomicrograph of Cetacean Calicivirus Tursiops - 1
(CCVTur-1). Negative staining using phosphotungstic acid on a carbon-coated
grid showing typical surface cup morphologic features as commonly seen by
electron microscopy. Bar = 100 nm.
Tissue Trophisms
The broad host range and diverse mechanisms of transmission and survival of
marine caliciviruses are expected of an RNA virus quasispecies (12). If
structural simplicity associated with a capsid made up of a single protein
species and replicative strategies conserved across rather broad tissue and
phylogenetic distances is a measure, caliciviruses are primitive RNA
viruses. Caliciviral RNA replicative mechanisms are thus expected to
generate numerous mutants (perhaps as high as one to 10 per template copy
(12), which will come in contact with many pelagic and terrestrial biota.
Opportunity exists to form clusters of virus adapted across a diversity of
life forms. Actual mutation rates have not been demonstrated for the
Caliciviridae, but plaque-size reversion studies have found that the
mutation rate for this phenomenon is one per 106 replicates (14,15). In
addition, the expected versatility from RNA virus replicative infidelity
and the resulting successful adaptive mechanisms are manifested in the wide
spectrum of calicivirus tissue trophisms.
Disease conditions involving calicivirus tissue trophisms include
blistering of the skin (particularly on the appendages and around the
mouth), pneumonia, abortion, encephalitis, myocarditis, myositis,
hepatitis, diarrhea, and coagulation/hemorrhage (1,3,5,7; Table 1).
Caliciviruses have the inherent potential and adaptive mechanisms to
successfully parasitize essentially all organ systems of the many animal
species that have been examined in detail.
The Future
Calicivirus disease manifestations in animals will likely continue but will
only become well defined with improved diagnostic means. With cultivatable
marine caliciviruses as models, the role of disease-causing caliciviruses
can be further defined. Now caliciviruses infecting humans can only be
visualized by electron microscopy or histochemistry but cannot be
propagated in vitro. Thus, miscarriage and birth defects in human patients,
hepatitis other than types A through G, hand-foot-and-mouth-like diseases,
viral myocarditis, viral encephalitis of unknown etiology, and joint and
muscle disease, for example, should be examined for caliciviruses when
other causes of disease are not found.
Table 1. Calicivirus tissue trophisms
---------------------------------------------------------------------------
Disease conditions       Species affected        Calicivirus groupa
---------------------------------------------------------------------------
Skin  blistering         Cattle, cats, dogs,      VESV, SMSV,  FCV, CCV
                          humans, primates,
 			      seals, swine
Pneumonia                Cats, cattle, swine      FCV, SMSV
Abortion                 Seals, swine             VESV, SMSV
Encephalitis             Cats, primates, seals,   VESV, SMSV
                          swine
Myocarditis              Seals, swine             VESV, SMSV
Hepatitis                Humans, rabbits, swine   VESV, RHDV, HEV
Diarrhea                 Cattle, dogs, humans,    VESV, SMSV, CCV, SRSV,
                          reptiles, swine          Sapporo
Coagulation/hemorrhage   Humans, rabbits, seals,  RHDV, VESV, HEV
                          swine
---------------------------------------------------------------------------
aThe family Caliciviridae has been tentatively divided into five groups,
each proposed to be a genus. Group 1: Vesicular exanthema of swine (VESV),
San Miguel sea lion virus (SMSV), Feline calicivirus (FCV), Canine
calicivirus (CCV); Group 2: Sapporo calicivirus (Sapporo); Group 3: Rabbit
hemorrhagic disease virus (RHDV); Group 4: Hepatitis E virus (HEV); Group
5: Small round structured virus (SRSV), which includes Norwalk virus.
In the absence of data, extrapolating from cultivatable caliciviruses to
predict future effects of poorly characterized caliciviruses should be
useful, particularly when there is an urgent need to assess possible human
risk. The calicivirus implicated in RHD is a case in point for it might be
expected to infect humans.
Additional evidence exists. An anecdotal account mentions a Mexican worker
who developed antibodies to RHD while eradicating the disease in Mexico
(31). An Australian study designed to assess the risk for illness after RHD
escaped from Wardang Island (32) examined a group of 269 persons (153
reporting exposure to rabbits or samples infected with RHD virus and 116
reporting no known RHD virus contact) from two Australian states with the
greatest amount of RHD virus activity in rabbits. Exposure was categorized
by degree of skin exposure to infected materials. Date of first exposure
was noted, but no cumulative exposure index was developed. A "high"
exposure category could derive from one exposure, and "low" exposure
categories could include multiple exposures, each with low exposure.
Symptoms were assessed by recall of illness over the previous 13 months.
Because the RHD agent was in high security containment facilities for the
first 3 months of the recall period and geographically confined for the
following 3 months, that period was considered a low exposure period.
Because of the rapid spread of the virus in the two states, the last 6
months of the recall period were considered the high exposure period.
The data (Table 2) show the rate ratios for the occurrence of different
illness in the two periods. All rate ratios were considerably greater than
1.00, and the rate ratios for any illness, diarrhea/gastroenteritis,
flu/fever, and neurologic illness are significant (p < 0.005). Because each
group contained health histories for 3 spring months or 3 autumn months, 1
summer month, and 2 winter months, the data are seasonally adjusted; hence,
winter illness does not explain the excess symptoms observed in the high
exposure group, and RHD virus exposure remains a plausible explanation for
increased disease incidence.
It is difficult to produce pure cultures of noncultivatable caliciviruses
to carry out Koch's postulates and establish cause and effect for a single
pathogen strain or species. For RHD, both a calicivirus and a parvovirus
have been identified in ill rabbits, and a parvovirus has been isolated in
vitro and reported to fulfill Koch's postulates (33-35). Yet, caliciviruses
have been purified from infected organs to the limits of purity by physical
means, and those preparations also cause RHD (35). The caliciviruses
purified by physical means cannot be proven to be free of contaminating
agents, such as parvovirus (35). If RHD is parvovirus-driven, extrapolation
from what is known of other small DNA viruses suggests a rather stable
genome and a reduced host range with less likelihood of new host
relationships (12). On the other hand, if calicivirus is the primary
pathogen, the genomic infidelity that occurs during small RNA virus
replication and the documented cross-species transmission of the
cultivatable caliciviruses suggest that RHD might also move across species
barriers (1,12).
Table 2. Population incidence of rabbit hemorrhagic disease (RHD) virus for
seasonally equalized periods (July-December and February-July), derived
from Mead et al. (32)
---------------------------------------------------------------------------
                  Jul-Dec        Feb-Jul          Rate      95% Confidence
                    1995           1996           Ratio        Interval
---------------------------------------------------------------------------
Exposure to             Low         High
  RHD  virus
Any illness            112          210           1.88         1.49-2.36
Flu/fever               94          189           2.01         1.57-2.57
Diarrhea/               41          73            1.78         1.21-2.61
  gastroenteritis
Neurologic              18          49            2.72         1.58-4.67
  symptoms
Rashes/skin             3           10            3.33         0.92-12.1
Bleeding/hepatitis      2            4            2.00         0.18-22.1
---------------------------------------------------------------------------
Adequate diagnostic reagents for epidemiologic studies need to be made
available; they include antigens, monoclonal antibodies, polymerase chain
reaction primer sets, and cDNA probes based on group epitopes. In addition,
biotype- or pathotype-specific reagents are needed to differentiate
pathogenic from nonpathogenic infections.
The future also holds the confounding problem of vaccines. Although
vaccines can be produced, because of calicivirus antigenic diversity, their
efficacy would be predictably short-lived and marginal. Other approaches
will need to be sought. Conserved traits that render the Caliciviridae
viable as a virus with certain predictable genomic expressions must be
sought, and if they exist, targeted for immune attack.
Conclusions
Only one of the five known calicivirus groups can be grown in vitro and
subjected to the full range of host-parasite tests and conditions necessary
to more fully define a virus in nature. Therefore, extrapolations developed
from this group, the cultivatable marine caliciviruses, should provide
insights as a predictive model to help answer questions for the
noncultivatable caliciviruses such as small round structured virus, Sapporo
virus, hepatitis E virus, and rabbit caliciviruses. From the replicative
strategy of the Caliciviridae (as RNA viruses), one would predict
considerable diversity. In vitro cultivation has shown that caliciviruses
exhibit survivability and plasticity in nature. Many of the factors
regarding host spectrum, zoonotic potential, disease conditions, transport,
intermediate hosts, and abrupt appearance or disappearance, which may be
unknown in newly emerging calicivirus diseases (e.g., RHD), may be more
reliably predicted with an established model such as the cultivatable
marine caliciviruses. New and better biologic tools for diagnostic and
epidemiologic assessments must be developed. This should be augmented by
recognizing the zoonotic potential of the cultivatable caliciviruses of
ocean origin and then examining them as possible models to help solve many
unanswered questions for pathogenic Caliciviridae.
Acknowledgments
We thank Ms. Christine Robinette for manuscript preparation. This study was
funded by the Oregon Agricultural Experiment Station through the College of
Veterinary Medicine.
Alvin W. Smith is professor of veterinary virology and head, Laboratory for
Calicivirus Studies at the College of Veterinary Medicine, Oregon State
University, Corvallis, Oregon. Dr. Smith is interested in mechanisms for
the preservation and movement of pathogenic viruses in nature, particularly
those contained in ocean reservoirs. His research has focused on the marine
caliciviruses.
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Perspectives
Outbreak Investigations-A Perspective
Arthur L. Reingold
University of California, Berkeley, California, USA
---------------------------------------------------------------------------
      Outbreak investigations, an important and challenging component
      of epidemiology and public health, can help identify the source
      of ongoing outbreaks and prevent additional cases. Even when an
      outbreak is over, a thorough epidemiologic and environmental
      investigation often can increase our knowledge of a given
      disease and prevent future outbreaks. Finally, outbreak
      investigations provide epidemiologic training and foster
      cooperation between the clinical and public health communities.
Investigations of acute infectious disease outbreaks are very common, and
the results of such investigations are often published; however,
surprisingly little has been written about the actual procedures followed
during such investigations (1,2). Most epidemiologists and public health
officials learn the procedures by conducting investigations with the
initial assistance of more experienced colleagues. This article outlines
the general approach to conducting an outbreak investigation. The approach
applies not only to infectious disease outbreaks but also to outbreaks due
to noninfectious causes (e.g., toxic exposure).
How Outbreaks Are Recognized
Possible outbreaks of disease come to the attention of public health
officials in various ways. Often, an astute clinician, infection control
nurse, or clinical laboratory worker first notices an unusual disease or an
unusual number of cases of a disease and alerts public health officials.
For example, staphylococcal toxic shock syndrome and eosinophilia myalgia
syndrome were first noted by clinicians (3,4). Frequently, it is the
patient (or someone close to the patient) who first suspects a problem, as
is often the case in foodborne outbreaks after a shared meal and as was the
case in the investigation of a cluster of cases of apparent juvenile
rheumatoid arthritis near Lyme, Connecticut, which led to the discovery of
Lyme disease (5). Review of routinely collected surveillance data can also
detect outbreaks of known diseases, as in the case of hepatitis B infection
among the patients of an oral surgeon in Connecticut and patients at a
weight reduction clinic (6,7). The former outbreak was first suspected when
routinely submitted communicable disease report forms for several patients
from one small town indicated that all of the patients had recently had
oral surgery. However, it is relatively uncommon for outbreaks to be
detected in this way and even more uncommon for them to be detected in this
way while they are still in progress. Finally, sometimes public health
officials learn about outbreaks of disease from the local newspaper or
television news.
Reasons for Investigating Outbreaks
The most compelling reason to investigate a recognized outbreak of disease
is that exposure to the source(s) of infection may be continuing; by
identifying and eliminating the source of infection, we can prevent
additional cases. For example, if cans of mushrooms containing botulinum
toxin are still on store shelves or in homes or restaurants, their recall
and destruction can prevent further cases of botulism.
However, even if an outbreak is essentially over by the time the
epidemiologic investigation begins-that is, if no one is being further
exposed to the source of infection-investigating the outbreak may still be
indicated for many reasons. Foremost is that the results of the
investigation may lead to recommendations or strategies for preventing
similar future outbreaks. For example, a Legionnaires' disease outbreak
investigation may produce recommendations for grocery store misting machine
use that may prevent other outbreaks (8). Other reasons for investigating
outbreaks are the opportunity to 1) describe new diseases and learn more
about known diseases; 2) evaluate existing prevention strategies, e.g.,
vaccines; 3) teach (and learn) epidemiology; and 4) address public concern
about the outbreak.
Once a decision is made to investigate an outbreak, three types of
activities are generally involved-the epidemiologic investigation; the
environmental investigation; and the interaction with the public, the
press, and, in many instances, the legal system. While these activities
often occur simultaneously throughout the investigation, it is conceptually
easier to consider each of them separately.
Epidemiologic Investigation
Outbreak investigations are, in theory, indistinguishable from other
epidemiologic investigations; however, outbreak investigations encounter
more constraints. 1) If the outbreak is ongoing at the time of the
investigation, there is great urgency to find the source and prevent
additional cases. 2) Because outbreak investigations frequently are public,
there is substantial pressure to conclude them rapidly, particularly if the
outbreak is ongoing. 3) In many outbreaks, the number of cases available
for study is limited; therefore, the statistical power of the investigation
is limited. 4) Early media reports concerning the outbreak may bias the
responses of persons subsequently interviewed. 5) Because of legal
liability and the financial interests of persons and institutions involved,
there is pressure to conclude the investigation quickly, which may lead to
hasty decisions regarding the source of the outbreak. 6) If detection of
the outbreak is delayed, useful clinical and environmental samples may be
very difficult or impossible to obtain.
Outbreak investigations have essential components as follows: 1) establish
case definition(s); 2) confirm that cases are "real"; 3) establish the
background rate of disease; 4) find cases, decide if there is an outbreak,
define scope of the outbreak; 5) examine the descriptive epidemiologic
features of the cases; 6) generate hypotheses; 7) test hypotheses; 8)
collect and test environmental samples; 9) implement control measures; and
10) interact with the press, inform the public. While the first seven
components are listed in logical order, in most outbreak investigations,
many occur more or less simultaneously. The importance of these components
may vary depending on the circumstances of a specific outbreak.
Case Definition
In some outbreaks, formulating the case definition(s) and exclusion
criteria is straightforward; for example, in an outbreak of gastroenteritis
caused by Salmonella infection, a laboratory-confirmed case would be
defined as a culture-confirmed infection with Salmonella or perhaps with
Salmonella of the particular serotype causing the outbreak, while a
clinical case definition might be new onset of diarrhea. In other
outbreaks, the case definition and exclusion criteria are complex,
particularly if the disease is new and the range of clinical manifestations
is unknown (e.g., in a putative outbreak of chronic fatigue syndrome). In
many outbreak investigations, multiple case definitions are used (e.g.,
laboratory-confirmed case vs. clinical case; definite vs. probable vs.
possible case; outbreak-associated case vs. nonoutbreak-associated case,
primary case vs. secondary case) and the resulting data are analyzed by
using different case definitions. When the number of cases available for
study is not a limiting factor and a case-control study is being used to
examine risk factors for becoming a case, a strict case definition is often
preferable to increase specificity and reduce misclassification of disease
status (i.e., reduce the chance of including cases of unrelated illness or
no illness as outbreak-related cases).
Case Confirmation
In certain outbreaks, clinical findings in reported cases should be
reviewed closely, either directly, by examining the patients, or
indirectly, by detailed review of the medical records and discussion with
the attending health-care provider(s), especially when a new disease
appears to be emerging (e.g., in the early investigations of Legionnaires'
disease, AIDS, eosinophilia myalgia syndrome, and hantavirus pulmonary
syndrome) (4,9-11). Clinical findings should also be examined closely when
some or all of the observed cases may be factitious, perhaps because of
laboratory error (12); a discrepancy between the clinical and laboratory
findings generally exists, which may be discernible only by a detailed
review of the clinical findings.
Establishing the Background Rate of Disease and Finding Cases
Once it is clear that a suspected outbreak is not the result of laboratory
error, a set of activities should be undertaken to establish the background
rate of the disease in the affected population and to find all the cases in
a given population in a certain period. This set of activities should prove
that the observed number of cases truly is in excess of the "usual" number
(i.e., that an outbreak has occurred), define the scope of the outbreak
geographically and temporally, find cases to describe the epidemiologic
features of those affected and to include them in analytic epidemiologic
studies (see below) or, most often, accomplish a combination of these
goals.
When hundreds of acute onset diarrhea cases are suddenly seen daily in a
single outpatient setting (10), an outbreak is clearly occurring. On the
other hand, when too many hospitalized patients are dying unexpectedly of
cardiac arrest (13) or the number of cases of listeriosis in a given county
in recent months is moderately elevated, it may be necessary to establish
the background rates in the population to determine whether an outbreak is
occurring. In such situations, the period and geographic areas involved
would provide the most useful baseline data, keeping in mind that the labor
and time required to collect such information is often directly
proportional to the length of the period and the size of the geographic
area selected. Because disease incidence normally fluctuates by season,
data from comparable seasons in earlier years should be included.
Establishing the background rate of a disease is generally more
straightforward if confirmatory tests are available than if laboratory
tests are unavailable or infrequently used. The rate of certain invasive
bacterial infections (e.g., listeriosis and meningococcal infections) in a
given area can be easily documented by reviewing the records of hospital
clinical microbiology laboratories; however, cases for which specimens were
not submitted to these laboratories for testing will go undetected. When a
disease is less frequently laboratory-confirmed because health-care
providers may not have considered the diagnosis or ordered the appropriate
laboratory tests (e.g., for Legionnaires' disease), establishing the
background rate of disease in a community or a hospital suspected of having
an outbreak generally requires alternative case-finding strategies and is
almost invariably more labor intensive. In an outbreak of a new disease,
substantial effort is often necessary to determine whether or not cases of
that disease had been occurring but had gone unrecognized.
Once data concerning the background rate of a disease (including
case-finding for the current period) have been collected, it is generally
possible to determine whether or not an outbreak is occurring or has
occurred, although in some situations it may remain unclear whether or not
the number of cases observed exceeds the background rate. In part, the
problem may relate to how an outbreak is defined. To paraphrase a U.S.
Supreme Court justice speaking about pornography, "I can't define an
outbreak, but I know one when I see one." Thus, it may be difficult to
detect and prove the existence of small outbreaks, but large ones are
self-evident.
An outbreak can also be difficult to identify when during the period under
study changes occur in the care-seeking behavior and access to care of
patients; the level of suspicion, referral patterns, and test-ordering
practices of health-care providers; the diagnostic tests and other
procedures used by laboratories; and the prevalence of underlying
immunosuppressive conditions or other host factors in the population. All
these factors, which can affect the apparent incidence of a disease and
produce artifactual changes perceived as increases (or decreases) in the
actual incidence, need to be considered when interpreting the findings.
Descriptive Epidemiology
By collecting patient data, the case-finding activities provide extremely
important information concerning the descriptive epidemiologic features of
the outbreak. By reviewing and plotting on an "epidemic curve" the times of
onset of the cases and by examining the characteristics (e.g., age, sex,
race/ethnicity, residence, occupation, recent travel, or attendance at
events) of the ill persons, investigators can often generate hypotheses
concerning the cause(s)/source(s) of the outbreak. While linking the sudden
onset of gastroenteritis among scores of persons who attended a church
supper to the single common meal they shared is generally not a challenge,
an otherwise cryptic source can be at least hinted at by the descriptive
epidemiologic features of the cases involved. For example, in a
particularly perplexing outbreak of _Salmonella_ Muenchen infections
ultimately traced to contaminated marijuana, the age distribution of the
affected persons and of their households was markedly different from that
typically seen for salmonellosis (14). Or, similarly, in the outbreak of
legionellosis due to contaminated misting machines in the produce section
of a grocery store, before the link to this exposure was even suspected, it
was noted that women constituted a substantially higher proportion of the
cases usually seen with this disease (5). The shape of the epidemic curve
can also be very instructive, suggesting a point-source epidemic, ongoing
transmission, or a combination of the two.
Generating a Hypothesis
The source(s) and route(s) of exposure must be determined to understand why
an outbreak occurred, how to prevent similar outbreaks in the future, and,
if the outbreak is ongoing, how to prevent others from being exposed to the
source(s) of infection. In some outbreaks, the source and route are obvious
to those involved in the outbreak and to the investigators. However, even
when the source of exposure appears obvious at the outset, a modicum of
skepticism should be retained because the obvious answer is not invariably
correct. For example, in an outbreak of nosocomial legionellosis in Rhode
Island, the results of an earlier investigation into a small number of
hospital-acquired cases at the same hospital had demonstrated that
_Legionella pneumophila_ was in the hospital potable water supply, and a
sudden increase in new cases was strongly believed to be related to the
potable water (15). However, a detailed epidemiologic investigation
implicated a new cooling tower at the hospital as the source of the second
outbreak.
While the true source of exposure, or at least a relatively short list of
possibilities, is apparent in many outbreaks, this is not the case in the
more challenging outbreaks. In these instances, hypotheses concerning the
source/route of exposure can be generated in a number of ways beyond a
detailed review of the descriptive epidemiologic findings. A review of
existing epidemiologic, microbiologic, and veterinary data is very useful
for learning about known and suspected sources of previous outbreaks or
sporadic cases of a given infection or disease, as well as the ecologic
niche of an infectious agent. Thus, in an outbreak of invasive
_Streptococcus zooepidemicus_ infections in New Mexico due to consumption
of soft cheese made from contaminated raw milk, the investigation focused
on exposure to dairy products and animals because of previous microbiologic
and veterinary studies (16).
A review of existing data generally only helps confirm what is already
known about a particular disease and is far less helpful in identifying
totally new and unsuspected sources or routes of infection (i.e., marijuana
as a source of Salmonella). When neither review of the descriptive
epidemiologic features of the cases nor review of existing scientific
information yields the correct hypothesis, other methods can be used to
generate hypotheses about what the patients have in common. Open-ended
interviews of those infected (or their surrogates) are one such method in
which investigators try to identify all possibly relevant exposures (e.g.,
a list of all foods consumed) during a given period. For example, in an
investigation of _Yersinia enterocolitica_ infections in young children in
Belgium, open-ended interviews of the mothers of some of the ill children
showed that many gave their children raw pork sausage as a weaning food,
providing the first clue as to the source of these infections (17).
Similarly, in two outbreaks of foodborne listeriosis, a variant of this
process led to the identification of the source of the outbreak. In one of
these outbreaks, a search of the refrigerator of one of the case-patients
who, as a visitor to the area, had had very limited exposure to foods
there, suggested cole slaw as a possible vehicle of infection (18). In the
other outbreak, an initial case-control study found no differences between
cases and controls regarding exposure to a number of specific food items
but showed that case households were more likely than control households to
buy their food at a particular foodstore chain. To generate a list of other
possible food sources of infection, investigators shopped with persons who
did the shopping for case households and compiled a list of foods purchased
at that foodstore chain that had not been reported in the previous study.
This approach implicated pasteurized milk from that chain as the source of
the outbreak (19).
In some particularly perplexing outbreaks, bringing together a subset of
the patients to discuss their experiences and exposures in a way that may
reveal unidentified links can be useful.
Testing the Hypothesis
Whether a hypothesis explaining the occurrence of an outbreak is easy or
difficult to generate, an analytic epidemiologic study to test the proposed
hypothesis should be considered. While in many instances a case-control
study is used, other designs, including retrospective cohort and
cross-sectional studies, can be equally or more appropriate. The goal of
all these studies is to assess the relationship between a given exposure
and the disease under study. Thus, each exposure of interest (e.g., each of
the meals eaten together by passengers on a cruise ship and each of the
foods and beverages served at those meals) constitutes a separate
hypothesis to be tested in the analytic study. In outbreaks where
generating the correct hypothesis is difficult, multiple analytic studies,
with additional hypothesis-generating activities in between, are sometimes
needed before the correct hypothesis is formed and tested (19).
In interpreting the results of such analytic studies, one must consider the
possibility that "statistically significant" associations between one or
more exposures and the disease may be chance findings, not indicative of a
true relationship. By definition, any "statistically significant"
association may have occurred by chance. (When the standard cut point of p
< 0.05 is used, this occurs 5% of the time.) Because many analytic
epidemiologic studies of outbreaks involve testing many hypotheses, the
problem of "multiple comparisons" arises often.
While there are statistical methods for adjusting for multiple comparisons,
when and even whether to use them is controversial. At a minimum, it is
important to go beyond the statistical tests and examine the magnitude of
the effect observed between exposure and disease (e.g., the odds ratio,
relative risk) and the 95% confidence intervals, as well as biologic
plausibility in deciding whether or not a given "statistically significant"
relationship is likely to be biologically meaningful. Evidence of a
dose-response effect between a given exposure and illness (i.e., the
greater the exposure, the greater the risk for illness) makes a causal
relationship between exposure and disease more likely. Whether the time
interval between a given exposure and onset of illness is consistent with
what is known about the incubation period of the disease under study must
also be assessed. When illness is "statistically significantly" related to
more than one exposure (e.g., to eating each of several foods at a common
meal), it is important to determine whether multiple sources of infection
(perhaps due to cross-contamination) are plausible and whether some of the
noted associations are due to confounding (e.g., exposure to one potential
source is linked to exposure to other sources) or to chance.
When trying to decide if a "statistically significant" exposure is the
source of an outbreak, it is important to consider what proportion of the
cases can be accounted for by that exposure. One or more of the patients
may be classified as "nonexposed" for various reasons: incorrect
information concerning exposure status (due to poor memory, language
barriers); multiple sources of exposure or routes of transmission (perhaps
due to cross-contamination); secondary person-to-person transmission that
followed a common source exposure; or patients without the suspected
exposure, representing background cases of the disease unrelated to the
outbreak. The plausibility of each of these explanations varies by
outbreak. While there is no cutoff point above or below which the
proportion of exposed case-patients should fall before an exposure is
thought to account for an outbreak, the lower this proportion, the less
likely the exposure is, by itself, the source.
Other possibilities need to be considered when the analytic epidemiologic
study finds no association between the hypothesized exposures and risk for
disease. The most obvious possibility is that the real exposure was not
among those examined, and additional hypotheses should be generated.
However, other possibilities should also be considered, particularly when
the setting of the outbreak makes this first explanation unlikely (e.g.,
when it is known that those involved in the outbreak shared only a single
exposure or set of exposures, such as eating a single common meal). Two
other explanations for failing to find a "statistically significant" link
between one or more exposures and risk for illness also need to be
considered-the number of persons available for study and the accuracy of
the available information concerning the exposures. Thus, if the outbreak
involves only a small number of cases (and non-ill persons), the
statistical power of the analytic study to find a true difference in
exposure between the ill and the non-ill (or a difference in the rate of
disease among the exposed and the unexposed) is very limited. If the
persons involved in the outbreak do not provide accurate information about
their exposure to suspected sources or vehicles of infection because of
lack of knowledge, poor memory, language difficulty, mental impairment, or
other reasons, the resulting misclassification of exposure status also can
prevent the epidemiologic study from implicating the source of infection.
Studies have documented that even under ideal circumstances, memory
concerning such exposures is faulty (20). However, given the usually
enormous differences in rates of disease between those exposed and those
not exposed to the source of the outbreak, even small studies or studies
with substantial misclassification of exposure can still correctly identify
the source.
Environmental Investigation
Samples of foods and beverages served at a common meal believed to be the
source of an outbreak of gastroenteritis or samples of the water or drift
from a cooling tower believed to be the source of an outbreak of
Legionnaires' disease can support epidemiologic findings. In the best
scenario, the findings of the epidemiologic investigation would guide the
collection and testing of environmental samples. However, environmental
specimens often need to be obtained as soon as possible, either before they
are no longer available, as in the case of residual food from a common
meal, or before environmental interventions are implemented, as in the case
of treating a cooling tower to eradicate Legionella. Because laboratory
testing of environmental samples is often expensive and labor-intensive, it
is sometimes reasonable to collect and store many samples but test only a
limited number. Collaborating with a sanitarian, environmental engineer, or
other professional during an environmental inspection or collection of
specimens is always beneficial.
While finding or not finding the causative organism in environmental
samples is often perceived by the public, the media, and the courts as
powerful evidence implicating or exonerating an environmental source,
either positive or negative findings can be misleading for several reasons.
For example, finding Legionella in a hospital potable water system does not
prove that the potable water (rather than a cooling tower or some other
source) is responsible for an outbreak of Legionnaires' disease (21).
Similarly, not finding the causative organism in an environmental sample
does not conclusively rule out a source as the cause of the problem, in
part because the samples obtained and tested may not represent the source
(e.g., because of error in collecting the specimens, intervening changes in
the environmental source) and in part because the samples may have been
mishandled. Furthermore, in some outbreaks caused by well-characterized
etiologic agents, laboratory methods of detecting the agent in
environmental samples are insensitive, technically difficult, or not
available, as in the case of recent outbreaks of _Cyclospora_ infections
associated with eating imported berries (22,23).
Control Measures
Central to any outbreak investigation is the timely implementation of
appropriate control measures to minimize further illness and death. At
best, the implementation of control measures would be guided by the results
of the epidemiologic investigation and possibly (when appropriate) the
testing of environmental specimens. However, this approach may delay
prevention of further exposure to a suspected source of the outbreak and
is, therefore, unacceptable from a public health perspective. Because the
recall of a food product, the closing of a restaurant, or similar
interventions can have profound economic and legal implications for an
institution, a manufacturer or owner, and the employees of the
establishments involved, acting precipitously can also have substantial
negative effects. The recent attribution of an outbreak of _Cyclospora_
infections to strawberries from California demonstrates the economic impact
that can result from releasing and acting on incorrect information (22,23).
Thus, the timing and nature of control measures are difficult. Balancing
the responsibility to prevent further disease with the need to protect the
credibility and reputation of an institution is very challenging.
Interactions with the Public and Press
While the public and the press are not aware of most outbreak
investigations, media attention and public concern become part of some
investigations. Throughout the course of an outbreak investigation, the
need to share information with public officials, the press, the public, and
the population affected by the outbreak must be assessed. While press,
radio, and television reports can at times be inaccurate, overall the media
can be a powerful means of sharing information about an investigation with
the public and disseminating timely information about product recalls.
Dr. Reingold worked as an epidemiologist at the Centers for Disease Control
and Prevention for 8 years before joining the faculty of the School of
Public Health at the University of California, Berkeley. He is currently
professor of epidemiology and head of the Division of Public Health Biology
and Epidemiology.
Address for correspondence: Arthur L. Reingold, Division of Public Health
Biology and Epidemiology, School of Public Health, University of
California, Berkeley, 140 Warren Hall, Berkeley, CA 94720-7360, USA; fax:
510-643-5163; e-mail: reingold@uclink3.berkeley.edu.
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     Schrader JS, et al. Salmonellosis associated with marijuana: a
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     Validity of food consumption histories in a foodborne outbreak
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     al. Large community outbreak of cryptosporidiosis due to contamination
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Synopses
Genetic Diversity of Wild-Type Measles Viruses: Implications for Global
Measles Elimination Programs
William J. Bellini and Paul A. Rota
Centers for Disease Control and Prevention, Atlanta, Georgia,  USA
---------------------------------------------------------------------------
Text Version
      Wild-type measles viruses have been divided into distinct
      genetic groups according to the nucleotide sequences of their
      hemagglutinin and nucleoprotein genes. Most genetic groups have
      worldwide distribution; however, at least two of the groups
      appear to have a more limited circulation. To monitor the
      transmission pathways of measles virus, we observed the
      geographic distribution of genetic groups, as well as changes
      in them in a particular region over time. We found evidence of
      interruption of indigenous transmission of measles in the
      United States after 1993 and identified the sources of imported
      virus associated with cases and outbreaks after 1993. The
      pattern of measles genetic groups provided a means to describe
      measles outbreaks and assess the extent of virus circulation in
      a given area. We expect that molecular epidemiologic studies
      will become a powerful tool for evaluating strategies to
      control, eliminate, and eventually eradicate measles.
Until the advent of a live-attenuated vaccine in the early 1960s, measles
was an epidemic disease worldwide. Today many countries have controlled
measles, but the disease remains endemic on most continents. Development of
a live-attenuated measles vaccine and implementation of laws that required
proof of vaccination upon school entry dramatically reduced the incidence
of measles in the United States. The number of reported cases plummeted
from approximately 500,000 before vaccine introduction in 1963 to fewer
than 1,500 in 1983. Despite these measures, a reemergence or resurgence of
measles in the United States from 1989 to 1991 resulted in more than 55,000
cases of measles and approximately 120 measles-associated deaths (Figure 1;
1). In exploring the reasons for the resurgence, our laboratory genetically
characterized measles viruses isolated from wild-type virus-infected
persons from the same outbreak or temporally and geographically distinct
outbreaks in the United States; in the regions examined, all measles
viruses isolated during the period of resurgence were almost identical in
nucleotide sequence and genetically distinct from vaccine strains (2).
Measles isolates from many regions of the world have been characterized in
parallel studies by our laboratory and by others. In conjunction with
classic epidemiologic investigations, these well-characterized viruses have
formed a picture of the distribution of wild-type measles viruses in most
areas of the world. Eight distinct genotypes have been identified, and
undoubtedly more will be added. Some are localized to specific regions,
while most are widely distributed. The assembly of these sequences into a
large database, which includes their geographic distribution, has become a
new means by which measles transmission pathways can be traced and control
measures can be assessed. This "molecular epidemiology" has affected the
U.S. measles elimination program, and if used appropriately with standard
epidemiologic methods, it will affect global measles elimination and
eradication. This article summarizes the status of the known measles
genotype distribution throughout the world and describes how molecular
epidemiologic information has been used to assess the effectiveness of
measles elimination in the United States.
                          [graph.gif (6842 bytes)]
Figure 1. Incidence of U.S. measles cases and measles deaths between 1980
and 1997. Total number of measles cases for years 1993-1997 (week 35) are
indicated above each bar.
Global Distribution of Measles Genotypes
In most cases, genetic characterization of wild-type measles viruses has
been conducted by sequencing the genes coding for the hemagglutinin (H)
protein or the nucleoprotein (N). Of the six genes on the viral genome, the
H and N genes are the most variable. Over their protein coding regions, the
H and N genes vary by up to 7% at the nucleotide level. The single most
variable part of the measles genome is the 450 nucleotides that code for
the COOH-terminus of the N protein, where nucleotide variability between
various wild-type viruses can approach 12%. Several laboratories have
analyzed the sequences of wild-type measles viruses and assigned the
viruses to various genetic groups(2, 14).
Many of our studies have focused on the genetic characterization of measles
viruses associated with cases and outbreaks in the United States during the
last 10 years (2, 11). These viruses can be separated into at least eight
distinct genetic groups (Table; Figure 2). Phylogenetic analyses using
various computer programs (15, 17) indicated good statistical support for
each of the groups described below. Actually, more than eight genetic
groups are listed when viruses from groups not yet found in the United
States are included (e.g., Zambia: 1993, Germany: 1992) (Figure 2). The
number of genetic groups is likely to increase since the true extent of
genetic heterogeneity among wild-type measles viruses is still unknown, and
virologic surveillance has not been conducted or has only just begun in
many areas of the world.
Table. Sources of genotypes isolated in the United States, 1995-1997
	  No. of  	    Imports     		     Also
Group   isolates         from                    circulating in
-------------------------------------------------------------------
4         13         Germany, Spain,     	 Central Europe,
                       United Kingdom,      	   Canada, Brazil,
                       Brazil, Austria,           United Kingdom
                       Italy,Greece,
		          Ukraine
3         9          Japan              	  Japan, Thailand
5         8          Italy, Germany        	  Central Europe,
                                                   United Kingdom,
                                                   Brazil
7         3          Paskistan, Kenya   	  South Africa,Canada
8         2          China, Vietnam 		  China
2         1          Philippines        	  United States
						          1989-1992,
                                                  Micronesia
1         1          Unknown           	  	  United Kingdom,
                                                  Russia, China,
                                                  Argentina
6         1          Kenya              	   The Gambia,
                                                   Cameroon, Gabon,
                                                   Zambia
------------------------------------------------------------------------
Group 1 contains the prototype, Edmonston strain, which was isolated in
1954. This group also contains all vaccine viruses sequenced regardless of
whether they were derived from Edmonston (Attenuvax, Edmonston-Zagreb,
AIKC, Schwarz) or from temporally and geographically independent wild-type
isolates (Shanghai-191: China, Changchun-47: China, CAM-70: Japan,
Leningrad-16: Russia) (18). Relatively few wild-type viruses from the
prevaccine era are available for molecular characterization. These viruses,
which were isolated in Japan, Russia, Finland, Romania, and the United
States during the 1950s and 1960s, are in group 1 (9). Therefore, while
viruses belonging to the other genetic groups may have been present, group
1 viruses must have had widespread distribution during the prevaccine era.
Group 1 viruses continue to circulate, and viruses from group 1 were
isolated from patients with clinical measles in the United States, United
Kingdom, Russia, China, and Argentina during the last 7 years (5, 11, 19,
20, and unpub. observations). These recent group 1 wild-type viruses have
several nucleotide substitutions that distinguish them from vaccine
viruses. In contrast, measles vaccine viruses reisolated from
immunosuppressed patients with giant cell pneumonia had nucleotide
sequences nearly identical to those of the vaccine virus found in the
vaccine vial (unpub. observations). This suggests that vaccine viruses are
very stable even after prolonged replication in a human host. Therefore, it
is unlikely that the group 1 wild-type viruses represent laboratory
contamination of cultures with vaccine virus or reisolation of vaccine
virus from recently vaccinated persons. Sequence studies have failed to
identify a distinct set of genetic markers that consistently differentiate
wild-type and presumably virulent viruses from attenuated viruses. Current
studies are focusing on the analysis of the noncoding regions of the viral
genome. More studies are needed to compare attenuated strains with their
more virulent or reactogenic precursors. The recent development of an
infectious clone for measles (21) will, no doubt, contribute to those
studies.
                        [fig]
Figure 2. Phylogenetic tree showing genetic relationships between the eight
genetic groups of measles virus associated with U.S. outbreaks and cases
since 1988. The location and year of isolation is given for each virus.
Viruses not assigned to one of the eight groups are labeled in brown. The
unrooted tree is based on the sequence of the protein coding region of the
H gene (1854 nt). Wt-Edmonston = low passage seed of the original Edmonston
isolate. SSPE = sequences obtained
from cases of subacute sclerosing panencephalitis.
Group 2 viruses were associated with the resurgence of measles in the
United States between 1989 to 1991, an epidemic that had an unusually high
incidence of deaths and hospitalizations(Figure 1). The circulation of
group 2 viruses within the United States was interrupted in 1993, and this
will be described in more detail below. Among these viruses, the Illinois-1
(Chicago-1) strain has become a representative of recent wild-type viruses,
and almost the entire genome has been sequenced. Group 2 viruses were first
isolated in Japan during the early 1980s; more recently, they were isolated
in Japan, the Philippines, and Micronesia (2,11-13,22).
The group referred to as group 3 viruses can actually be divided into two
distinct groups with a common geographic distribution. These viruses have
been isolated from outbreaks in Japan and Thailand and from sporadic cases
following importation into North  America and Europe (2,11). Although virus
from groups 2 and 3 cocirculated in Japan during the late 1980s and early
1990s, the group 3 viruses have recently become the predominant genotype
(23).
Groups 4 and 5 appear to be circulating widely in many countries in western
Europe, particularly Germany, Spain, and the United Kingdom, where
virologic surveillance has been conducted (6,7,24). Viruses from this group
are also circulating in France, Italy, Austria, and Greece since they have
been associated with multiple importations from these areas into the United
States (2,11; Table).
All representatives of the group 6 viruses have been isolated in the
African countries of The Gambia (4), Cameroon, Gabon, and Zambia or
associated with importations into the United States from Kenya. There is
more genetic variability within the group 6 viruses than among most of the
other genetic groups, yet all group 6 viruses contain a subset of
nucleotide substitutions that places them on this African lineage.
Relatively few viruses from central Africa, where most measles infections
are occurring, have been isolated for genetic analysis, and it will be
interesting to determine if other genotypes are also present in this area.
Group 7 viruses were first isolated during an epidemic in Montreal, Canada,
in 1988 (2,11). Group 7 was the predominant group among a number of
recently isolated viruses from Johannesburg, South Africa (9,25,26). The
identification of a group 7 virus in association with an importation to the
United States from Pakistan suggests (11) that the viruses in this group
may be circulating widely in Africa and Asia.
The group 8 viruses form a highly distinct group isolated in four provinces
within the People's Republic of China during the early 1990s (19). Like
group 6, group 8 viruses have more nucleotide variability within the group
(up to 3%) than the other groups. Recent evidence also suggests that group
8 viruses are circulating in other parts of China (Hong Kong) and in
Vietnam.
Several recently isolated viruses do not fit into the eight genetic groups
that, so far, contain most recent isolates (Figure 2). Some outliers
represent single isolations of a unique genetic type. However, preliminary
analysis of a number of wild-type viruses from Zambia indicates that these
viruses belong to a genetic group that is distinct from the eight groups
described thus far (unpub. observations).
If viruses isolated during the early to mid-1980s were included into the
genetic analysis (not shown), it would be apparent that more genetic groups
exist. However, viruses representing these groups have not been isolated in
the last 10 to 15 years, and it must be assumed that these groups are
circulating in restricted geographic regions, are circulating at such a low
frequency as to escape surveillance, or are extinct.
A summary of genetic groups (Figure 3) represents a static picture that
simply identifies where particular genetic groups have been isolated, with
no accounting for frequency of isolation or source of the virus. Certain
regions of the world (including much of Africa and most of southern Asia)
are still vastly underrepresented. A survey of recent Australian isolates
is in progress. The pattern of genotypes in the United Kingdom and the
United States is very complex because of relatively good strain
surveillance and the frequency of international travel to these locations.
                        [fig]
Figure 3. Global distribution of measles genetic groups. Colored circles
indicate areas where measles viruses from various genetic groups have been
isolated. Viruses not assigned to one of the eight groups are labeled in
brown.
Molecular Epidemiology
Measles has long been considered one of the most communicable of diseases.
The resurgence of disease from 1989 to 1991 in the United States (Figure 1)
provides a good example of the rapid transmissibility of the virus. During
this resurgence only group 2 viruses were isolated, and the sequences from
these viruses were highly related (2). With continued molecular
surveillance, we were able to document the interruption of transmission of
the group 2 viruses and monitor the change in measles genotypes associated
with outbreaks and sporadic cases from 1994 to the present. Molecular
surveillance of measles viruses was most useful when the change in
genotypes was observed over time. Without that information, it would not
have been possible to describe the transition from an apparently
"indigenous" lineage to importation of multiple lineages (Figure 4). This
is in contrast to the situation in South and Central America. In these
areas, viral surveillance was not conducted before mass vaccination
campaigns were initiated, so the identity of the prevailing genotype could
not be determined. Therefore, it is difficult to interpret the genetic data
obtained from viruses currently causing outbreaks in these regions.
The molecular surveillance of wild-type viruses in the United States
between 1989 and 1997 provides the best example of dynamic molecular
surveillance (Figure 4). Viruses isolated over a 4-year period from major
outbreaks in New York, Philadelphia, Chicago, Los Angeles, Houston, and
southern Texas varied by less than 0.4% at the nucleotide level in the H
and N genes (2). Analysis of the few wild-type measles viruses isolated in
the United States before 1988 indicates lineages other than group 2. This
suggests that the group 2 viruses were probably imported during the late
1980s and were rapidly transmitted to the entire country. While numerous
importations occurred during the resurgence, apparently these viruses did
not circulate widely enough to be detected by molecular surveillance.
Perhaps the number of measles-susceptible persons in the U.S. population
during the resurgence was high enough to sustain continuous transmission
without accumulation of variants or displacement by other imported viruses.
                         [fig]
Figure 4. Change in genetic groups of measles viruses associated with U.S.
cases and outbreaks between 1988 and September 1997. Arrows indicate
sources of virus, if known.
More aggressive childhood vaccination programs, the introduction of a
two-dose schedule, and successful mass vaccination campaigns conducted by
the Pan American Health Organization in South and Central America greatly
reduced the number of reported measles cases in the United States in 1993
(Figure 1). During a 6-week period at the end of 1993, no indigenous cases
of measles were reported (27). Molecular surveillance of measles viruses
associated with cases and outbreaks in the United States during 1994, 1995,
1996, and 1997 documented this interruption of transmission of what had
been the indigenous genotype (2,11). Only one group 2 virus was detected in
the United States after 1993, and this was directly linked to importation
from the Philippines (11).
Molecular surveillance data allow us to draw several conclusions about the
transmission of measles virus in the United States. The first is that
increasing the level of population immunity by vaccination can interrupt
the transmission of measles virus. This is hardly new information, and
interruption of transmission was described for The Gambia in 1983 and more
recently in Finland. However, our studies are the first in which genetic
analysis of measles strains has been used to document interruption of
transmission. Secondly, long-term asymptomatic transmission of virus is
unlikely since no group 2 viruses were detected in the United States after
1993 that were not directly linked to importation. Finally, measles will
not be fully controlled anywhere until it is controlled globally. Virus
introduced by importation will continue to fuel sporadic outbreaks and
epidemics even in areas with relatively good control measures. These
observations should strengthen our resolve to accelerate measles control
activities on a global level.
The molecular data imply that under conditions of continuous indigenous
transmission of measles virus, the number of circulating genotypes is
limited. As population immunity increases, the pattern of genotypes becomes
more complex to reflect the multiple sources of imported virus. We hope to
test this model further by conducting molecular surveillance of wild-type
measles viruses circulating in areas that still have endemic measles.
Conclusions
Genetic characterization of wild-type measles viruses provides a valuable
means to measure the level of virus circulation in areas just beginning to
implement measles control plans. In areas that already achieved good
measles control, molecular epidemiologic studies provide a means to
describe outbreaks and cases. Identifying the source of the virus can lead
to improved control measures. To be maximally effective, molecular
epidemiologic studies must include surveys of viral genetic groups from all
areas of the world. Specimens for viral isolations should be obtained from
as many chains of transmission as possible. Obtaining specimens must become
an integral part of measles surveillance and be included in the standard
operating procedures for investigating measles cases. If we can establish a
large database to describe the indigenous genetic groups before large-scale
control measures are enacted, we can closely monitor the ability of these
control measures to reduce or interrupt transmission of measles. Molecular
epidemiology will greatly enhance measles elimination and eradication
efforts.
Dr. Bellini is chief of the Measles Section, Respiratory and Enteric
Viruses Branch, Division of Viral and Rickettsial Diseases, National Center
for Infectious Diseases, CDC. Dr. Rota is a microbiologist in the Measles
Section, CDC. The authors work closely with national and international
public health agencies to support global measles eradication activities.
They provide expertise and training on the genetic characterization of
measles viruses and laboratory diagnosis of measles infections. Research
interests include molecular virology, viral pathogenesis, and immune
response to viral infections.
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Synopses
Diversity among Multidrug-Resistant Enterococci
Barbara E. Murray, M.D.
University of Texas Houston-Medical School, Houston, Texas,  USA
------------------------------------------------------------------------
Enterococci are associated with both community- and
hospital-acquired infections. Even though they do not cause
severe systemic inflammatory responses, such as septic shock,
enterococci present a therapeutic challenge because of their
resistance to a vast array of antimicrobial drugs, including
cell-wall active agents, all commercially available
aminoglycosides, penicillin and ampicillin, and vancomycin. The
combination of the latter two occurs disproportionately in
strains resistant to many other antimicrobial drugs. The
propensity of enterococci to acquire resistance may relate to
their ability to participate in various forms of conjugation,
which can result in the spread of genes as part of conjugative
transposons, pheromone-responsive plasmids, or broad host-range
plasmids. Enterococcal hardiness likely adds to resistance by
facilitating survival in the environment of a
multidrug-resistant clone, thus enhancing potential spread from
person to person. The combination of these attributes within
the genus Enterococcus suggests that these bacteria and their
resistance to antimicrobial drugs will continue to pose a
challenge.
Enterococci, which have been known as a cause of infective endocarditis for
close to a century, more recently have been recognized as a cause of
nosocomial infection and "superinfection" in patients receiving
antimicrobial agents (1). The enterococcus is now receiving increased
attention because of its resistance to multiple antimicrobial drugs, which
probably explains a large part of its prominence in nosocomial infections.
The most common enterococci-associated nosocomial infections are infections
of the urinary tract, followed by surgical wound infections and bacteremia
(1-3). Enterococci are often present in intraabdominal and pelvic
infections, although not all patients with such infections require specific
antienterococcal therapy. Other enterococcal infections include infections
(including meningitis and bacteremia) in very ill neonates; central nervous
system infections in adults, typically with a history of central nervous
system surgery or intrathecal chemotherapy; and rarely, osteomyelitis and
pulmonary infections. Enterococci frequently arise from colonization of
indwelling T tubes, causing liver or biliary infection in liver transplant
patients (1).
Antimicrobial Resistance
Most enterococci have naturally occurring or inherent resistance to various
drugs, including cephalosporins and the semisynthetic
penicillinase-resistant penicillins (e.g., oxacillin) and clinically
achievable concentrations of clindamycin and aminoglycosides. Compared with
streptococci, most enterococci are relatively resistant to penicillin,
ampicillin, and the ureidopenicillins, with MICs of 1 g/ml to 8 g/ml for
most Enterococcus faecalis and even higher for most E. faecium. Many
enterococci are also tolerant to the killing effects of cell-wall active
agents, including ampicillin and vancomycin; recent data suggest that this
property may not be inherent, but rather acquired after exposure to
antibiotics (4). Inherent in vivo resistance of E. faecalis to
trimethoprim-sulfamethoxazole may explain the lack of efficacy in animal
models. In vitro, trimethoprim-sulfamethoxazole readily inhibits most
enterococci at low concentrations, but this activity is lessened by
exogenous folates (5). Moreover, bactericidal activity against E. faecalis
seems unreliable and very method dependent (6). In animal models, this
combination has not shown good activity and is not generally accepted as an
effective antienterococcal therapy, especially for systemic infections
(7,8).
In addition to natural resistance to many agents, enterococci have also
developed plasmid- and transposon-mediated resistance to tetracycline (as
well as minocycline and doxycycline), erythromycin (plus the newer
compounds azithromycin and clarithromycin), chloramphenicol, high levels of
trimethoprim, and high levels of clindamycin.
The propensity of E. faecalis to acquire multiple antibiotic-resistance
traits may result from a variety of distinctly different mechanisms for
conjugation, i.e., bacterial mating. The best studied system of conjugation
involves oligopeptides called pheromones and pheromone-responsive plasmids
(9;Figure 1). Briefly, strains of E. faecalis typically secrete into the
culture medium a number of different small peptide sex pheromones specific
for different types of plasmids. When a cell containing a
pheromone-responsive plasmid (the potential donor cell) comes into contact
with its corresponding pheromone, transcription of a gene on the plasmid is
turned on, resulting in the synthesis of a sticky substance (called
aggregation substance) on its surface. When the donor cell bumps into
another E. faecalis, aggregation substance, which contains two Arg-Gly-Asp
motifs, sticks to the binding substance on the surface of most E. faecalis
cells, causing them to clump together. In the test tube, clumps of cells
actually fall to the bottom of the tube, resulting in a visible aggregate.
By a process not yet well understood, the pheromone-responsive plasmid can
then transfer from the donor bacterium to the other (recipient) bacterium.
Once the recipient cell has acquired this particular plasmid, the synthesis
of the corresponding sex pheromone is shut off to prevent self-clumping.
This system of conjugation, which occurs primarily in E. faecalis, is
highly efficient and results in transfer of plasmids in both filter and
broth matings.
Another system of conjugation, also not well understood, involves broad
host-range plasmids that can transfer among species of enterococci and
other gram-positive organisms such as streptococci and staphylococci (10).
The transfer frequency is generally much lower than with the pheromone
system and is much more efficient with filter than with broth matings.
Since staphylococci, streptococci, and enterococci share a number of
resistance genes, these broad host-range plasmids may be a mechanism by
which some of these resistance genes have spread among different genera.
                                 [Fig]
Figure 1. Enterococcus faecalis pheromone-responsive conjugative system.
Pheromone A released from the potential recipient cell (right) interacts
with plasmid A in the potential donor cell (left) to induce synthesis of
aggregation substance. Attachment of aggregation substance to binding
substance causes the cells to clump into visible aggregates. Once the
pheromone-responsive plasmid A has transferred from donor to recipient
cell, synthesis of pheromone A is shut off.
A third type of conjugation, which involves conjugative transposons, may
also explain the spread of resistance genes to many different species (11).
As opposed to ordinary transposons, which can jump within a cell from one
DNA location to another, conjugative transposons also encode the ability to
bring about conjugation between different bacterial cells. Since plasmids
typically require rather complex machinery for replication (often depending
on successful interactions with host proteins) and must face additional
problems of surface exclusion and incompatibility, conjugative transposons
(which do not replicate, but instead insert into the chromosome or into a
plasmid of the new host) appear to be an even more efficient and
far-reaching way of disseminating a resistance gene. This may explain why
the tetM gene of the conjugative transposon Tn916 has spread beyond the
gram-positive species into gram-negative organisms, including gonococci,
meningococci, and Haemophilus ducreyi, as well as into mycoplasma and
ureaplasma, among others (12,13). Other resistance genes, including those
encoding resistance to erythromycin and kanamycin, are also found on
conjugative transposons; these frequently contain or are related to Tn916.
Such transposons may have evolved from a Tn916 ancestor; their emergence
suggests the possibility of further dissemination of resistance among
gram-positive organisms. Particularly ominous are reports of the vanB gene
cluster within large conjugative chromosomal elements that appear similar,
at least in function, to conjugative transposons (14).
High-Level Aminoglycoside Resistance
Although some acquired resistance of enterococci is not clinically
important because the agents involved are not commonly used, other
resistance greatly affects enterococcal therapy; high-level resistance
(HLR) to aminoglycosides is an example. This resistance is added onto the
normal low-level resistance of enterococci to aminoglycosides and typically
results in MICs of > 2,000 >/= [mu]g/ml. This degree of resistance predicts,
without exception, resistance to synergism between cell-wall active agents
and the aminoglycoside to which the organism is highly resistant (1).
High-level aminoglycoside resistance is most often due to
aminoglycoside-modifying enzymes; HLR to streptomycin can also be
ribosomal, that is, due to a mutation that results in ribosomes resistant
to streptomycin inhibition. HLR to kanamycin (without gentamicin) is a
fairly common trait and is due to the production of a
3'-phosphotransferase, APH(3')-III. This enzyme is important because it
also eliminates synergism between cell-wall active agents and amikacin
(through phosphorylation of the 3'-hydroxyl group), although it does not
necessarily confer HLR to amikacin. HLR to gentamicin results from the
bifunctional protein (AAC(6')-I/APH(2")-I), encoded by a single gene with
two active sites, one with 6'-acetyltransferase activity and the other,
2"-phosphotransferase activity (15). The combination of these activities
results in HLR or resistance to synergism for all commercially available
aminoglycosides except streptomycin, which is not modified by this enzyme.
However, HLR to streptomycin (due to either ribosomal resistance or a
streptomycin adenylyltransferase) is also common and can coexist with the
gene(s) for HLR to other aminoglycosides. Spectinomycin is also not
modified by the bifunctional enzyme, but this agent, which is not a true
aminoglycoside, is not generally bactericidal against enterococci and does
not appear to show synergism with cell-wall active agents.
Strains of enterococci from patients with endocarditis and other serious
infections for whom combination therapy is desired should be screened for
HLR to streptomycin and gentamicin. HLR screening for tobramycin is not
generally performed or advisable It could, in principle, be used for E.
faecalis, but E. faecium isolates have a chromosomally encoded, naturally
occurring gene for a 6'-acetyltransferase that eliminates synergism with
tobramycin, although it does not cause HLR (MICs are typically 128-500
g/ml); a probe for this gene has been used to confirm the identification
of E. faecium isolates to species. In addition, HLR of an E. faecium
isolate (without HLR to gentamicin) to tobramycin, due to an
adenylyltransferase, was recently described (16). Therefore, the use of
tobramycin for possible synergism in serious enterococcal infections would
need to be preceded by screening for HLR to tobramycin (a test that is not
commonly available), as well as for identification to species, neither of
which is practical.
More recently, veterinary and human isolates resistant to moderate levels
of gentamicin (256 g/ml) were found to have a new gentamicin-modifying
enzyme encoded by a gene designated aph(2")-Ic (17). This gene conferred
resistance to synergism between gentamicin and cell-wall active agents and
may be less easily detected than strains producing the bifunctional enzyme.
Beta-Lactamase-- and non-Beta--Lactamase--Associated Penicillin Resistance
The first known penicillinase-producing isolate of enterococcus was an
isolate of E. faecalis recovered from a patient in Houston, Texas, in 1981
(18). Although rare, these isolates have been reported from the United
States (Texas, Florida, North Carolina, Delaware, Pennsylvania, New York,
Massachusetts, and Connecticut), Lebanon, Canada, and Argentina (19). Like
other enterococci, beta-lactamase-producing strains have been found as
colonizers, as in the large "outbreak" of colonization in a Boston
children's hospital, but they have also been associated with true
infections, as was demonstrated by cases at a Virginia Veterans
Administration hospital, by isolates from Argentina, and in other reports
(20,21). The enterococcal penicillinase gene, identical to the gene
encoding staphylococcal type A penicillinase, almost always occurs in
strains with HLR to gentamicin and is often found on a transferable plasmid
that also contains aph(2")-Ia/aac(6')-Ie . The relatively low levels of
beta-lactamase produced by enterococci result in a marked inoculum effect
with these strains so that at low and even moderate inocula (10[sup]3-10[sup]5
CFU/ml), penicillinase-producing enterococci usually appear no more
resistant than other enterococci, while at high inocula (> 10[sup 7] CFU/ml),
these organisms are usually highly resistant to penicillin, ampicillin, and
ureidopenicillins. The activity of the penicillinase is reversed by the
beta-lactamase inhibitors clavulanate, sulbactam, and tazobactam; in animal
models of endocarditis, beta-lactamase inhibitors have been shown to
markedly enhance the therapeutic efficacy of ampicillin or penicillin. In
the clinical laboratory, penicillinase-producing enterococci are generally
not detected by routine laboratory susceptibility testing, such as MICs or
disk diffusion. For this reason, if a penicillin is to be used for therapy,
enterococcal isolates from patients with endocarditis or other serious
infections should be tested for penicillinase production by using a
specific beta-lactamase test such as the chromogenic cephalosporin
nitrocefin.
Nonpenicillinase-producing, penicillin-resistant enterococci have been
reported for decades and usually are E. faecium. Until recently, MICs of
penicillin typically ranged from 8 g/ml to 64 g/ml, with an occasional
isolate having higher levels of resistance. However, increasingly, strains
with much higher levels of penicillin resistance have been reported (22).
Whether a large number of strains have converted from low-level to
high-level resistance or a more limited number of strains have been
disseminated is unclear. The mechanisms involved in this resistance are
overproduction of a low-affinity penicillin-binding protein (a cell-wall
synthesis enzyme) and a further decrease in the affinity of one of these
enzymes for penicillin (23). As a possible explanation for why many
vancomycin-resistant E. faecium also have very high levels of resistance to
ampicillin, Rice and colleagues (24) showed that transfer of vancomycin
resistance from one strain to another was linked to transfer of ampicillin
resistance.
Vancomycin Resistance
Most surprising in recent years has been the emergence among enterococci of
acquired resistance to vancomycin. Vancomycin had been in clinical use
since the 1950s, although it was not heavily used until the late 1970s and
particularly the 1980s. Because multiple genes are involved in generating
vancomycin resistance, the development of resistance was neither easy nor
recent. Three phenotypes of vancomycin resistance (types A, B, and C) are
now well described; a fourth, type D, has been recently reported (25).
VanA-type strains are typically highly resistant to vancomycin and
moderately to highly resistant to teicoplanin. This phenotype is often
plasmid or transposon mediated and is inducible (i.e., exposure of bacteria
to vancomycin results in the induction of the synthesis of several proteins
that together confer resistance) (26).
In vancomycin-susceptible enterococci, D-alanyl-D-alanine (formed by an
endogenous D-alanine-D-alanine ligase) is added to a tripeptide precursor
to form a pentapeptide precursor. The D-Ala-D-Ala terminus is the target of
vancomycin; once vancomycin has bound, the use of this pentapeptide
precursor for further cell-wall synthesis is prevented. In the VanA
phenotype, one of the proteins whose synthesis is induced by exposure of
bacterial cells to vancomycin is called VanA; VanA is a ligase and
resembles the D-alanine-D-alanine ligase from Escherichia coli and other
organisms, including vancomycin-susceptible enterococci (27). VanA
generates D-Ala-D-X, where X is usually lactate; the formation of D-lactate
is due to the presence of VanH, a dehydrogenase encoded by vanH. The
depsipeptide moiety, D-Ala-D-Lac, is then added to a tripeptide precursor,
resulting in a depsipentapeptide precursor. Vancomycin does not bind to the
D-Ala-D-Lac terminus, so this depsipentapeptide can be used in the
remaining steps of cell-wall synthesis. However, when the normal
pentapeptide precursor ending in D-Ala-D-Ala is also present, cells are not
fully vancomycin resistant, despite the presence of D-Ala-D-Lac containing
precursors. This apparent problem is taken care of in large part by vanX,
which encodes a dipeptidase, VanX, that cleaves D-Ala-D-Ala, preventing its
addition to the tripeptide precursor. Should any D-Ala-D-Ala escape
cleavage and result in a normal pentapeptide precursor, vanY encodes an
ancillary or back-up function. That is, it codes for a carboxypeptidase,
VanY, which cleaves D-alanine and D-lactate from D-Ala-D-Ala and
D-Ala-D-Lac termini, respectively, resulting in tetrapeptide precursors, to
which vancomycin does not bind. The other genes involved in the VanA
resistance complex include vanR and vanS, whose encoded proteins are
involved in somehow sensing the presence of extracellular vancomycin or its
effect and signaling intracellularly to activate transcription of vanH,
vanA, and vanX (27). A final gene in the vanA cluster is vanZ, which
encodes VanZ, the role of which is not known.
VanB, encoded by vanB in the vanB gene cluster, is also a ligase that
stimulates the formation of D-Ala-D-Lac. The VanB phenotype is typically
associated with moderate to high levels of vancomycin resistance but is
without resistance to teicoplanin. This is explained by the observation
that vancomycin, but not teicoplanin, can induce the synthesis of VanB and
of VanH[sub beta] and VanX[sub beta]. However, because mutants resistant to
teicoplanin can readily be selected from VanB strains on teicoplanin-containing
agar, clinical resistance would likely occur among VanB strains if teicoplanin
were widely used. Most of the proteins encoded by the vanA gene cluster
have homologues encoded by the vanB gene cluster, except for VanZ. The vanB
gene cluster has an additional gene, vanW, of unknown function.
The VanC phenotype (low-level resistance to vancomycin, susceptible to
teicoplanin) is an inherent (naturally occurring) property of E. gallinarum
and E. casseliflavus. This property is not transferable and is related to
the presence of species-specific genes vanC-1 and vanC-2, respectively
(28); a third possible species, E. flavescens and its gene vanC-3, are so
closely related to E. casseliflavus and vanC-2 that different names are
probably not warranted (29). These species appear to have two ligases; the
cell-wall pentapeptide, at least in E. gallinarum, ends in a mix of
D-Ala-D-Ala and D-Ala-D-Ser (29,30). The genes vanC-1 and vanC-2 apparently
lead to the formation of D-Ala-D-Ser containing cell-wall precursors, while
D-Ala-D-Ala ligases, also present in these organisms, result in
D-Ala-D-Ala. The presence of both D-Ala-D-Ala and D-Ala-D-Ser precursors
may explain why many isolates of these species test susceptible to
vancomycin and why even those isolates with decreased susceptibility
display only low-level resistance.
VanD-type glycopeptide resistance has been recently described in an E.
faecium isolate from the United States (25). The organism was
constitutively resistant to vancomycin (MIC > 64 g/ml) and to low levels
(4 g/ml) of teicoplanin. Following polymerase chain reaction amplification
with primers that amplify many D-Ala-D-Ala ligases, a 605-bp fragment was
identified whose deduced amino acid sequence showed 69% identity to VanA
and VanB and 43% identify to VanC.
Molecular Epidemiology of Newer Resistance Traits
High-Level Gentamicin Resistance
The DNA sequence of the gene encoding HLR to gentamicin in E. faecalis is
the same as the sequence of the gentamicin resistance gene of staphylococci
(15). Since this gene was well established in staphylococci by the 1970s
but HLR to gentamicin was not reported in enterococci until 1979, the
seemingly obvious conclusion is that this gene spread from staphylococci to
enterococci rather than vice versa or, at least, staphylococci acquired it
first. However, the disk diffusion method used in the 1970s and microtiter
dilution MICs done later are capable of detecting gentamicin resistance in
staphylococci but do not distinguish enterococci with high-level gentamicin
resistance from those with low-level, inherent resistance. Therefore, since
laboratories were not screening enterococci by special techniques for
high-level gentamicin resistance, it cannot be definitively stated that
this resistance did not appear in enterococci earlier or coincident with
its emergence in staphylococci. However, several observations support the
likelihood that gentamicin resistance appeared and disseminated in
staphylococci before it did in enterococci. In 1971, Moellering et al.
reported the lack of high-level gentamicin resistance among enterococci
(31). Watanakunakorn reported the absence of high-level gentamicin
resistance among 126 enterococci from 1980 to 1984, with HLR subsequently
appearing in 1985 (32). Phillips et al. from the United Kingdom reported no
highly gentamicin-resistant strains in 1969 to 1979 or 1980 to 1985 and
appearance of strains in 1986 (33). Zervos et al. reported that only one
(0.04%) of 269 isolates of E. faecalis had high-level gentamicin resistance
in 1981; this figure gradually increased to 7.7% in 1984 (34). High-level
gentamicin resistance in E. faecium appears to have occurred after its
appearance in E. faecalis, with the first report occurring in 1988 (35).
Delineation of the molecular epidemiology of strains of enterococci was
limited in the past by the lack of an easy, reliable, and widely accessible
method for subspecies strain differentiation. Zervos and Schaberg reported
the use of plasmid patterns in enterococci to suggest the intrahospital
spread of strains with high-level gentamicin resistance. Pulsed-field gel
electrophoresis (PFGE) of E. faecalis with HLR to gentamicin found that
different isolates from both the same and different locations had markedly
different restriction endonuclease digestion patterns. That is, it found no
evidence of a common strain or strains that predominated among
gentamicin-resistant organisms (36). Strains isolated between 1981 and 1984
at the University of Michigan demonstrated that plasmids encoding
high-level gentamicin resistance were heterogeneous, which again argues
against clonal dissemination of a limited number of strains or plasmids to
account for spread of this property (34,37). We have subsequently shown
that gentamicin resistance in enterococci can be encoded on a transposon
identical to that in staphylococci (38). In addition, the enterococcal
gentamicin-resistance gene has been found in other genetic settings, one of
which has also been found in North American Staphylococcus aureus with
gentamicin resistance (39). Since all enterococci with HLR to gentamicin
(MIC >/= 2,000 g/ml) that have been tested have hybridized with the same
gene probe, this property could be termed a "gene epidemic." However, by
the time gentamicin resistance was discovered in enterococci, this gene was
already widespread with no evidence of either a common plasmid or a common
or predominant strain.
Other genetic elements encoding HLR to gentamicin have also been described.
Thal et al. have described a 27-kb element designated Tn924 that encodes
HLR to gentamicin and could be mobilized from the chromosome of an E.
faecalis by a coresident plasmid (40). Rice et al. have described a large
(ca. 60 kb) transferable element, tentatively named Tn5385, which appears
to contain within it an 18-kb conjugative transposon (Tn5381) encoding
tetracycline resistance and a 26-kb IS256-based composite transposon
(Tn5384) encoding resistance to gentamicin and to erythromycin (41).
Penicillin-Resistant Penicillinase-Producing Enterococci
PFGE analyses of penicillinase-producing enterococci have shown that a
common penicillinase-producing strain (or "clone"), defined as having an
identical or related chromosomal digestion pattern, was present in Texas,
Florida, North Carolina (unpub. observation), Delaware, Pennsylvania, and
Virginia, which had a large outbreak with numerous infections (42,43).
Moreover, at each of these locations, all isolates of
penicillinase-producing E. faecalis examined were derivatives of this
strain; in the hospital in which this strain has been endemic for many
years, a single penicillinase-producing isolate of E. faecium, the only
such isolate ever reported, was also found (44). Among numerous non-Bla+ E.
faecalis, a PFGE pattern similar to that of Bla+ E. faecalis has been found
only once from an isolate from a Philadelphia hospital that had one of the
Bla+ isolates (43 and unpub. obs.). Penicillinase-producing isolates from
Connecticut, Boston, Canada (unpub. observations), Lebanon, and Argentina
represent different strains (19). However, clonal relatedness of three
isolates with an almost identical pattern was demonstrated in Connecticut
(despite one of these lacking HLR to gentamicin) (45), and all six isolates
in Argentina appeared to be a single strain. The Boston isolates have not
been studied by PFGE, but a number of isolates from the same hospital had a
single shared plasmid, suggesting that these also represent clonal
dissemination of a single strain. While all of these
penicillinase-producing E. faecalis could be detected by nitrocefin, a
vancomycin-resistant E. faecalis that tested negative for penicillinase by
nitrocefin but showed a marked inoculum effect with penicillin and
destroyed penicillin in a bioassay has been reported (46). Thus, in
contrast to high-level gentamicin resistance in enterococci, which appeared
to be widely disseminated on different plasmids and in different strains by
the time this phenotype was studied, penicillinase production in
enterococci is still largely associated with a limited number of strains;
moreover, in locations known to have more than one isolate, oligoclonal
spread within each setting remains the rule.
NonpenicillinaseProducing Penicillin-Resistant Strains
PFGE analyses of highly penicillin-resistant E. faecium from the Medical
Colleges of Virginia and Pennsylvania have shown that within each location,
most highly resistant strains represented a single clone (47). Analysis of
the PFGE patterns also raised the possibility, on the basis of similarities
between patterns of isolates in these two different locations, that a
strain may have spread from one institution to the other (47); this
conclusion was supported by the finding that these isolates belonged to the
same multilocus enzyme cluster (unpub. observations). Circumstantial
evidence of intrahospital spread of highly penicillin-resistant E. faecium
also comes from the study of Grayson et al. (22), who reported a sudden
increase in more highly penicillin-resistant isolates of E. faecium at the
Massachusetts General Hospital in 1988. Although no genetic analysis was
done, the fact that gentamicin resistance also simultaneously appeared and
most of the E. faecium highly resistant to penicillin were also highly
resistant to gentamicin suggests clonal dissemination of one or a few
strains within that hospital (22).
Vancomycin Resistance
Vancomycin resistance in enterococci is heterogeneous on many levels. For
example, three different, well-described types of vancomycin resistance are
known, each associated with different ligase genes, (vanA, vanB, vanC1, and
vanC2), and a fourth type, VanD, has been reported recently (25). VanA and
VanB type resistance is encoded by gene clusters that are acquired (i.e.,
not part of the normal genome of enterococci) and are often transferable.
In contrast, vanC1 and vanC2 are normally occurring genes that are
endogenous species characteristics of E. gallinarum and E. casseliflavus,
respectively, and are not transferable. The acquired gene clusters
associated with vanA and vanB are found in different genetic surroundings.
The vanA gene cluster has been found in a small Tn3-like transposon,
Tn1546, and in elements that appear to be closely related (e.g., Tn5488,
which has an insertion sequence [IS1251] within Tn1546 [48,49]) or lacking
vanZ (50). These elements have in turn been found on both transferable and
nontransferable plasmids, as well as on the chromosome of the host strain.
VanB type resistance was initially not found to be transferable, but at
least in some instances, the vanB gene cluster has been found on large (90
kb to 250 kb) chromosomally located transferable elements, one of which
contains within it a 64-kb composite transposon (Tn1547) ((Figure 2;14).
More recently, vanB has been found as part of plasmids.
In addition to being found in different genetic surroundings, the vanA and
vanB gene clusters have also been found in a number of different bacterial
species. vanA has been found in multiple enterococcal species as well as in
lactococci, Orskovia, and Arcanobacteria (51). The distribution of the vanB
gene cluster seems somewhat more restricted, having been found primarily in
E. faecium and E. faecalis, although it has recently been found in
Streptococcus bovis (51).
                                 [Fig]
Figure 2. Potential modes of spread of vancomycin-resistant genes. Adapted
in part from Quintiliani and Courvalin (14).
The vanB gene cluster (shown on the left) on a 64-kb transposon is part of
a 250-kb mobile element shown to move from the chromosome of one
enterococcus and insert into the chromosome of another. Although not
demonstrated, circularization of the vanB containing large mobile elements
resembles the mechanism described for conjugative transposons that can
excise from the chromosome of one strain, circularize, transfer from one
enterococcus to another, and reinsert into the chromosome of the recipient
(such as the one on the right). The 64-kb transposon shown on the left can
also jump to another plasmid within the host enterococcus. If it is a
conjugative (Tra+) plasmid, that plasmid can then transfer by conjugation
to other bacteria, taking the van resistance genes with it. In one
instance, the vancomycin resistance transposon was shown to transpose to a
plasmid encoding the virulence factor hemolysin (Hly).
When vancomycin-resistant enterococci (VRE) from patients in a given
hospital have been examined, particularly after the first recovery of VRE,
evidence is often found of a single or predominant strain (53-58). Finding
isolates with identical or highly related PFGE patterns in different
hospitals indicates interhospital clonal transmission (59-61). Some reports
do not find a single or a predominant strain, especially when VRE have been
present in a hospital or area for some time (62-63). This was also true of
two reports from France in which all of 16 and all of 24
vancomycin-resistant E. faecium were different (50,64,65). The reports from
France likely reflect another observation regarding the diversity of
vancomycin resistance. vanA has also been shown to be present in normal
fecal enterococci of healthy, nonhospitalized persons in different parts of
Europe (68); in one study, 20 different strains (identified by PFGE) of
vancomycin-resistant E. faecium were found in the fecal flora of 17 persons
in two areas in Belgium (68). vanA containing E. faecium have also been
found in the feces of healthy animals as well as from animal products in
Europe (50,67,69-71); in one study, VRE were found in the feces of healthy
meat-eaters but not vegetarians (72). VRE have not, however, been found as
part of the normal fecal flora in the United States (73) possibly because
glycopeptides, often used in animal feed in Europe, are not used in the
United States. For example, 24,000 kg annually of the glycopeptide
avoparcin were reportedly used in recent years in Denmark (74). Reports of
VRE in the feces of animals on farms using glycopeptides, but not in those
without such use, support this hypothesis (71,75). While oral glycopeptide
use markedly increases the numbers of VRE per gram of stool in humans (76)
and by analogy, presumably does so in animals, glycopeptide use does not
explain the origin of these gene clusters.
The problem of multidrug-resistant enterococci promises to be with us for
the foreseeable future. The enterococcus has likely emerged as a major
nosocomial pathogen in part because of its resistance to multiple
antibiotics, which allows it to survive and subsequently infect patients.
With its propensity to acquire new traits, such as high-level gentamicin,
penicillin, and vancomycin resistance, the enterococcus continues to create
new therapeutic problems and dilemmas; its ability to transfer some of its
plasmids to streptococci and staphylococci and the implications of a
possible spread of penicillin and vancomycin resistance to these and other
gram-positive species are also of concern.
Dr. Murray is professor and director, Division of Infectious Diseases, and
codirector, Center for the Study of Emerging and Re-emerging Pathogens,
University of Texas Houston-Medical School. Her main interests are in the
areas of antibiotic resistance, molecular epidemiology, and bacterial
pathogenesis. Recent work has been focused on possible mechanisms of
virulence of enterococci.
Address for correspondence: Barbara E. Murray, Division of Infectious
Diseases, University of Texas Houston-Medical School, 6431 Fannin, 1.728
JFB, Houston, TX 77030, USA; fax: 713-500-6761; e-mail:
iminfdis@heart.med.uth.tmc.edu.
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     Vancomycin-resistant enterococci from nosocomial, community, and
     animal sources in the United States. Antimicrob Agents Chemother
     1996;40:2605-9.
 74. Kjerulf A, Pallesen L, Westh H. Vancomycin-resistant enterococci at a
     large university hospital in Denmark. APMIS 1996;104:475-9.
 75. Klare I, Heier H, Claus H, Witte W. Environmental strains of
     Enterococcus faecium with inducible high-level resistance to
     glycopeptides. FEMS Microbiol Lett 1993;106:23-30.
------------------------------------------------------------------------
Synopses
Proteases of Malaria Parasites: New Targets for Chemotherapy
Philip J. Rosenthal
San Francisco General Hospital and University of California, San Francisco,
California, USA
---------------------------------------------------------------------------
      The increasing resistance of malaria parasites to antimalarial
      drugs is a major contributor to the reemergence of the disease
      as a major public health problem and its spread in new
      locations and populations. Among potential targets for new
      modes of chemotherapy are malarial proteases, which appear to
      mediate processes within the erythrocytic malarial life cycle,
      including the rupture and invasion of infected erythrocytes and
      the degradation of hemoglobin by trophozoites. Cysteine and
      aspartic protease inhibitors are now under study as potential
      antimalarials. Lead compounds have blocked in vitro parasite
      development at nanomolar concentrations and cured
      malaria-infected mice. This review discusses available
      antimalarial agents and summarizes experimental results that
      support development of protease inhibitors as antimalarial
      drugs.
Hundreds of millions of cases of malaria occur annually, and infections
with _Plasmodium falciparum_, the most virulent human malaria parasite,
cause more than one million deaths per year (1). Despite extensive control
efforts, the incidence of the disease is not decreasing in most
malaria-endemic areas of the world, and in some it is clearly increasing
(2). Malaria also remains a major risk to travelers from industrialized to
developing countries. Because malaria parasites are increasingly resistant
to antimalarial drugs, appropriately counseled travelers to malaria-endemic
regions are more likely to contract malaria now than they were 40 years
ago.
Malaria control efforts include attempts to develop an effective vaccine,
eradicate mosquito vectors, and develop new drugs (2,3). However, the
development of a vaccine has proven very difficult, and a highly effective
vaccine will probably not be available in the near future (4). Efforts to
control _Anopheles_ mosquitoes have had limited success, although the use
of insecticide-impregnated bed nets does appear to reduce malaria-related
death rates (5). In addition, methods to replace natural vector populations
with mosquitoes unable to support parasite development are under study and
may contribute to malaria control in the long term (6). However, the
current limitations of vaccine and vector control, as well as the
increasing resistance of malaria parasites to existing drugs, highlight the
continued need for new antimalarial agents.
Established Antimalarial Drugs
Antimalarial drugs have been used for centuries. Early natural products,
including the bark of the cinchona tree in South America and extracts of
the wormwood plant in China, were among the first effective antimicrobial
agents to be used. Cinchona bark was used in Europe beginning in the 17th
century, and upon its isolation from bark in 1820, quinine became widely
used. In the last 50 years, extensive efforts, including the screening of
hundreds of thousands of compounds, have led to the development of a number
of effective synthetic antimalarial drugs. The most important of these,
chloroquine, has been the mainstay of antimalarial chemotherapy for the
last 50 years. The compound eradicates parasites rapidly, has minimal
toxicity, is widely available at low cost throughout the world, and needs
to be taken only once a week for chemoprophylaxis. However, resistance to
chloroquine has been steadily increasing since the drug's initial use in
South America and Southeast Asia in the late 1950s. Chloroquine resistance
is now widespread in most _P. falciparum_ -endemic areas of the world (3).
Thus, the use of chloroquine for presumptive treatment of falciparum
malaria or for chemoprophylaxis is usually no longer appropriate (7).
Moreover, resistance to chloroquine of _P. vivax_, the second most lethal
human malaria parasite, is increasing in South Asia (8).
No other antimalarial drug (9-12) is as efficacious and safe as chloroquine
(Table 1). The best antimalarial drug for treating chloroquine-resistant
falciparum malaria remains quinine (or intravenous quinidine), which is
fairly toxic; quinine resistance is increasing in Southeast Asia,
particularly in the border areas of Thailand (9). Amodiaquine, used to
treat chloroquine-resistant malaria in developing countries, is also quite
toxic, and resistance to it is also common (13). Mefloquine (14) is widely
used for chemoprophylaxis against chloroquine-resistant _P. falciparum_,
but its use is limited by toxicity (15) and (in the developing world) high
cost. Mefloquine is not approved for treatment of malaria in the United
States because of the neurotoxicity of doses required for the treatment.
Fansidar, a combination of sulfadoxine and pyrimethamine, is no longer
recommended for chemoprophylaxis because of its dermatologic toxicity (15).
Fansidar is also not an ideal drug for treatment because it is slow acting,
but it is increasingly important in treating chloroquine-resistant malaria
in developing countries because economic constraints limit the use of other
agents (16). The use of both mefloquine and Fansidar will increasingly be
limited by drug resistance, already widespread in parts of Southeast Asia
(9,17).
Table 1. Established antimalarial drugs (sup a)
---------------------------------------------------------------------------
Drug               Role               Best Feature(s)   Limitations
---------------------------------------------------------------------------
Chloroquine        TX of and CP       Very safe; low    Widespread R
                   against non- Pf    cost; long
                   and sensitive Pf   half-life
                   parasites
Quinine/quinidine  Best TX for Pf     Limited R;        Fairly toxic
                   malaria; low cost  rapidly acting    (cinchonism,
                                                        cardiac)
Amodiaquine (sup   TX of R Pf malaria Low cost          Toxicity (bone
b)                                                      marrow, liver); R
                                                        common
Mefloquine         CP against R Pf    Relatively little Moderately toxic
                   malaria; not       R, though         (mostly CNS);
                   approved for TX in increasing; long  high  cost; R in
                   United States      half-life         SE Asia
Fansidar           TX of Pf malaria;  Relatively low    Skin toxicity (can
                   no longer          cost; long        be  fatal);
                   recommended for CP half-life         increasing R
Primaquine         Eradication of     Only drug for     Hemolysis with
                   chronic liver      this indication   G6PD deficiency;
                   stage Pv, Po                         increasing R
                   malaria
Proguanil (sup b)  CP only (often     Low cost;         R common
                   with chloroquine)  nontoxic
Maloprim (sup b)   CP only (often     Low cost          R common; skin
                   with chloroquine)                    rashes
Tetracyclines      CP; TX of Pf       Low cost          Skin and
                   malaria in                           gastrointestinal
                   combination with                     toxicity
                   quinine
---------------------------------------------------------------------------
(sup a) TX, therapy; CP, chemoprophylaxis; R, resistance/resistant; Pf,
_Plasmodium falciparum_; Pv, _P. vivax_; Po, _P. ovale_; CNS, central
nervous system; G6PD, glucose 6-phosphate dehydrogenase.
(sup b) Not available in the United States.
---------------------------------------------------------------------------
Other antimalarial drugs have specialized uses. Tetracyclines and some
other antibiotics (clindamycin, sulfas) are slow acting and generally best
used as an adjunct to quinine therapy in treating falciparum malaria (9).
Doxycycline is also used for chemoprophylaxis in regions with high levels
of drug resistance, especially Southeast Asia (10,17). Other drugs for
chemoprophylaxis include proguanil, which remains effective in combination
with chloroquine in many areas other than Southeast Asia, and Maloprim, a
combination of dapsone and pyrimethamine (10,17). Resistance to these drugs
is fairly common, however. Primaquine has a well-defined specific role:
eradicating chronic liver stages of _P. vivax_ and _P. ovale_ after
treating the acute blood infection with chloroquine.
New Antimalarial Drugs
Relatively few new antimalarial drugs are undergoing clinical testing
(Table 2). Halofantrine, identified in the 1940s, was not developed until
the 1980s; its use has been limited by variable oral absorption and cardiac
toxicity (12,18). The drug is approved in the United States for treatment
of chloroquine-resistant _P. falciparum_ infection, although in most cases
quinine (or intravenous quinidine) is preferable. The most effective new
drugs are artemisinin and related compounds. Artemisinin was isolated in
1972 from _Artemisia annua_, a plant used in China for centuries to treat
fever (19). Artemisinin derivatives (artesunate, artelinate, artemether,
arteether, dihydroartemisinin) have been synthesized and are undergoing
extensive clinical testing. These compounds, which are already widely used
in some areas, are potent, rapidly acting antimalarials that are effective
against chloroquine-resistant _P. falciparum_ (20). Because recrudescences
of infection after treatment are common, however, artemisinin and related
compounds might best be used in combination with another drug.
Table 2. New antimalarial drugs
---------------------------------------------------------------------------
 Drug               Role              Best Feature(s)   Limitations
 --------------------------------------------------------------------------
 Halofantrine       TX of Pf malaria; Usually effective Variable
                    not approved for  against R Pf      bioavailability,
                    CP                malaria           cardiac toxicity
 Artemisinin and    TX of Pf malaria  Rapidly acting;   Recurrence after
 related compounds                    effective against TX fairly common
 (sup a)                              multidrug-R
                                      strains
 Atovaquone         ? TX of Pf        Limited toxicity  Limited studies so
                    malaria; ? CP                       far show frequent
                    (probably in                        recurrence after
                    combination with                    TX
                    proguanil)
 Pyronaridine (sup  ? TX of Pf        Effective against Studies limited to
 a)                 malaria           R strains         date
 Desferrioxamine    ? TX of severe Pf Well tolerated    Studies limited to
                    malaria           when used for     date
                                      iron overload
 Azithromycin       ? CP              Limited toxicity  Studies limited to
                                                        date
 --------------------------------------------------------------------------
For abbreviations, see Table 1, footnote a.
(sup a) Not available in the United States.
Other compounds are under evaluation. Atovaqone (21), which is approved for
treating patients with _Pneumocystis_ infections, appears to be effective
against malaria in combination with proguanil (22), but its use has been
limited by recrudescence after treatment. Pyronaridine, an acridine
derivative used to treat malaria in China, has shown efficacy against
falciparum malaria (23). The iron chelator desferrioxamine enhances the
clearance of parasites in mild malaria (24) and, in conjunction with
quinine and Fansidar, hastens recovery from deep coma in severe falciparum
malaria (25). Azithromycin, a quinolone antibiotic, appears efficacious in
malaria chemoprophylaxis (26).
Malarial Proteases: New Targets for Chemotherapy
The limitations of antimalarial chemotherapy underscore the need for new
drugs, ideally directed against new targets. Potential targets for
chemotherapy include malarial proteases (27). The erythrocytic life cycle,
which is responsible for all clinical manifestations of malaria, begins
when free merozoites invade erythrocytes. The intraerythrocytic parasites
develop from small ring-stage organisms to larger, more metabolically
active trophozoites and then to multinucleated schizonts. The erythrocytic
cycle is completed when mature schizonts rupture erythrocytes, releasing
numerous invasive merozoites. Proteases appear to be required for the
rupture and subsequent reinvasion of erythrocytes by merozoite-stage
parasites and for the degradation of hemoglobin by intraerythrocytic
trophozoites figure.
 [fig]
 Figure. Protease targets in erythrocytic malaria parasites. The _Plasmodium
 falciparum_ erythrocytic life cycle is shown schematically, and data supporting
 cysteine (CP), serine (SP), and aspartic (AP) proteases of the different
 parasite stages as chemotherapeutic targets are provided in italics.
Proteases and Erythrocyte Rupture and Invasion
The rupture of erythrocytes by mature schizonts and the subsequent invasion
of erythrocytes by free merozoites appear to require malarial protease
activity, possibly to breach the erythrocyte cytoskeleton, a complex
network of proteins. In addition, a number of malarial proteins are
proteolytically processed during the late schizont and merozoite life-cycle
stages; for example, merozoite surface protein-1 is processed in a manner
inhibited by serine protease inhibitors (28), presumably to facilitate the
complex series of events involved in erythrocyte rupture and invasion (29).
Although the specific roles of different classes of proteases are not
completely clear, inhibitors of cysteine and serine proteases have
consistently blocked erythrocyte rupture and invasion (27).
Candidate _P. falciparum_ rupture/invasion proteases have been identified,
but none has been fully characterized biochemically or molecularly: 1) a 68
kD cysteine protease was identified in schizonts and merozoites and
localized to the merozoite apex, suggesting that it may be released from
the rhoptry organelle during invasion (30); 2) a cysteine protease of
mature schizonts and a serine protease of merozoites were identified in
highly synchronized parasites (31); 3) a serine protease was shown to be
bound to the schizont/merozoite membrane by a glycosyl-phosphatidylinositol
anchor, to be activated by phosphatidylinositol-specific phospholipase C
during the merozoite stage, and to be capable of cleaving the erythrocyte
cytoskeletal protein band 3 (32,33); 4) another protease, inhibited by both
cysteine and serine protease inhibitors, hydrolyzed the erythrocyte
cytoskeletal proteins spectrin and band 4.1 (34); and 5) the serine repeat
antigen (35,36) and the related protein SERP H (37), both expressed in
mature schizonts, have important similarities in their sequences with
cysteine proteases. Further research should identify the specific biologic
roles of the proteases mentioned and better characterize these enzymes,
thus fostering the development of specific inhibitors.
Host proteases may also play a role in erythrocyte rupture by _P.
falciparum_. In recent studies, host urokinase was shown to bind to the
surface of _P. falciparum_-infected erythrocytes, and the depletion of
urokinase from parasite culture medium inhibited erythrocyte rupture by
mature schizonts (38). This inhibition was reversed by exogenous urokinase.
Drug Development Efforts
Synthetic peptide inhibitors of the _P. falciparum_ schizont cysteine
protease Pf 68 inhibited erythrocyte invasion by cultured parasites
(39,40). The most effective peptide, GlcA-Val
-Leu-Gly-Lys-NHC (sub 2)H(sub
5), inhibited the protease and blocked parasite development at high
micromolar concentrations (40; Table 3). Although these results do not
demonstrate levels of inhibition expected to be therapeutically relevant,
they suggest that a specific protease activity is required for erythrocyte
invasion by malaria parasites and thus is a potential target for
antimalarial drugs.
Proteases and Malarial Hemoglobin Degradation
Extensive evidence suggests that the degradation of hemoglobin is necessary
for the growth of erythrocytic malaria parasites, apparently to provide
free amino acids for parasite protein synthesis (27,50). In _P.
falciparum_, hemoglobin degradation occurs predominantly in trophozoites
and early schizonts, the stages at which the parasites are most
metabolically active. Trophozoites ingest erythrocyte cytoplasm and
transport it to a large central food vacuole. In the food vacuole,
hemoglobin is broken down into heme, a major component of malarial pigment
(51), and globin, which is hydrolyzed to its constituent amino acids. The
food vacuole is an acidic organelle analogous to lysosomes. Several
lysosomal proteases are well characterized, including cysteine (cathepsins
B, H, and L) and aspartic (cathepsin D) proteases (52), and malaria
parasites contain analogous food vacuole proteases that degrade hemoglobin.
At least two aspartic proteases and one cysteine protease have been
isolated from purified _P. falciparum_ food vacuoles (53).
Malarial aspartic protease activities have been identified (54-60). Two
recently characterized aspartic proteases (plasmepsin I and plasmepsin II)
are located in the food vacuole, have acid pH optima, and share sequence
homology with other aspartic proteases (41,53,61,62). Furthermore, the
aspartic proteases can cleave hemoglobin. One of the enzymes, plasmepsin I,
cleaves native hemoglobin (53,59). Plasmepsin II appears to prefer
denatured globin as a substrate (53). On the basis of these data,
plasmepsin I is thought to be responsible for initial cleavages of
hemoglobin after the molecule is transported to the food vacuole (53).
Incubation of cultured _P. falciparum_ parasites with the protease
inhibitor leupeptin caused trophozoite food vacuoles to fill with
apparently undegraded erythrocyte cytoplasm (63-65). Analysis of the
leupeptin-treated parasites showed that they contained large quantities of
undegraded globin, while minimal globin was detectable in control parasites
(64,66). Leupeptin inhibits both cysteine and some serine proteases, but
the highly specific cysteine protease inhibitor E-64 also caused undegraded
globin to accumulate. After parasites were incubated with inhibitors of
other classes of proteases including the aspartic protease inhibitor
pepstatin (63-67), globin did not accumulate. More recent studies that used
nondenaturing electrophoretic methods demonstrated that cysteine protease
inhibitors not only blocked malarial globin hydrolysis, but also inhibited
earlier steps in hemoglobin degradation, including denaturation of the
hemoglobin tetramer and the release of heme from globin (68). Another study
showed that E-64, but not pepstatin, inhibited the production of hemozoin
(the malarial end product of heme) by cultured parasites (69). These
results suggest that a cysteine protease is required for initial steps in
hemoglobin degradation by _P. falciparum_.
A _P. falciparum trophozoite cysteine protease with biochemical features
expected for a food vacuole hemoglobinase has been identified (31) and
biochemically (70-72) and molecularly (73) characterized. This protease,
called falcipain, degraded denatured and native hemoglobin in vitro; its
acid pH optimum, substrate specificity, and inhibitor sensitivity indicated
that it was a papain family cysteine protease (64,70,71). Specific
inhibitors of falcipain blocked hemoglobin degradation and prevented
parasite development. The degree of inhibition of falcipain by fluoromethyl
ketones (44) and vinyl sulfones (46) correlated with their inhibition of
hemoglobin degradation and parasite development, supporting the hypothesis
that falcipain is the cysteine protease required for hemoglobin
degradation.
The specific mechanism for hemoglobin degradation in the malarial food
vacuole remains unclear. As noted above, both the aspartic protease
plasmepsin I and the cysteine protease falcipain have been identified in
parasite food vacuoles and shown to cleave denatured and native hemoglobin
in vitro (53,71). Results showing that only cysteine protease inhibitors
block hemoglobin processing and globin hydrolysis in cultured parasites
suggest that falcipain is required for initial steps of hemoglobin
degradation (66-68,74). However, other studies have shown that native
hemoglobin is cleaved by plasmepsin I, but not falcipain, in nonreducing
conditions that may be present in the food vacuole (53,59,72). In any
event, regardless of the exact sequence of hemoglobin processing, multiple
enzymes, including at least the three proteases already identified, appear
to participate in the degradation of hemoglobin. These proteases are thus
logical targets for antimalarial drug development.
Aminopeptidase activity has also been described in malaria parasites
(75-77). This activity, with a neutral pH optimum, was not found in food
vacuole lysates (77). When these lysates were incubated with hemoglobin,
discrete peptide fragments, but not free amino acids, were identified (77).
These results suggest that hemoglobin is degraded to small peptides in the
food vacuole, that these peptides are transported to the parasite cytosol,
and that additional processing of hemoglobin peptides is mediated by
cytosolic aminopeptidase activity (77).
Drug Development Efforts
Both the cysteine protease inhibitor E-64 and the aspartic protease
inhibitor pepstatin blocked _P. falciparum_ development (63-67).
Administered together, the two inhibitors acted synergistically (67).
However, only E-64 blocked globin hydrolysis (64-67). Numerous
peptide-based cysteine protease inhibitors, including fluoromethyl ketones
(44,70,78) and vinyl sulfones (46), inhibited falcipain at low nanomolar
concentrations and inhibited _P. falciparum_ development and hemoglobin
degradation at concentrations below 100 nanomolar (Table 3). In a malaria
animal model, a fluoromethyl ketone that inhibited falcipain at low
nanomolar concentrations blocked _P. vinckei_ protease activity in vivo
after a single subcutaneous dose, and, when administered for 4 days, cured
80% of murine malaria infections (45). Thus, despite the theoretical
limitations of potentially rapid degradation in vivo and inhibition of host
proteases, peptide protease inhibitors show promise as candidate
antimalarial drugs. Fluoromethyl ketones have subsequently shown toxicity
in animal studies, but evaluations of related, apparently nontoxic
inhibitors of falcipain as antimalarial drugs are under way.
Table 3. Protease targets for chemotherapy
---------------------------------------------------------------------------
                                              Effective inhibitors (sup a)
                         ------------------------------------------------------------------------
Protease    Biologic     Compound (Reference)                 In vitro (sup In vivo (sup
            role                                              b)            c)
                                                                            (mg/kg/day)
                                                              (IC(sub 50);
                                                              (Microgram M)
-------------------------------------------------------------------------------------------------
Pf68        Erythrocyte  GlcA-Val-Leu-Gly-Lys-NHC(sub         	900
            invasion     2)H(sub 5) (40)
Plasmepsin  Hemoglobin   SC-50083 (41)                            2-5
I           degradation                                           0.25
                         Ro 40-4388 (42)
Plasmepsin  Hemoglobin   Compound 7 (43)                        	20
II          degradation
Falcipain   Hemoglobin   Z-Phe-Arg-CH2F (44)                      0.064
            degradation
                         Mu-Phe-HPh-CH2F (45)                    ~0.03 400
                         Mu-Leu-HPh-VSPh (46)                     0.01
                         Oxalic bis ((2-hydroxy-1-naph-           7
                         thylmethylene)hydrazide) (47)
                         1-(2,5-dichlorophenyl)-3-                0.23
                         (4-quinolinyl)-2-propen-1-one (48)
                         7-chloro-1,2-dihydro-2-(2,3-di-          2
                         methoxy-phenyl)-5,5-dioxide-
                         4-(1H,10H)-phenothiazinone (49)
-------------------------------------------------------------------------------------------------
(sup a) The structures of these compounds and details of the described studies are in the
references noted.
(sup b) Assays compared the development of new ring-form parasites or the uptake of [ (sup 3)
H]hypoxanthine by treated and control parasites.
(sup c) Cure of _Plasmodium vinckei_-infected mice.
A computer model for the structure of falcipain was used to identify
nonpeptide inhibitors (47). Screening of potential nonpeptide inhibitors
identified a low micromolar lead compound (47; Table 3). Subsequent
synthesis and testing of small molecules based on the structure of the lead
compound have identified biologically active falcipain inhibitors,
including chalcones that block parasite metabolism at submicromolar
concentrations (48) and phenothiazines that block parasite metabolism and
development at low micromolar concentrations (49).
Peptidelike aspartic protease inhibitors are potent inhibitors of
plasmepsins I and II. In independent studies SC-50083 (41), Ro 40-4388
(42), and "compound 7" (43) inhibited plasmepsin I or II at nanomolar
concentrations and blocked parasite development at high nanomolar to
micromolar concentrations (Table 3). Drug development efforts should be
assisted by the recent determination of the structure of plasmepsin II
(43). Inhibitors of aspartic and cysteine proteases have synergistic
effects in inhibiting the growth of cultured malaria parasites (67), and
these proteases also act synergistically to degrade hemoglobin in vitro
(41). Therefore, the combination of inhibitors of malarial cysteine and
aspartic proteases may provide the most effective chemotherapeutic regimen
and best limit the development of parasite resistance to protease
inhibitors. Ultimately, a better understanding of the biochemical
properties and biologic roles of malarial proteases will foster the
development of protease inhibitors that specifically inhibit parasite
enzymes and thus are the most suitable candidates for chemotherapy.
Acknowledgments
Work in the author's laboratory was supported by grants from the National
Institutes of Health, the UNDP/World Bank/WHO Special Programme for
Research and Training in Tropical Diseases, and the American Heart
Association.
Dr. Rosenthal is associate professor, Department of Medicine, Division of
Infectious Diseases, San Francisco General Hospital and the University of
California, San Francisco. His research interests include the evaluation of
proteases of malaria parasites as chemotherapeutic targets.
Address for correspondence: Philip Rosenthal, Box 0811, University of
California, San Francisco, CA 94143-0811, USA; fax: 415-206-6015; e-mail:
rosnthl@itsa.ucsf.edu.
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---------------------------------------------------------------------------
Synopses
Zoonotic Tuberculosis due to Mycobacterium bovis in Developing Countries
O. Cosivi,* J.M. Grange, C.J. Daborn, M.C. Raviglione,* T. Fujikura,  D.
Cousins, R.A. Robinson,** H.F.A.K. Huchzermeyer,   I. de Kantor, and
F.-X. Meslin*
*World Health Organization, Geneva, Switzerland; Imperial College School
of Medicine, National Heart and Lung Institute, London, United Kingdom;
Edinburgh University, Edinburgh, Scotland; The University of Zambia,
Lusaka, Zambia; Department of Agriculture, South Perth, Australia;
**College of Veterinary Medicine, Veterinary Teaching Hospital,
---------------------------------------------------------------------------
      The World Health Organization (WHO) estimates that human
      tuberculosis (TB) incidence and deaths for 1990 to 1999 will be
      88 million and 30 million, respectively, with most cases in
      developing countries. Zoonotic TB (caused by Mycobacterium
      bovis) is present in animals in most developing countries where
      surveillance and control activities are often inadequate or
      unavailable; therefore, many epidemiologic and public health
      aspects of infection remain largely unknown. We review
      available information on zoonotic TB in developing countries,
      analyze risk factors that may play a role in the disease,
      review recent WHO activities, and recommend actions to assess
      the magnitude of the problem and control the disease in humans
      and animals.
Tuberculosis (TB), one of the most widespread infectious diseases, is the
leading cause of death due to a single infectious agent among adults in the
world. Mycobacterium tuberculosis is the most common cause of human TB, but
an unknown proportion of cases are due to M. bovis (1). In industrialized
countries, animal TB control and elimination programs, together with milk
pasteurization, have drastically reduced the incidence of disease caused by
M. bovis in both cattle and humans. In developing countries, however,
animal TB is widely distributed, control measures are not applied or are
applied sporadically, and pasteurization is rarely practiced. The direct
correlation between M. bovis infection in cattle and disease in the human
population has been well documented in industrialized countries. Whereas
little information is available from developing countries (2,3), risk
factors for M. bovis in both animals and humans are present in the tropics.
TB is a major opportunistic infection in HIV-infected persons (4). The vast
majority of people carrying this dual infection live in developing
countries; however, dual HIV and M. bovis infection has been reported in
industrialized countries (5-11). The epidemic of HIV infection in
developing countries, particularly countries in which M. bovis infection is
present in animals and the conditions favor zoonotic transmission, could
make zoonotic TB a serious public health threat to persons at risk
(3,12-14).
We summarize available epidemiologic information on TB and zoonotic TB,
examine risk factors that can influence the occurrence of zoonotic TB in
developing countries, and describe the most recent TB activities of the
World Health Organization (WHO) (15-18).
Human TB: Global Situation and Trends
The global incidence of TB is greatly underestimated. In 1995, 3.3 million
cases were reported to the Global Tuberculosis Programme of WHO, whereas a
more likely number is 8.8 million. Of the reported cases, 62% occurred in
the Southeast Asian and Western Pacific regions, 16% in sub-Saharan Africa,
and 7% to 8% in each of the regions of the Americas, Eastern Mediterranean,
and Europe. Many countries, especially those with few resources, are unable
to report all TB cases because of difficulties in identifying suspected
cases, establishing a diagnosis, and recording and reporting cases.
In 1995, an estimated 8.8 million new TB cases occurred-5.5 million (62%)
in the Southeast Asian and Western Pacific regions and 1.5 million (17%) in
sub-Saharan Africa. The annual global incidence is predicted to increase to
10.2 million by the year 2000, an increase of 36% from 1990. Southeast
Asia, Western Pacific regions, and sub-Saharan Africa will account for 81%
of these new cases (Table 1). For 1990 to 1999, in the absence of effective
control, global TB incidence and deaths will reach 88 million and 30
million, respectively (19); 70% of the new cases will occur in patients 15
to 59 years of age, the most economically productive segment of the
population.
As a result of the HIV epidemic, the crude incidence rate of TB is expected
to increase in sub-Saharan Africa from 191 cases per 100,000 in 1990 to 293
in 2000. However, the total number of new cases will double by the year
2000. Because of the HIV epidemic, the decline of the crude incidence rate
in the Southeast Asian and Central and South American regions is expected
to be slower than in previous years. In industrialized countries, a small
increase in crude incidence rate and total cases is expected as the result
of immigration from countries with a high prevalence of dual HIV and TB
infection.
The worldwide incidence of HIV-attributable TB cases is estimated to
increase from 315,000 (4% of the total TB cases) in 1990 to 1.4 million
(14% of the total TB cases) by the year 2000. In 2000, approximately 40% of
these HIV-attributable cases will occur in sub-Saharan Africa and 40% in
Southeast Asia. Ten percent of the total number of TB cases expected during
1990 to 1999 are estimated to be attributable to HIV infection.
While demographic factors, such as population growth and changes in
population structure, will largely account for the expected increase in TB
incidence worldwide, the HIV epidemic in sub-Saharan Africa will have a
greater role than demographic factors.
By the year 2000, 3.5 million persons will be dying of TB annually, an
increase of 39% from 1990. In Southeast Asia alone, 1.4 million deaths will
occur annually. During 1990 to 1999, an estimated 30 million will die of
TB, with 9.7% of the cases attributable to HIV infection. M. tuberculosis
will be largely responsible for the new TB cases and deaths, but an
unknown, and potentially important, proportion will be caused by M. bovis.
Table 1. Estimated human tuberculosis and HIV-attributable tuberculosis
cases in 1990, 1995, and 2000, by region (19)
                	1990                  		 1995                     2000
             ---------------------------   ------------------------- --------------------------
TB            	    HIV-      TB          	HIV-        TB              HIV-
Region      cases   Rate(supa) attributed  cases   Rate  attributed    cases    Rate  attribut
-----------------------------------------------------------------------------------------------
Southeast
 Asia	   3,106,000    237 	  66,000   3,499,000   241   251,000    3,952,000  247   571,000
Western
 Pacific
 (supb)    1,839,000  	 136 	  19,000   22,045,000  140    31,000    2,255,000  144    68,000
Africa       992,000 	 191  	 194,000    1,467,000  242   380,000    2,079,000  293   604,000
Eastern
 Mediter-
 ranean      641,000 	 165 	   9,000      745,000  168    16,000      870,000  168    38,000
Americas(supc)569,000 	 127 	  20,000      606,000  123    45,000     645,000   120    97,000
Eastern
 Europed     194,000   	  47    1,000       202,000   47     2,000      210,000   48      6,000
Industr-
 ialized
 countries
 (supe)      196,000 	 23 	 6,000      204,000    23    13,000      211,000    24    26,000
Total TB
 cases     7,537,000 	 143 	 315,000    8,768,000  152   738,000   10,222,000   163 1,410,000
  Attributed
   to HIV 			    4.2%			  8.4% 				13.8%
  Increase
   since 1990 					  16.3% 			35.6%
-------------------------------------------------------------------------------------------------
(supa)Rate: incidence of new cases per 100,000 population.
(supb)Western Pacific Region of WHO except Japan, Australia, and New Zealand.
(supc)American Region of WHO, except USA and Canada.
(supd)Eastern European countries and independent states of the former USSR.
(supe)Western Europe, USA, Canada, Japan, Australia, and New Zealand.
Bovine TB in Developing Countries
Although prevalence data on animal TB in developing countries are generally
scarce, information on bovine TB occurrence and control measures exists
(20,21).
Africa
Of 55 African countries, 25 reported sporadic/low occurrence of bovine TB;
six reported enzootic disease; two, Malawi and Mali, were described as
having a high occurrence; four did not report the disease; and the
remaining 18 countries did not have data (Figure 1).
Of all nations in Africa, only seven apply disease control measures as part
of a test-and-slaughter policy and consider bovine TB a notifiable disease;
the remaining 48 control the disease inadequately or not at all (Figure 2).
Almost 15% of the cattle population are found in countries where bovine TB
is notifiable and a test-and-slaughter policy is used. Thus, approximately
85% of the cattle and 82% of the human population of Africa are in areas
where bovine TB is either partly controlled or not controlled at all.
Asia
Of 36 Asian nations, 16 reported a sporadic/low occurrence of bovine TB,
and one (Bahrain) described the disease as enzootic; ten did not report
bovine TB; and the remaining nine did not have data (Figure 3). Within the
Asian region, seven countries apply disease control measures as part of a
test-and-slaughter policy and consider bovine TB notifiable. In the
remaining 29 countries, bovine TB is partly controlled or not controlled at
all (Figure 4).
Of the total Asian cattle and buffalo populations, 6% and less than 1%,
respectively, are found in countries where bovine TB is notifiable and a
test-and-slaughter policy is used; 94% of the cattle and more than 99% of
the buffalo populations in Asia are either only partly controlled for
bovine TB or not controlled at all. Thus, 94% of the human population lives
in countries where cattle and buffaloes undergo no control or only limited
control for bovine TB.
Latin American and Caribbean Countries
Of 34 Latin American and Caribbean countries, 12 reported bovine TB as
sporadic/low occurrence, seven reported it as enzootic, and one (Dominican
Republic) described occurrence as high. Twelve countries did not report
bovine TB. No data were available for the remaining two countries (Figure
5).
In the entire region, 12 countries apply disease control measures as part
of a test-and-slaughter policy and consider bovine TB a notifiable disease.
In the remaining 22 nations, the disease is partly controlled or not
controlled at all (Figure 6). The regional prevalence of bovine TB has been
estimated at 1% and higher in 67% of the total cattle population and 0.1%
to 0.9% in a further 7%; the remaining 26% are free of the disease or are
approaching the point of elimination (22).
Of the total Latin American and Caribbean cattle population, almost 76% is
in countries where bovine TB is notifiable and a test-and-slaughter policy
is used. Thus, approximately 24% of the cattle population in this region is
either only partly controlled for bovine TB or not controlled at all. It is
also estimated that 60% of the human population live in countries where
cattle undergo no control or only limited control for bovine TB.
 		[Fig] 					[Fig]
      Figure 1. Bovine tuberculosis   Figure 2. Control measures for
      occurrence, Africa (21).        bovine tuberculosis based on
                                      test-and-slaughter policy and
                                      disease notification, Africa
                                      (21).
 		[Fig]                         	 [Fig]
   Figure 3. Bovine tuberculosis     Figure 4. Control measures for bovine
   occurrence, Asia (21). 		    test-and-slaughter tuberculosis based on
 					    policy and disease notification, Asia (21).
     		[Fig]          			[Fig]
      Figure 5. Bovine tuberculosis  Figure 6. Control measures for
      occurrence, Latin America and  bovine tuberculosis based on
      the Caribbean (21).            test-and-slaughter policy and
                                     disease notification, Latin
                                     America and the Caribbean (21).
Zoonotic TB in Humans
TB caused by M. bovis is clinically indistinguishable from TB caused by M.
tuberculosis. In countries where bovine TB is uncontrolled, most human
cases occur in young persons and result from drinking or handling
contaminated milk; cervical lymphadenopathy, intestinal lesions, chronic
skin TB (lupus vulgaris), and other nonpulmonary forms are particularly
common. Such cases may, however, also be caused by M. tuberculosis. Little
is known of the relative frequency with which M. bovis causes nonpulmonary
TB in developing nations because of limited laboratory facilities for the
culture and typing of tubercle bacilli.
Agricultural workers may acquire the disease by inhaling cough spray from
infected cattle; they develop typical pulmonary TB. Such patients may
infect cattle, but evidence for human-to-human transmission is limited and
anecdotal.
In regions where bovine TB has been largely eliminated, a few residual
cases occur among elderly persons as a result of the reactivation of
dormant lesions. These are fewer than 1% of all TB cases. Surveys in the
United States, Scandinavia, and South England have shown that approximately
half of these postprimary cases are pulmonary, a quarter involve the
genitourinary tract (a rare occurrence in primary disease), and the
remainder involve other nonpulmonary sites, notably cervical lymph nodes
(23). In the same regions, approximately 10% of cases caused by M.
tuberculosis are nonpulmonary, although, for reasons that are not clear,
the incidence is higher, approximately 20%, in ethnic minority populations.
Information on human disease due to M. bovis in developed and developing
countries is scarce. From a review of a number of zoonotic tuberculosis
studies, published between 1954 and 1970 and carried out in various
countries around the world, it was estimated that the proportion of human
cases due to M. bovis acccounted for 3.1% of all forms of tuberculosis:
2.1% of pulmonary forms and 9.4% of extrapulmonary forms (24). Table 2
summarizes the findings of more recent reports of TB caused by M. bovis in
industrialized countries.
   Table 2. Human tuberculosis due to Mycobacterium bovis, industrialized
                                 countries
    Cases
 					------------------------------------
 							   % of     Pulmonary
 							   total   (% of total
Country(ref.)   	Years	    No.    TB      M. bovis)
              --------------------------------------------------------
              Australia(25) 	1970-94   240	  0.43-3.1	 71.69(supa)
              England (23)		1977-90   232	    1.2	  40.0
              Germany (26)		1975-80   236	    4.5	  73.7
              Ireland
                 Rural (27)	1986-90    17	    6.4	  70.6
                 Urban (28)	1982-85     9	    0.9	  88.8
              New Zealand(29)	1983-90    22	    7.2	  31.8
 Spain (30)		1986-90    10	    0.9         50.0
              Sweden (17)		1983-92    96	    2.0	    -
              Switzerland(31)	1994	    18	    2.6	    -
 U.S.(32)		1954-68     6	    0.3	  33.3
              U.S. (9)		1980-91    73	    3.0      52.0(supb)
                                                      	12.0(supc)
 		---------------------------------------------------------
              (supa) Overall percentage includes 80.6 % males and 51.2% females
              (supb)Adults.
              (supc)Children
Human disease caused by M. bovis has been confirmed in African countries.
In an investigation by two Egyptian health centers, the proportions of
sputum-positive TB patients infected with M. bovis, recorded during three
observations, were 0.4%, 6.4%, and 5.4% (33). In another study in Egypt,
nine of 20 randomly selected patients with TB peritonitis were infected
with M. bovis, and the remaining with M. tuberculosis (34).
Isolation of M. bovis from sputum samples of patients with pulmonary TB has
also been reported from Nigeria. Of 102 M. tuberculosis complex isolates, 4
(3.9%) were M. bovis (35). Another study in Nigeria reported that one of 10
mycobacteria isolated from sputum-positive cultures was M. bovis (36).
In a Zaire study, M. bovis was isolated from gastric secretions in two of
five patients with pulmonary TB (37). In the same study, the prevalence of
the disease in local cattle was approximately 8% by tuberculin testing and
isolation of M. bovis.
In a recent investigation in Tanzania, seven of 19 lymph node biopsies from
suspected extrapulmonary TB patients were infected with M. tuberculosis and
four with M. bovis (14). No mycobacteria were cultured from the remaining
eight (Table 3). Although the number of samples was low, the high
proportion (36%) of M. bovis isolates is of serious concern.
   Table 3. Isolates from suspected extrapulmonary tuberculosis patients,
                            Tanzania, 1994 (14)
                          No. of  M. tuber-    M.
              Occupation samples  culosis     bovis     Neg
             -------------------------------------------------
              Livestock      4        0        2        2
               keeper
              Farmers        6        2        1        3
              Children       3        2        1        0
              Unknown        6        3        0        3
              Total         19        7        4        8
             -------------------------------------------------
In an epidemiologic study in Zambia (38), an association between
tuberculin-positive cattle and human TB was found. Households that reported
a TB case within the previous 12 months were approximately seven times more
likely to own herds containing tuberculin-positive cattle (odds ratio =
7.6; p = 0.004). Although this could be explained by zoonotic TB
transmission, other factors such as transient sensitivity to tuberculin of
cattle exposed to TB patients and coincidental environmental factors
favoring both human clinical TB and sensitivity to bovine tuberculin should
also be considered.
In Latin America, a conservative estimate would be that 2% of the total
pulmonary TB cases and 8% of extrapulmonary TB cases are caused by M.
bovis. These cases would therefore account for 7,000 new TB cases per year,
a rate of nearly 2 per 100,000 inhabitants. From a nationwide study in
Argentina during 1982 to 1984, 36 (0.47%) of 7,672 mycobacteria cultured
from sputum samples were M. bovis (39). However, in another study in Santa
Fe province (where most of the dairy cattle industry is concentrated)
during 1984 to 1989, M. bovis caused 0.7% to 6.2% of TB cases (40).
Very limited data on the zoonotic aspects of M. bovis are available from
Asian countries. However, cases of TB caused by M. bovis were not reported
in early investigations in India (41).
Epidemiology
Much information on the epidemiologic patterns of zoonotic TB has been
obtained in this century from industrialized countries. However, some
striking epidemiologic differences related to both animal and human
populations in developing countries require particular attention.
    Risk Factors: Animal Population
Animal reservoirs. The widespread distribution of M. bovis in farm and wild
animal populations represents a large reservoir of this microorganism. The
spread of the infection from affected to susceptible animals in both
industrialized and developing countries is most likely to occur when wild
and domesticated animals share pasture or territory (42). Well-documented
examples of such spread include infection in badgers (Meles meles) in the
United Kingdom and possums (Trichosurus vulpecula) in New Zealand. Wild
animal TB represents a permanent reservoir of infection and poses a serious
threat to control and elimination programs.
Milk production and animal husbandry. Milk production has increased in most
developing countries as a consequence of greater demand for milk for human
consumption (43; Figure 7). This increased demand for milkestimated at 2.5%
per year for 1970 to 1988 for sub-Saharan Africa (44) led to increases in
the number of productive animals and milk imports and intensification of
animal production through the introduction of more productive exotic
breeds.
Although the prevalence of the disease within a country varies from area to
area, the highest incidence of bovine TB is generally observed where intensive
dairy production is most common, notably in the milksheds of larger cities (1).
This problem is exacerbated where there is inadequate veterinary supervision,
as is the case in most developing countries. In addition, in some industrialized
countries such as the United States, where bovine TB is close to elimination,
large dairy herds (i.e., 5,000 or more cows) that are crowded together represent
the main source of infection (45).
                         [Fig]
          Figure 7. Cow milk production by region (43).
In developing countries, bovine TB infects a higher proportion of exotic
dairy breeds (Bos taurus) than indigenous zebu cattle (Bos indicus) and
crossbred beef cattle (1). However, under intensive feedlot conditions, a
death rate of 60% and depression of growth have been found in tuberculous
zebu cattle (46). In those areas where extensive management is more common,
animal crowding (e.g., near watering ponds, dip tanks, markets, and
corrals) still plays a major role in the spread of the disease.
Control measures and programs. The basic strategies required for control
and elimination of bovine TB are well known and well defined (47). However,
because of financial constraints, scarcity of trained professionals, lack
of political will, as well as the underestimation of the importance of
zoonotic TB in both the animal and public health sectors by national
governments and donor agencies, control measures are not applied or are
applied inadequately in most developing countries.
Successful conduct of a test-and-slaughter policy requires sustained
cooperation of national and private veterinary services, meat inspectors,
and farmers, as well as adequate compensation for services rendered. Only a
few developing countries can adhere to these requirements.
In addition, bovine TB does not often justify the emergency measures
required for other zoonotic diseases (e.g., Rinderpest, East Coast fever,
and foot and mouth disease). The full economic implications of zoonotic TB
are, however, overlooked in many developing nations where the overall
impact of the disease on human health and animal production needs to be
assessed. According to recent estimates, annual economic loss to bovine TB
in Argentina is approximately 63 million US dollars (48). In a study
recently conducted in Turkey, the estimated socioeconomic impact of bovine
TB to both the agriculture and health sectors was approximately 15 to 59
million US dollars per year (49).
Several Latin American countries, through agreements between governments
and cattle owners associations, have made the decision to control and
eliminate bovine TB. Where foot and mouth disease has been eliminated,
bovine TB and other existing infections such as brucellosis become
important because of their impact on the meat and live animal export trade.
Bovine TB and brucellosis also limit the development of the dairy industry
and its expansion at the regional level.
Risk Factors: Human Population
Close physical contact. Close physical contact between humans and
potentially infected animals is present in some communities, especially in
developing regions. For example, in many African countries cattle are an
integral part of human social life; they represent wealth and are at the
center of many events and, therefore, gatherings. In addition, with 65% of
African, 70% of Asian, and 26% of Latin American and Caribbean populations
working in agriculture, a significant proportion of the population of these
regions may be at risk for bovine TB.
Food hygiene practices. Consumption of milk contaminated by M. bovis has
long been regarded as the principal mode of TB transmission from animals to
humans (1). In regions where bovine TB is common and uncontrolled,
milkborne infection is the principal cause of cervical lymphadenopathy
(scrofula) and abdominal and other forms of nonpulmonary TB. Although
proper food hygiene practices could play a major role in controlling these
forms of TB, such practices are often difficult to institute in developing
countries.
In all countries of sub-Saharan Africa, there is active competition between
large-scale, often state-run, processing and marketing enterprises and the
informal sector. The informal sector can ignore standards of hygiene and
quality, and producers often sell directly to the final consumers. In
addition, an estimated 90% of the total milk produced is consumed fresh or
soured (44). Although it has been stated that Africans generally boil milk
and that the souring process destroys M. bovis (44), other sources strongly
contradict these statements (39). M. bovis was isolated from seven (2.9%)
of 241 samples of raw milk in Ethiopia (17). Both M. bovis and M.
tuberculosis have also been found in milk samples in Nigeria (36) and Egypt
(34). Thus, serious public health implications of potentially contaminated
milk and milk products should not be underestimated.
HIV/AIDS. According to recent WHO global estimates, of the 9.4 million
people infected with both HIV and TB in mid-1996, 6.6 million (70%) live in
sub-Saharan Africa (4). The greatest impact of HIV infection on TB is in
populations with a high prevalence of TB infection among young adults. The
occurrence of both infections in one person makes TB infection very likely
to progress to active disease.
In many developing countries, TB is the most frequent opportunistic disease
associated with HIV infection. HIV seroprevalence rates greater than 60%
have been found in TB patients in various African countries (4). Persons
infected with both pathogens have an annual risk of progression to active
TB of 5% to 15%, depending on their level of immunosuppression;
approximately 10% of non-HIV infected persons newly infected with TB become
ill at some time during their lives. In the remaining 90%, effective host
defenses prevent progression from infection to disease.
TB cases due to M. bovis in HIV-positive persons also resemble disease
caused by M. tuberculosis. Thus, they manifest as pulmonary disease,
lymphadenopathy, or, in the more profoundly immunosuppressed, disseminated
disease.
M. bovis has been isolated from HIV-infected persons in industrialized
countries. In France, M. bovis infection accounted for 1.6% of TB cases in
HIV-positive patients. All isolated strains were resistant to isoniazid
(7). Taking into consideration the intrinsic resistance of M. bovis to
pyrazinamide, two of the first-line anti-TB drugs were not effective.
WHO-recommended standard treatment for new TB cases includes, in the
initial phase, isoniazid, rifampicin, pyrazinamide, and streptomycin or
ethambutol. In situations of high primary resistance to isoniazid and
streptomycin, the intrinsic resistance of M. bovis to pyrazinamide may
severely limit the efficacy of treatment of TB caused by M. bovis.
In a Paris hospital, a source patient with pulmonary TB due to a
multidrug-resistant strain of M. bovis led to active disease in five
patients. Disease occurred 3 to 10 months after infection (10). This
observation led to three concerns: 1) human-to-human M. bovis transmission
leading to overt disease, 2) a short interval between infection and overt
disease, and 3) disseminated multidrug-resistant M. bovis.
In another study, conducted in San Diego, California, one of 24 adults with
pulmonary TB and 11 of 24 adults with nonpulmonary TB due to M. bovis had
AIDS. One of 25 children, a 16-year-old boy with abdominal TB, was also
HIV-positive (9).
It is commonly believed that M. bovis is less virulent than M. tuberculosis
in humans and therefore less likely to lead to overt postprimary disease
and that human-to-human transmission leading to infectious disease is rare.
However, if the apparent difference in virulence is the result of
differences in responsiveness of the host defense mechanisms, HIV-induced
immunosuppression could well lower host defenses leading to overt disease
after infection.
Surveillance of TB due to M. bovis
The use of direct smear microscopy as the only method for diagnosis of
suspected TB, although an essential requirement of any national TB program,
could partly explain the relatively low notification rate of disease caused
by M. bovis in developing countries. Direct smear microscopy does not
permit differentiation between species of the M. tuberculosis complex; in
addition, culture and speciation are often not carried out, and even when
culture facilities are available, M. bovis grows poorly in standard
Lwenstein-Jensen medium, one of the most widely used culture media (50).
In some countries, human disease caused by M. bovis is merely reported as
TB to avoid inquiries from disease control agencies, which might generate
problems of patient confidentiality (2).
The collection of representative data on the incidence of TB due to M.
bovis from most laboratories in developing countries has additional
problems. For example, the location and coverage of laboratories are often
biased towards city populations; sputum specimens may predominate, with
relatively few specimens from extrapulmonary lesions, particularly among
children. Specimens from children with TB are frequently negative on
culture, and biopsies are difficult to take from lesions.
Recent outbreaks of multidrug-resistant TB in some parts of the world
underscore the need for surveillance through wider application of reliable
culture and drug susceptibility tests.
Control Measures and Programs in Developing Countries
Bovine TB can be eliminated from a country or region by implementing a
test-and-slaughter policy, if no other reservoir host of infection exists.
While the test-and-slaughter policy is likely to remain the backbone of
national elimination bovine TB programs, the policy has numerous
constraints in developing countries. Alternative strategies (e.g., programs
based on slaughterhouse surveillance and traceback of tuberculous animals
to herds of origin) may be technically and economically more appropriate in
these countries.
Measures to prevent transmission of infection should be the primary
objective to be achieved with trained public health personnel, public
education, and proper hygienic practices. Test-and-slaughter programs may
be feasible and appropriate in areas with low bovine TB prevalence and
effective control of animal movement.
Animal Vaccination and Research Developments
Although not usually considered relevant to elimination programs in
livestock (47), vaccination of animals against TB would be a viable
strategy in two disease control situations: in domesticated animals in
developing countries and in wildlife and feral reservoirs of disease in
industrialized countries where test-and-slaughter programs have failed to
achieve elimination of the disease.
Many issues need to be addressed before vaccination becomes a realistic
option for control of disease in cattle and other animals. First, a highly
effective vaccine needs to be developed. The results obtained globally with
bacillus Calmette-Gurin (BCG) have been suboptimal, and efficacy has
varied considerably from region to region (42,51). Secondly, the delivery
of the vaccine poses few problems in domesticated animals, but it is
fraught with difficulties in wild animals. Thirdly, vaccination may
compromise diagnostic tests. A vaccine that induces tuberculin reactivity
would invalidate the key diagnostic tool used in control programs.
Fourthly, short of performing lengthy and expansive field studies,
evaluation of the protective efficacy of a new vaccine will pose serious
difficulties. Traditionally, the guinea pig and mouse have been used for
this purpose, but the information gained has been of little value. Recent
work has, however, indicated that deer may well prove a suitable mammal for
evaluating new vaccines and optimum delivery systems (52).
Enzyme-linked immunosorbent assay and gamma-interferon tests may prove to
be more sensitive and specific than the tuberculin test and may facilitate
diagnostic procedures. Nucleic acid-based technology, notably polymerase
chain reaction and related methods, may provide more rapid, sensitive, and
specific diagnostic tools. Multicenter studies of the applicability of
these techniques to the diagnosis of human TB have, however, shown that
their sensitivity and specificity are not as high as originally expected
and that many problems need to be solved before the techniques are
introduced into routine laboratory practice (53). Restriction fragment
length polymorphism analysis (DNA fingerprinting) could be useful in
epidemiologic studies that trace the spread of disease between cattle,
other animals, and humans (54) or in the rapid differentiation of M. bovis
within the M. tuberculosis complex (55). The use of these techniques is
limited by resources in most developing countries.
WHO and Zoonotic TB
The public health importance of animal TB was recognized early by WHO,
which in its 1950 report of the Expert Committee on Tuberculosis (56)
stated: "The committee recognizes the seriousness of human infection with
bovine tuberculosis in countries where the disease in cattle is prevalent.
There is the danger of transmission of infection by direct contact between
diseased cattle and farm workers and their families, as well as from
infected food products." Since then, TB in animals has been controlled and
almost eliminated in several industrialized countries but in very few
developing countries.
More recently, WHO has been involved in zoonotic TB through the activities
of the Division of Emerging and other Communicable Diseases Surveillance
and Control at WHO in Geneva (WHO/EMC) and the Veterinary Public Health
program of the WHO Regional Office for the Americas, Pan American Health
Organization (PAHO/HCV).
WHO/EMC has organized and coordinated a working group of experts from
countries worldwide (15-17). Their subjects are epidemiology, public health
aspects, control, and research on zoonotic TB. In addition, a joint WHO,
Food and Agriculture Organization of the United Nations (FAO), and Office
International des Epizooties (OIE) Consultation on Animal Tuberculosis
Vaccines was held to review current knowledge on TB vaccine development for
humans and animals and make recommendations for animal TB vaccine research
and development (57). Promising results of cattle vaccination with low
doses of BCG were reported. It is also planned for field trial cattle
vaccination to commence early in 1998 in Madagascar in collaboration with
national and international research institutions, OIE and WHO. In the
framework of the working group activities, the guidelines for speciation
within the Mycobacterium tuberculosis complex (50) have been prepared to
respond to the growing need for reliable differentiation between M.
tuberculosis, M. africanum, and M. bovis and to promote and strengthen
surveillance.
A Plan of Action for the Eradication of Bovine Tuberculosis in the Americas
(18) has been developed by PAHO in collaboration with member countries of
the region. PAHO/HCV, in cooperation with the Pan American Institute for
Food Protection and Zoonosis (INPPAZ), Buenos Aires, Argentina, and other
technical institutions (e.g., FAO), provides technical support to the
regional plan. PAHO/HCV activities train specialists in diagnosis,
reporting, surveillance systems, and quality control of reagents, as well
as supporting the planning and implementation of national programs. INPPAZ
acts as a reference center for these activities. The first phase of the
regional plan is expected to lead, in the next 10 years, to the elimination
of bovine TB from countries with more advanced national programs. In the
remaining countries, the objectives will be to strengthen epidemiologic
surveillance, defining areas at risk and setting up control and elimination
programs.
Conclusions
Although the epidemiology of bovine TB is well understood and effective
control and elimination strategies have been known for a long time, the
disease is still widely distributed and often neglected in most developing
countries. Its public health consequences, although well documented from
the past experiences of industrialized countries, have scarcely been
investigated and are still largely ignored in these regions. Because of the
animal and public health consequences of M. bovis, disease surveillance
programs in humans should be considered a priority, especially in areas
where risk factors are present. The increase of TB in such areas calls for
stronger intersectoral collaboration between the medical and veterinary
professions to assess and evaluate the scale of the problem, mostly when
zoonotic TB could represent a significant risk, for example, in rural
communities and in the workplace.
Industrialized countries, where the test-and-slaughter policies have not
completely eliminated infection in cattle because of wild animal
reservoirs, are now reconsidering wild animal vaccination. Any vaccination
research and development program should therefore also take into account
the possible application of vaccines to cattle, particularly in developing
countries.
In developing countries, where HIV and bovine TB are likely to be common,
particularly in young persons, the ability of HIV infection to abrogate any
host factors that prevent the progression of infection by M. bovis to overt
disease may lead to higher incidence and case-fatality rates for human TB
caused by this species and increased human-to-human transmission of this
disease. This should be of great concern in those developing countries
where bovine TB is present and measures to control spread of infection are
not applied or are applied inadequately. Research is needed to determine
when M. bovis is of zoonotic importance and what the underlying mechanisms
of transmission are. Locally operative risk factors for zoonotic TB should
therefore be identified to determine persons at risk and develop
appropriate control measures. International cooperation in all aspects of
zoonotic TB remains essential in the fight against this disease.
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Synopses
What Makes Cryptococcus neoformans a Pathogen?
Kent L. Buchanan and Juneann W. Murphy
University of Oklahoma Health
Sciences Center, Oklahoma City, Oklahoma, USA
------------------------------------------------------------------------
      Life-threatening infections caused by the encapsulated fungal
      pathogen Cryptococcus neoformans have been increasing steadily
      over the past 10 years because of the onset of AIDS and the
      expanded use of immunosuppressive drugs. Intricate
      host-organism interactions make the full understanding of
      pathogenicity and virulence of C. neoformans difficult. We
      discuss the current knowledge of the characteristics C.
      neoformans must possess to enter the host and establish
      progressive disease: basic growth requirements and virulence
      factors, such as the polysaccharide capsule; shed products of
      the organism; melanin production; mannitol secretion;
      superoxide dismutase; proteases; and phospholipases.
Cryptococcus neoformans is an encapsulated fungal organism (Figure 1) that
can cause disease in apparently immunocompetent, as well as
immunocompromised, hosts (1,2). Most susceptible to infection are patients
with T-cell deficiencies (1,2). C. neoformans var. neoformans causes most
cryptococcal infections in humans, so this review will focus on information
from the neoformans variety of this basidiomycetous fungus. C. neoformans
var. neoformans is found worldwide; its main habitats are debris around
pigeon roosts and soil contaminated with decaying pigeon or chicken
droppings (1,3). Not part of the normal microbial flora of humans, C.
neoformans is only transiently isolated from persons with no pathologic
features (2,4). It is generally accepted that the organism enters the host
by the respiratory route in the form of a dehydrated haploid yeast or as
basidiospores. After some time in the lungs, the organism hematogenously
spreads to extrapulmonary tissues; since it has a predilection for the
brain, infected persons usually contract meningoencephalitis (1). If
untreated, cryptococcal meningoencephalitis is 100% fatal, and even when
treated with the most effective antifungal drugs, cryptococcal infections
can be fatal if the host does not have adequate T-celldependent immune
function (2).
To be classified as a pathogen, an organism must be able to cause infection
under certain conditions. By this definition, C. neoformans can certainly
be classified as a pathogen. Because the immunodeficient are more
susceptible than the immunocompetent to infection with this yeast-like
organism, C. neoformans is frequently referred to as an opportunistic
pathogen. The factors that make C. neoformans a pathogen can be divided
into two major groups. The first comprises the basic characteristics needed
to establish an infection and survive in the human host; the second
 	comprises the virulence factors that affect the degree of pathogenicity.
                          [Fig]
Figure 1. Transmission electron micrograph of budding C. neoformans showing
the characteristic polysaccharide capsule.
Basic Characteristics for Pathogenicity
The Infectious Particle
To enter the alveolar spaces of the lungs and establish pulmonary
infection, an organism must produce viable forms smaller than 4 m in
diameter. The typical vegetative form of C. neoformans is the yeast form
with a cell diameter of 2.5 m to 10 m. The organism can also undergo
sexual reproduction, and since it is a basidiomycete (Filobasidiella
neoformans), it forms basidiospores. Sexual reproduction appears to occur
much less frequently in nature than asexual or vegetative reproduction. The
sexual spores (basidiospores) are approximately 1.8 m to 3 m in diameter
and result from crosses of the and  a-mating types on an
appropriate medium (1). Although the exact nature of the infectious
particles of C. neoformans is not known, they are presumed to be the
dehydrated yeast cells or basidiospores of the appropriate size range to
get into the lungs. Once inside the lungs, the yeast cells become
rehydrated and acquire the characteristic polysaccharide capsule (Figure
2). In the case of basidiospores, they would convert to encapsulated
blastoconidia.
                                 [Fig]
Figure 2. Proposed means of infection by C. neoformans.
Recently, Wickes et al. (5) reported that the [Alpha]-mating type of C.
neoformans can produce monokaryotic hyphae on a solid medium without a
nitrogen source or water. The monokaryotic hyphae contain clamp connections
and basidia with short chains of basidiospores and are similar in all
respects except in nuclei number to the dikaryotic hyphae of the sexual
forms (5). The haploid fruiting bodies formed by nitrogen-starved
[Alpha]-mating type isolates also produce abundant amounts of
blastoconidia, which on the appropriate medium can produce haploid hyphae
(5). None of the a-mating type strains tested produced the haploid fruiting
bodies. Backcross studies indicated that the ability to undergo haploid
fruiting lies within (or is tightly linked to) the MAT[Alpha] locus, which
is responsible for the [Alpha]-mating type phenotype (5). These results
suggest basidiospores from the monokaryotic hyphae may be another source of
the infectious particle in nature. Regardless of the nature of the
infectious particle (yeast cell or basidiospore), if a cryptococcal isolate
is not able to produce small infectious particles, it cannot be pathogenic
under the usual conditions for establishing the disease.
Mating Types
Kwon-Chung and Bennett (6) surveyed the mating types of C. neoformans
isolates from environmental and clinical sources and found a 30- to 40-fold
higher frequency of [Alpha]-mating type than [Alpha]-mating type. The
skewed ratio of [Alpha]-mating type to a-mating type was not thought to be
due to a genetic preponderance towards [Alpha]-mating type progeny because
crosses between two test strains resulted in a 1:1 ratio of [Alpha]-mating
and [Alpha]-mating type progeny (7). To examine the reason for the
disparate ratio, Kwon-Chung and colleagues (8) constructed a pair of
congenic strains of C. neoformans that differed only in their mating type.
Survival studies with these congenic strains demonstrated that the
[Alpha]-mating type (B-4500) was significantly more virulent than the
a-mating strain (B-4476) when injected intravenously into mice (8). Eighty
percent of mice infected with 10 [sup6] B-4500 ([Alpha]-mating type) were dead
within 36 days; whereas, it took 93 days for 80% of mice infected with 10 [sup6]
B-4476 (a-mating type) to die (8). Although the mating type locus of C.
neoformans has been cloned and partially characterized, the genes or gene
products that contribute to the increased virulence of [Alpha]-mating type
isolates are not known.
The mating type locus (MAT[Alpha]) cloned from an [Alpha]-mating type C.
neoformans isolate is 35 kb to 45 kb long (9). Moore and Edman (9)
demonstrated that introducing a 2.1-kb fragment of the MAT[Alpha] locus
into an a-mating strain isolate stimulated filament formation on starvation
medium. Basidia and basidiospores were not produced on the filaments. In
contrast, electroporation of an [Alpha]-mating strain with the same MAT
[Alpha] DNA fragment was ineffective in stimulating filament formation
under the same growth conditions (9). Sequence analysis of the 2.1-kb
fragment from the MAT[Alpha] locus identified a 114-bp open reading frame
that encodes a 38-amino acid propheromone peptide (9). The nature and the
effects of the active pheromone peptide on mating and possibly virulence of
C. neoformans are unknown; consequently, how the mating type relates to
virulence at the genetic level is also unknown. However, the observation of
haploid fruiting by [Alpha]-mating type isolates of C. neoformans on
nitrogen- and water-depleted medium (9) may help explain why the
[Alpha]-mating type isolates from clinical specimens are more frequent than
the a-mating type strains (6).
Growth In Vivo
To cause infection in humans, a C. neoformans isolate must grow at 37 degrees C in
an atmosphere of approximately 5% CO2 and at a pH of 7.3 to 7.4. To survive
at 37 degrees C, the organism must have an intact gene that encodes the C.
neoformans calcineurin A catalytic subunit (10). Calcineurin is a
serine-threonine specific phosphatase that is activated by Ca2+-calmodulin
and is involved in stress responses in yeasts (10). Although calcineurin A
mutant strains of C. neoformans can grow at 24 degrees C, they cannot survive in
vitro at 37 degrees C, in 5% CO2, or at alkaline pH (10). Since these are similar
to conditions in the host, one would predict that the calcineurin A mutant
would not survive in the human host. In support of that prediction, Odom et
al. have shown that such mutants are not pathogenic for immunosuppressed
rabbits (10). Calcineurin A appears to be a basic requirement for C.
neoformans survival in the host and consequently is a necessary factor for
the pathogenicity of the organism.
Virulence Factors
Virulence factors increase the degree of pathogenicity of a microbe. C.
neoformans has a number of virulence factors; generally, the virulence of
an isolate cannot be attributed to any single factor, but rather it is
attributed to many working in unison to cause progressive disease. As
virulence factors go, those of C. neoformans would be considered low-grade.
We will discuss each virulence factor separately; however, the severity of
the host's disease results from a combination of several virulence factors
superimposed on the host's innate and immune resistance status. The
virulence factors that will be discussed are capsule, cryptococcal
products, melanin production, mannitol production, and potential factors
such as superoxide dismutase, proteases, phospholipase B, and
lysophospholipase. The polysaccharide capsule and the soluble extracellular
constituents of C. neoformans (referred to here as cryptococcal products)
are probably the dominant virulence factors.
Capsule
C. neoformans has a capsule composed primarily of a high molecular weight
polysaccharide that has a backbone of [Alpha]-1,3-D-mannopyranose units
with single residues of -D-xylopyranosyl and -D-glucuronopyranosyl
attached (11-15). This polysaccharide is referred to as glucuronoxylomannan
(GXM) (11) and has four serotypes: A and D, produced by C. neoformans var.
neoformans, and B and C, produced by C. neoformans var. gattii. The
evidence indicates that the capsule is a key virulence factor for C.
neoformans; acapsular mutants are typically avirulent, whereas encapsulated
isolates have varying degrees of virulence (20). Similar observations have
been made with the CAP64 gene (21,22). Although their biochemical functions
have not been defined, it appears that the two genes are essential for
capsule formation and virulence.
Chemotactic Effects on Leukocytes
Some properties of the C. neoformans capsule enable the host to more
effectively clear the organism from tissues; however, others protect the
organism from host defenses. The capsules of serotype A and D isolates are
chemotactic for neutrophils (23). Moreover, complement is fixed by
cryptococcal capsules by the alternative pathway (24), and this process
produces chemotactic peptides such as C5a (23). Chemotaxis of leukocytes
induced by either of these mechanisms would be advantageous to the host.
Effects of Complement Interactions
Complement fixing by C. neoformans in tissue would result in chemotactic
factor production and attraction of leukocytes into the infection site.
Once in the infected tissue, the leukocytes would interact with and kill
the organism. As complement is fixed, C3b and C3bi are deposited on the
surface of the cryptococci (24). The capsule can mask the C3b and C3bi
deposits (24-26); however, if they are not completely masked, the deposited
complement components facilitate binding of the cryptococci to CR3
receptors on leukocytes (27). Such binding interactions are advantageous to
the host; they enhance the opportunity for the leukocytes to kill the
cryptococci either extracellularly or after phagocytosis. The organism can
also be opsonized by antibodies to GXM, but the capsule may block the Fc
portion of the antibody and prevent it from binding to Fc receptors on the
phagocytic host cells (28). Some of these functions of the capsule favor
the host; however, if the capsule is very large, the organism is protected
(24-26). The cryptococci could deplete complement in the host, creating an
environment that favors the cryptococci (29).
Effects on Phagocytosis
All considered, the capsule is more beneficial to the organism than to the
host. Encapsulated C. neoformans cells are not phagocytized or killed by
neutrophils, monocytes, or macrophages to the same degree as acapsular
mutants (25,30-33). Encapsulated C. neoformans have a stronger negatively
charged surface than acapsular cells or Saccharomyces cerevisiae cells
(34). The high negative charge could cause electrostatic repulsion between
the organism and the negatively charged host effector cells and reduce
intimate cell-cell interactions required for clearance of the cryptococci
(34).
Altered Antigen Presentation
The inability of macrophages to ingest the encapsulated organisms could
also diminish antigen presentation to T cells and consequently reduce
immune responses. This speculation has been experimentally demonstrated by
Collins and Bancroft (35). Other studies with human alveolar macrophages
have confirmed that antigen presentation is not as effective with
encapsulated cryptococci as with acapsular strains (33,36). Unlike
acapsular cells, encapsulated isolates cannot stimulate proliferative
responses in T cells because of the reduced secretion of interleukin-1
(IL-1) by the antigen-presenting cells stimulated with the encapsulated
yeasts (36).
Effects on Cytokine Production
In addition to being antiphagocytic, more resistant to killing, and less
able to stimulate T-cell proliferation (25,30-33,35,36), highly
encapsulated isolates of C. neoformans opsonized with a heat-labile serum
component, presumably complement, do not stimulate monocytes and
macrophages to produce proinflammatory cytokines such as TNF[Alpha], IL-1,
and IL-6 as effectively as similarly opsonized nonencapsulated or weakly
encapsulated isolates (37-41). The signal for induction of cytokine
production can be a direct result of the attachment of the monocyte or
macrophage to the acapsular cryptococci or can be an outcome of the
phagocytic process, induced by acapsular cryptococci. Since the capsule
blocks phagocytosis, any cytokines induced by the phagocytic process would
not be induced by the encapsulated C. neoformans cells. If the cryptococcal
cell wall materials must be exposed to induce cytokine production, the
capsule would block the direct induction of cytokine production. Regardless
of the mechanism of stimulation, the lack of production of proinflammatory
cytokines could have a bearing on protection. TNF[Alpha] is necessary for
the induction of the protective immune response against C. neoformans (42).
Consequently, the lack of or reduced production of TNF[Alpha] in infections
with highly encapsulated isolates of C. neoformans would prevent the
induction of protective immunity, resulting in progressive disease. The
roles of IL-1 and IL-6 in protecting against C. neoformans have not been
defined, but it is highly probable that the lack of these two cytokines
could compromise the protective responses of the host and give cryptococci
the advantage.
In contrast to the reduced TNF[Alpha], IL-1, and IL-6 produced by
stimulating monocytes and macrophages with highly encapsulated cryptococci,
IL-10 produced by these host cells increases after interaction with
encapsulated strains (43). Neutralization of IL-10 with anti-IL-10 in
cocultures of human peripheral blood mononuclear cells and encapsulated
cryptococci increased the amounts of TNF[Alpha] and IL-1 produced, which
indicates that the induction of IL-10 production by stimulating macrophages
with encapsulated C. neoformans downregulates the production of the
proinflammatory cytokines TNF[Alpha] and IL-1 (43). One might predict that
the induction of high levels of IL-10 would also preferentially stimulate
the induction of a T-helper 2 (Th2) response rather than a Th1 response in
the T cells (44). Since the Th1 response is associated with protection
against C. neoformans (45), increased levels of IL-10 would dampen
induction of the protective immune response.
Encapsulated cryptococcal cells do not affect the different types of
leukocytes in the same manner. Although encapsulated isolates do not
stimulate macrophages to produce proinflammatory cytokines, they do
stimulate neutrophils to produce proinflammatory cytokines and the
chemotactic factor IL-8 more effectively than weakly encapsulated or
acapsular organisms (46). As with the stimulation of macrophages by
acapsular cryptococci to produce proinflammatory cytokines, serum is
required for the encapsulated organisms to induce neutrophils to produce
cytokines (46). In the case of cytokine production by neutrophils in
response to encapsulated C. neoformans, it appears that the opsonization
process releases a factor into the supernatant that induces the neutrophils
to produce the cytokines (46). How these opposing in vitro observations
with macrophages and neutrophils relate to the in vivo system or
pathogenicity is yet to be determined.
Cryptococcal Products
In disseminated cryptococcosis, measurable levels of cryptococcal products
are present in the body fluids of the patients (47). Although GXM is the
major cryptococcal component in body fluids, it is highly probable that the
organism also sheds galactoxylomannan (GalXM) and mannoproteins (MP) in
vivo. This speculation is based on indirect evidence from in vivo studies
and on the fact that GalXM and MP are in culture medium when the organism
is grown in vitro (15,48). Cryptococcal antigens in serum or spinal fluid
are diagnostic for cryptococcosis (47). Furthermore, if disseminated
cryptococcosis patients have high cryptococcal antigen titers at the onset
of therapy, they are less likely to respond to therapy or more likely to
die before therapy is completed than patients with low cryptococcal antigen
titers (49). The direct relationship of cryptococcal antigen levels in body
fluids and the severity of disease suggests that the cryptococcal antigens
in the host's circulatory system or spinal fluid may have adverse effects
on host defenses.
Effects on Leukocyte Migration
It has been recently demonstrated in the mouse model that intravascular
cryptococcal antigens inhibit the migration of leukocytes from the
bloodstream into an inflammatory site (50). Intravascular cryptococcal
antigens significantly reduce leukocyte infiltration into a site of acute
inflammation induced by such stimuli as cryptococcal culture filtrate
antigen, TNFa, or the chemotactic peptide FMLP (formylmethionyl leucyl
phenylalanine) or into a delayed-type hypersensitivity reaction site
induced by purified protein derivative of Mycobacterium tuberculosis or by
C. neoformans antigen(s) (50). Each of the identified cryptococcal
products, GXM, GalXM, and MP, when given intravenously to mice, can inhibit
leukocyte migration (50). Considering that GXM is the dominant cryptococcal
component in the host's circulation, one might predict that GXM is mainly
responsible for the reduced leukocyte migration into inflammatory sites
(50). These observations imply that pulmonary cryptococcosis patients, who
have low to no cryptococcal antigen concentrations in their serum, would
have a normal influx of leukocytes into pulmonary sites of infections. On
the other hand, in severe pulmonary infections or in disseminated
cryptococcosis patients who have high levels of circulating cryptococcal
antigens, minimal inflammation would be expected in the infected tissues.
In fact, for years investigators have commented on the minimal host tissue
responses observed in patients with disseminated cryptococcosis (3). These
recent data demonstrate that the circulating cryptococcal antigens are
responsible, at least in part, for the lack of host tissue response.
Cryptococcal antigen(s) can potentially prevent leukocytes from migrating
into an inflammatory site in two nonexclusive ways (51,52). First, GXM can
stimulate neutrophils to shed L-selectin, a surface molecule necessary for
the first step in neutrophil movement into tissues (51). Without L-selectin
the neutrophils do not slow down and roll along the inflamed endothelial
cells lining the blood vessels. With this first step in extravasation
blocked, the numbers of neutrophils in infected tissues would be greatly
reduced. Second, GXM and GalXM can bind to CD18, the beta chain of the
adhesion molecule LFA-1, and prevent the binding of anti-CD18 to LFA-1
(52). Consequently, this binding of GXM and GalXM could prevent LFA-1 on
the neutrophils from binding to the LFA-1 ligand, ICAM-1 on the inflamed
endothelial cell surface. If leukocytes are inhibited from entering tissues
by either or both of these mechanisms, the organism is not effectively
eliminated, and the disease is more severe.
Induction of Immunomodulatory Cells
Cryptococcal antigens injected into the bloodstream of experimental animals
can induce regulatory T cells, which dampen or ablate the anticryptococcal
humoral as well as cell-mediated immune responses (53-71). Some clinical
correlates support the concept that the antigenemia seen in disseminated
cryptococcosis downmodulates the immune responses (72,73).
A long-lasting, specific immunologic unresponsiveness has been reported in
persons cured of cryptococcal meningitis (72-74). Although they made
antipneumococcal polysaccharide antibodies to the same degree as control
volunteers after vaccination with pneumococcal polysaccharide, cured
patients could not make antibodies to cryptococcal polysaccharides after
vaccination with cryptococcal antigens (72,73). Henderson et al. (72,73)
speculated that the intense, prolonged antigenemia associated with the
cryptococcosis may account for the observed unresponsiveness. It is not
unusual for patients with disseminated cryptococcosis to have depressed
cell-mediated immune (CMI) responses to cryptococcal antigens (75,76).
However, sufficient information is not available to determine whether the
patients had a generalized defect in CMI function or had a specifically
depressed CMI response because of the cryptococcal antigenemia.
Experimental animal models demonstrate convincingly that cryptococcal
antigens given intravenously can induce immunoregulatory T cells that
downmodulate the anticryptococcal CMI response (56-71). Serum from C.
neoformans-infected mice with a cryptococcal antigen titer of 10-4 when
transferred intravenously to naive mice induces regulatory T cells that
specifically depress the anticryptococcal CMI response (67). Similar
immunoregulatory T cells are induced by simulating the antigenemia with
intravenous injection of cryptococcal culture filtrate antigen
(56,57,60-63,66-69). The immunoregulatory CD4+ T cells, which appear in the
lymph nodes of the mice 7 days after antigen injection, diminish the
induction of T cells responsible for the anticryptococcal delayed-type
hypersensitivity response and reduce the ability of the mice to clear
cryptococci from tissues (56,57,60,68). A second immunoregulatory T cell, a
CD8+ cell, has been found in spleens of mice (57). The CD4+
immunoregulatory cells do not alter the numbers or types of leukocytes that
infiltrate an anticryptococcal delayed-type hypersensitivity reaction;
however, they do have a downregulating effect on IL-2 and IFN[Alpha]
production at the site (77). In contrast, the CD8+ immunoregulatory cells
appear to affect the numbers of neutrophils that infiltrate the
delayed-type hypersensitivity reaction site (Murphy, unpub. data). The
details of the characteristics and functions of these immunoregulatory T
cells have been reviewed (45). The available data strongly support the
notion that cryptococcal products in the circulation induce an array of
immunoregulatory T cells, which depress the anticryptococcal immune
responses and protection.
More consideration should be given to the impact of high levels of
cryptococcal antigen in the cerebrospinal fluid (CSF) on progression of
disease. As suggested by Denning et al. (78), high cryptococcal antigen
concentrations could change the osmolality of the CSF, thereby affecting
its outflow and adsorption and increasing intracranial pressure. The
increased pressure may cause headaches, visual loss, and early death (78).
It is also possible that release of mannitol by C. neoformans contributes
to increased intracranial pressure in cryptococcal meningitis patients
(78).
Melanin Synthesis
One characteristic that differentiates pathogenic isolates of C. neoformans
from nonpathogenic isolates and other Cryptococcus species is the
organism's ability to form a brown to black pigment on a medium (such as
birdseed or caffeic acid agar) that contains diphenolic compounds (1). This
pigment, first described by Staib (79), is a melaninlike compound produced
by C. neoformans isolates with phenoloxidase activity (80). The importance
of melanin production in C. neoformans virulence was first demonstrated in
the early 1980s. Rhodes, Polacheck, and Kwon-Chung (81) reported that
naturally occurring C. neoformans mutants lacking melanin (Mel-) were less
virulent in mice than melanin-producing strains. Others (82-84), using
chemically induced mutants or isogenic pairs of C. neoformans, have
confirmed and extended this observation.
Biochemical analyses suggest that in C. neoformans melanogenesis is
accomplished by conversion of dihydroxyphenols such as
3,4-dihydroxyphenylalanine (DOPA) to dopaquinone (Figure 3). This
conversion is catalyzed by a phenoloxidase and is the rate-limiting step,
presumably because subsequent steps in the pathway, such as dopaquinone
rearranging to dopachrome and ultimately autoxidation to melanin, are
spontaneous (85). C. neoformans lacks a tyrosinase enzyme required for
endogenous production of dihydroxyphenols (83); thus to produce melanin, a
C. neoformans isolate must be able to acquire diphenolic compounds from its
growth environment, and it must have the enzyme phenoloxidase to catalyze
conversion of these compounds into the subsequent melanin intermediates.
The brain is a tissue rich in catecholamines such as DOPA and is a favorite
target for infection by C. neoformans. However, the organism cannot use
catecholamines as a sole carbon source, which suggests that the brain is
not a preferred nutritional niche for growth of C. neoformans (86); rather,
it may serve as a survival niche as described below.
                                 [Fig]
Figure 3. Proposed pathway for melanin synthesis by C. neoformans.
Polacheck et al. (86) reported that melanin-producing isolates of C.
neoformans were resistant to damage by an in vitro epinephrine oxidative
system, whereas mutants lacking phenoloxidase activity were highly
susceptible, as evidenced by decreased survival. Jacobson and Tinnell (87)
found that melanin protected C. neoformans from damage by hypochlorite and
permanganate but not by hydrogen peroxide. In these experiments,
hypochlorite was 100 times more fungicidal than hydrogen peroxide, and C.
neoformans could produce sufficient levels of melanin to effectively
protect the organism from oxidative compounds produced by macrophages (87).
Wang and Casadevall (88) extended these findings by examining survival of
C. neoformans in the presence of nitric oxide and reactive nitrogen
intermediates as well as in the epinephrine oxidative system described by
Polacheck et al. (86). Wang and Casadevall cultured C. neoformans cells
with L-DOPA to melanize the yeast cells (88). Melanized cryptococci
survived damage by both nitrogen- and oxygen-derived oxidants significantly
better than nonmelanized organisms of the same strain. These results
support the hypothesis that C. neoformans uses catecholamines in the brain
to make melanin, thereby protecting the organism from oxidative damage by
scavenging free radicals (86).
Recently, cryptococcal diphenoloxidase has been purified, and the gene
CNLAC1 encoding this enzyme was cloned (89). The glycosylated protein has a
molecular weight of 75 kDa and contains copper. The substrate specificity
of the enzyme indicates that it is a laccase. CNLAC1 contains 14 introns, a
624 amino acid open reading frame; transcriptional activity is derepressed
in the absence of glucose (89), which confirms an earlier report of low
glucose requirements for melanin formation (90). Disruption of CNLAC1
resulted in loss of virulence of C. neoformans, whereas complementation
with CNLAC1 increased virulence of Mel- mutants in mice (84); these results
suggest that the laccase (phenoloxidase) encoded by CNLAC1 is a potential
virulence factor of C. neoformans. Furthermore, transcripts of the CNLAC1
gene have been detected by reverse transcription-polymerase chain reaction
(RT-PCR) in C. neoformans yeast cells isolated from CSF in a rabbit model
of cryptococcal meningitis (84).
Besides acting as an antioxidant, melanin production may help C. neoformans
survive in the host in other ways. Melanized yeast cells are less
susceptible to amphotericin B than nonmelanized yeast cells, and this may
contribute to the inability to effectively treat infections in
immunocompromised hosts (91). Furthermore, phagocytosis of melanized C.
neoformans by a macrophage cell line in the presence of an anti-GXM
antibody was decreased, which suggests that melanin deposition in the cell
wall may inhibit opsonization by specific antibodies (92). Recently,
Huffnagle and coinvestigators (93) reported that melanized heat-killed C.
neoformans strain 145 yeast cells stimulated less TNF[Alpha] production by
alveolar macrophages and less antigen-specific T-cell proliferation than
nonmelanized heat-killed 145 strain cells. The authors suggest that melanin
"cloaks" C. neoformans from recognition by host effector cells and inhibits
induction of a protective T-cell-mediated immune response (93); however, it
is possible that scavenging of host-produced oxidants and inhibition of
phagocytosis by melanin may contribute to the decreased TNF[Alpha]
production and lymphoproliferation observed.
Although melanin production is important in the virulence of C. neoformans,
there is very little evidence demonstrating the presence of melanin in
vivo. One report indicates that phenoloxidase activity in C. neoformans is
greatly diminished at 37 degrees C compared with activity at 25 degrees C,
which suggests that melanin production may be limited in vivo (94). Detection of melanin in vivo is hampered by the lack of specific antibodies or stains. A
modified Fontana-Masson stain has been used to detect a brown pigment in
the cell wall of C. neoformans cells in histologic brain sections; however,
the stain is not specific because Cryptococcus laurentii, which is Mel-,
also stains with Fontana-Masson (95).
In summary, accumulating evidence indicates that melanin production is an
effective survival (virulence) factor of C. neoformans, and melanin may
serve multiple roles in protecting this medically important fungus from
host defenses. Two caveats should be noted in labeling melanin as a major
virulence factor for C. neoformans. The C. neoformans isolate must be able
to internalize the melanin precursors, and the phenoloxidase enzyme must
make a sufficient amount of melanin precursor at 37 degrees C. Consequently, much
more work is needed to delineate the role of melanin production in
virulence of C. neoformans, especially in light of the apparent temperature
sensitivity of the cryptococcal phenoloxidase enzyme.
Mannitol Production
Accumulating evidence suggests that production of the hexitol D-mannitol
may contribute to survival of C. neoformans in the host. Wong et al. (96)
reported that of the 12 human isolates of C. neoformans examined, all
produced and secreted D-mannitol into growth medium. Further, by using a
rabbit cryptococcal meningitis model, they showed that C. neoformans can
produce D-mannitol in vivo. CSF from rabbits treated with cortisone and
infected intracisternally with C. neoformans contained more D-mannitol than
CSF from controls, cortisone-treated uninfected rabbits, or rabbits
infected with C. neoformans but not treated with cortisone (96). In the
latter group, cryptococcal infection was limited. The levels of D-mannitol
in infected rabbit CSF correlated well with both the numbers of culturable
C. neoformans and the cryptococcal antigen titer of the CSF, which suggests
that levels of D-mannitol in CSF may be prognostic (96); however, it is not
known whether different isolates of C. neoformans vary in mannitol
production. These authors suggested two means by which mannitol production
may contribute to C. neoformans pathogenesis. First, high concentrations of
D-mannitol in the CNS may contribute to brain edema. Second, mannitol is a
potent scavenger of hydroxyl radicals, and cryptococcal-produced D-mannitol
may help protect the organism from oxidative damage (96).
To investigate the role of mannitol in cryptococcosis, Chaturvedi and
co-workers (97) isolated a low mannitol producing mutant after UV
irradiation of C. neoformans strain H99. The mutant, mannitol low producer
(MLP), was similar to the parent strain H99 in many growth and morphology
characteristics and in known virulence factors such as capsule production
and phenoloxidase activity (97). However, the mutant MLP strain was more
susceptible to growth inhibition and killing by heat and high salt than the
parent strain. In addition, the mutant MLP strain was significantly less
virulent than the parent strain (MLP LD50 = 3.7 X 106 CFU, H99 LD50 = 6.9 X
102) (97). Further comparisons of H99 and MLP strains showed that
polymorphonuclear leukocytes (PMNL) killed MLP significantly better than
H99 at several effector-to-target ratios (98). Addition of superoxide
dismutase, mannitol, or dimethyl sulfoxide inhibited PMNL killing of both
strains, but addition of catalase did not alter killing, which suggests
that reactive oxygen intermediates such as the hydroxyl radical and
hypochlorous acid are potent effector molecules against C. neoformans and
mannitol may protect against oxidative killing by scavenging such compounds
(98).
The results above indicate that mannitol production by C. neoformans
correlates with increased resistance to heat stress, osmotic stress, and
damage by reactive oxygen intermediates, as well as increased pathogenicity
of this fungal agent. Additional studies are required to determine the role
of mannitol production in virulence of C. neoformans. Isogenic strains
lacking the enzyme required for mannitol production, namely mannitol
dehydrogenase, are not available because the gene for this enzyme has not
been found. However, a gene (Mtl) has been isolated from C. neoformans that
can induce expression of the S. cerevisiae mannitol dehydrogenase gene
(99). The 346 amino acid product encoded by the Mtl gene is thought to be
involved in regulating mannitol production in C. neoformans and may be
beneficial in future studies of mannitol production (99).
Other Potential Virulence Factors
Superoxide Dismutase
Jacobson and coinvestigators (100) examined superoxide dismutase (SOD)
production by C. neoformans to determine whether SOD levels increased at
37 degrees C to compensate for possible decreases in melanin production at this
temperature. These investigators observed an increase in SOD levels at
37 degrees C, which suggests that SOD may participate in free radical scavenging at
this higher temperature in vivo (100); however, there is no evidence that
SOD production serves as a virulence factor for C. neoformans.
Proteases
Muller and Sethi (101) reported that C. neoformans grown in culture
produced a protease that could digest human plasma proteins, and Brueske
(102) reported that C. neoformans culture supernatants contained a protease
capable of digesting casein. However, neither of these studies determined
whether the proteases were manufactured in vivo. Limited evidence
indicating in vivo production of proteases has been presented by Salkowski
and Balish (103). These investigators observed skin lesions on
T-cell-deficient mice after intravenous infections with C. neoformans
strain SLHA (103). Microscopic examination of the lesions suggested that
the cryptococcal yeast cells were degrading collagen bundles in the dermis
(103). Supernatants of SLHA strain cultures liquefied gelatin in vitro
indicating that this cryptococcal strain secreted a collagenaselike protein
(103). Thus, C. neoformans proteases possibly serve as virulence mechanisms
by initiating invasion of host tissues; however, more studies with isogenic
strains of C. neoformans are required before proteases can be listed as
virulence factors.
Phospholipases
Recently, Chen and co-workers (104,105) reported phospholipase,
lysophospholipase, and lysophospholipase-transacylase activity of C.
neoformans grown on egg yolk agar. Of 50 strains tested, 49 had
phospholipase activity (104), due to phospholipase B secreted into cultures
(105). Analysis of four strains with varying levels of phospholipase
activity indicated a correlation between phospholipase activity and
virulence in BALB/c mice (104). The authors suggest that extracellular
phospholipase activity produced by C. neoformans may disrupt mammalian cell
membranes and allow the yeast cells to penetrate into host tissues
(104,105); however, further investigations are necessary to establish the
role, if any, of these types of products in the virulence of C. neoformans.
Regulation of Virulence
Regulation of virulence factors such as capsule production and melanin
formation is not well understood. However, the gene GPA1, which encodes a
G-protein [Alpha]-subunit, is involved in the regulation of these virulence
factors as well as in C. neoformans mating (106). Disruption of GPA1
resulted in defects in mating in response to nitrogen starvation, capsule
production in response to iron limitation, and melanin synthesis in
response to glucose starvation (106). Furthermore, gpa1 mutants were much
less virulent in a rabbit model of cryptococcal meningitis (106).
Reconstitution of the gpa1 mutant with wild-type GPA1 restored mating,
capsule production, melanin synthesis, and virulence. Addition of cyclic
AMP also restored these phenotypes, which suggests that C. neoformans GPA1
regulates these factors by sensing the nutritional signals of the
environment and regulating cyclic AMP metabolism in the organism (106).
These findings along with results of other molecular studies are intriguing
and represent the initial steps in defining at the molecular level the
factors controlling virulence in C. neoformans.
Conclusions
Virulent isolates of C. neoformans must be able to produce small particles
that can get into the alveolar spaces, must be able to grow at 37 degrees C at a pH
of 7.3 to 7.4 in an atmosphere of approximately 5% CO2, must have an intact
calcineurin pathway, and (possibly) must be an [Alpha] -mating type. The
ability to produce a large capsule and shed great amounts of capsular
material into the body fluids makes the organism highly virulent. Other
factors, such as melanin, mannitol, superoxide dismutase, protease, and
phospholipase production, may enhance the pathogenicity of C. neoformans.
The effectiveness of many of these cryptococcal virulence factors depends
on the status of the host's defensive mechanisms. Although we have learned
much about the pathogenicity and virulence of C. neoformans, many gaps
still remain in our knowledge.
Acknowledgments
We thank Drs. J.A. Alspaugh, J.R. Perfect, and J. Heitman for making their
manuscript available before publication.
Dr. Buchanan is a research assistant professor in the Department of
Microbiology and Immunology, University of Oklahoma Health Sciences Center,
Oklahoma City, Oklahoma, USA. Dr. Murphy is a George Lynn Cross Research
Professor of Microbiology and Immunology in the same department.
Address for correspondence: Juneann W. Murphy, University of Oklahoma
Health Sciences Center, Oklahoma City, OK, USA; fax: 405-271-3117; e-mail:
kent-buchanan@ouhsc.edu or juneann-murphy@ouhsc.edu.
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[Emerging Infectious Diseases * Volume 4 * Number 1 * January - March 1998]
Dispatches
Hantavirus Infection in Children in Argentina
Noem C. Pini,* Amanda Resa, Gladys del Jess Laime, Gustavo Lecot,
Thomas G. Ksiazek, Silvana Levis,* and Delia A. Enria*
*Instituto Nacional de Enfermedades Virales Humanas "Dr. Julio I
Maiztegui," Pergamino, Argentina; Hospital de El Bolsn, Ro Negro,
Argentina; Hospital de Orn, Salta, Argentina; Hospital de Olavarra,
Buenos Aires, Argentina; and Centers for Disease Control and Prevention,
Atlanta, Georgia, USA
---------------------------------------------------------------------------
Text Version
      Clinical hantavirus infection was diagnosed in five Argentine
      children ages 5 to 11 years by immunoglobulin M (IgM)- capture
      enzyme-linked immunosorbent assay using Sin Nombre virus (SNV)
      antigens. Death in three of the children was associated with
      absence of detectable IgG to SNV antigens. An additional two
      cases in healthy children were studied: one, a breast-fed
      15-month-old whose mother died of suspected hantavirus
      pulmonary syndrome (HPS) 8 months previously, had hantavirus
      IgG(>/= 1:6400); a second, whose mother survived HPS during month
      three of pregnancy, apparently had maternal antibodies no
      longer detectable 1 year after birth.
In May 1993, a new hantaviral illness, hantavirus pulmonary syndrome (HPS),
was recognized in the southwestern region of the United States (1). HPS is
a viral zoonosis characterized by a febrile prodrome in young, healthy
adults; the disease progresses to respiratory failure with the clinical
picture of adult respiratory distress syndrome (ARDS). The striking
pulmonary involvement differentiates HPS from a previously described
hantaviral disease known as hemorrhagic fever with renal syndrome.
In the first 100 HPS cases in the United States, the average age was 34.9
years (range 11 to 69); eight cases were in children or adolescents under
16 years of age (2). In Argentina, from 1987 to July 1997, 114 cases were
diagnosed in three areas of the country where several strains of new world
hantaviruses are known to cause HPS diseases (3,4). Before 1995, no cases
were detected in Argentine children under 12 years of age. Ten cases were
reported among adolescents (13 to 19 years) with a case-fatality rate of
30% (Instituto Nacional de Enfermedades Virales Humanas, [INEVH], unpub.
data).
The initial case definition referred to ARDS and included adults and young
adults (5) as the affected population. The lack of HPS cases among children
in the original outbreaks led to a circulating hypothesis that children
were not at risk or were at a very low risk for HPS. Another hypothesis was
that children were protected from pulmonary involvement, perhaps by immune
system immaturity or a lack of other risk factors (such as cigarette
smoking) for lung injury.
In this report we describe five cases in children; in all of them the
etiologic diagnosis was established by the presence of immunoglobulin M
(IgM) antibody to Sin Nombre virus (SNV) antigens. Serologic results for
two of the children were also positive for SNV IgG antibody. Serum samples
were tested for IgM and IgG antibodies to SNV by enzyme-linked
immunosorbent assay (ELISA) (6). An ELISA titer greater than or equal to
1:400 was considered positive (Table 1)
Table 1. Hantavirus infection in children, Argentina, 1995-1997
---------------------------------------------------------------------------
                                          Serology(supa)
                                             Date
                                Date of      IgM
   Case       Sex    Age (yrs)   onset       IgG       Area(supb) Outcome
---------------------------------------------------------------------------
     1         M         9      4-19-95     5-3-95     South       Alive
                                            >6400
                                             1600
     2         F         5      3-21-97    3-23-97     North       Dead
                                            >6400
                                             Neg
     3         M         9      3-30-97    4-23-97     North       Alive
                                             1600
                                             400
     4         F        11      4-14-97    4-16-97     North       Dead
                                             1600
                                             Neg
     5         M         5      4-27-97    4-28-97    Central      Dead
                                             1600
                                             Neg
---------------------------------------------------------------------------
(supa)Titer expressed as the reciprocal of the serum dilution reactive in
enzyme-linked  immunosorbent assay.
(supb)Area of origin in Argentina.
Patient 1 was identified during the study of the first outbreak in southern
Argentina in 1995 (5). Four patients in this outbreak were from the same
family. During interviews of the family members, we found that a 9-year-old
boy had a febrile disease without respiratory involvement, beginning on
April 19. Serology performed on May 3, 14 days after the onset of symptoms,
demonstrated IgM and IgG antibodies to SNV antigens.
The other four cases in children were identified during routine
surveillance. From 1995 to 1997, samples from 25 children (ages 3 months to
12 years; mean = 5.8 years) were sent to INEVH for diagnosis. Table 2
summarizes the main clinical and laboratory findings. None of the children
had renal failure; patient 2 had uremia of 0.30 g/l, and patient 4 had a
serum creatinine level of 1.40 g/l. All patients who later died received
supplemental oxygen as part of their treatment.
Two special situations involving children arose during the study of the
first cases of HPS in El Bolsn in 1995. 1) A woman belonging to the family
of patient 1 contracted HPS during the first quarter of pregnancy. She had
a febrile syndrome, without respiratory failure; chest X-rays showed
bilateral interstitial infiltrates. Serologic tests showed both SNV IgM
(>/=1:6400) and IgG (>/=:6400) on April 22, 1995, 8 days after the onset of
symptoms. She delivered a healthy infant in October 1995. A sample of the
newborn's cord blood was positive for SNV IgG (>/=1:6400) and negative for
SNV IgM. A serum sample drawn from the mother at the same time had a SNV
IgG titer >/=1:6400 and was negative for SNV IgM. A second serum sample,
taken from the baby a year later during November 1996, had no detectable
SNV IgG or IgM. 2) During a retrospective search for cases fulfilling the
HPS case definition, a woman who died of ARDS in September 1994 was
considered to have a possible case. No serum samples or autopsy tissues
were available to make an etiologic diagnosis. Before dying, this woman
breast-fed a 7-month-old baby; when tested for antibodies 8 months later in
May 1995 at 15 months of age, the baby had SNV IgG >/=1:6400) and no
detectable SNV IgM antibodies. A second serologic sample, collected 18
months later in November 1996, still had SNV IgG antibodies, with a similar
titer. Both babies have continued to develop normally as of October 1997.
Table 2. Laboratory results and clinical features of children with
hantavirus infection, Argentina, 1995-1997
---------------------------------------------------------------------------
                                         Case
Tests and    --------------------------------------------------------------
features          1        2         3             4               5
---------------------------------------------------------------------------
Leukocytes     9,200    27,000     12,800        10,600          69,200
[/mm(sup3)]
Hematocrit       44      66          43            55              53
(%)
Thrombocytes    ND(supa)266,000    200,000       97,000            ND
[/mm(sup3)]
Sedimentation   ND        4         28             8               1
rate (mm/hr)
GOT/GPT(supb)   ND    Increased   Increased     Increased      Increased
                                                                (mild)
Chest X-ray   HI(supc) DII(supd)    DII           DII            DII
Respiratory    None    Distress    Slight      Tachypnea,        Acute
symptoms                          dyspnea     clinical and    respiratory
                                                 X-ray       insufficiency
                                             disassociation,
                                             hypoventilation
---------------------------------------------------------------------------
(supa)ND: Not done.
(supb)GOT/GPT: Glutamic oxalacetic transaminase/Glutamic pyruvic transaminase.
(supc)HI: Hilar indistinctness.
(supd)DII: Diffuse interstitial infiltrate.
A case similar to that of patient 1 was detected in New Mexico in June 1993
(7) in the course of the investigation of a fatal HPS case. In this case,
the patient also had a mild clinical course that did not meet the
surveillance case definition for HPS. This case definition (revised 9/96)
is as follows: "a febrile illness characterized by bilateral diffuse
interstitial edema that may radiographically resemble ARDS, with
respiratory compromise requiring supplemental oxygen, developing within 72
hours of hospitalization, and occurring in a previously healthy person; or
an unexplained respiratory illness resulting in death, with an autopsy
examination demonstrating noncardiogenic pulmonary edema without an
identifiable cause" (8).
Our remaining four cases were sporadic, in persons without previous contact
with other HPS patients, and were suspected because their clinical symptoms
were typical of HPS. Results of serologic testing with SNV antigens of the
household contacts in cases 2 and 5 (five persons each) were negative. The
clinical, radiologic, and laboratory findings were similar in children and
in adults; severely ill patients had greater variation in laboratory values
than mild cases, and in fatal cases, only SNV IgM was present.
The case-fatality rate in this series was 60%, but the small number of
cases does not permit conclusions. In previously reported cases in
adolescents 13 to 19 years of age, the case-fatality rate was 30%.
These cases originated in the three areas where the illness is endemic in
Argentina. This is an important point because an unusual case of HPS in
southern Argentina, with the possibility of person-to-person transmission,
had been reported (9,10). Patient 1 and the baby that was breast-feeding
when the mother died of suspected HPS could be further instances of
person-to-person transmission.
A case of hemorrhagic fever with renal syndrome and pregnancy was reported
in 1992 (11); the dynamics of serum antibody persistence were similar to
those found in the one instance where we believe antibody was passively
transferred from mother to baby. These results indicate that HPS should be
considered in the differential diagnosis of respiratory distress or
atypical bilateral pneumonia in children, at least in areas where these
diseases have been confirmed. Mild disease should be considered too,
especially in contacts of HPS patients and in younger age groups.
Our findings also suggest the transfer of passive antibodies from mother to
fetus (without fetal infection) and the possibility of transmission of
infection by maternal breast feeding.
References
  1. Centers for Disease Control and Prevention. Outbreak of acute illness:
     southwestern United States, 1993. MMWR Morb Mortal Wkly Rep
     1993;42:421-4.
  2. Khan AS, Khabbaz RF, Armstrong LR, Holman RC, Bauer SP, Graber J, et
     al. Hantavirus pulmonary syndrome: the first 100 US Cases. J Infect
     Dis 1996;173:1297-303.
  3. Parisi MN, Enria DA, Pini NC, Sabattini M. Deteccin retrospectiva de
     infecciones clnicas por hantavirus en la Argentina. Medicina (B
     Aires) 1996;56:1-13.
  4. Levis SC, Briggiler AM, Cacase M, Peters CJ, Ksiazek TG, Corts J, et
     al. Emergence of hantavirus pulmonary syndrome in Argentina
     [abstract]. Am J Trop Med Hyg 1995;53:233.
  5. Hughes JM, Peters CJ, Cohen ML, Mahy BWJ. Hantavirus pulmonary
     syndrome: an emerging infectious disease. Science 1993;262:850-1.
  6. Ksiazek TG, Peters CJ, Rollin PE, Zaki S, Nichol S, Spiropoulou C, et
     al. Identification of a new North American hantavirus that causes
     acute pulmonary insufficiency. Am J Trop Med Hyg 1995;52:117-23.
  7. Armstrong LR, Bryan RT, Sarisky J, Khan AS, Rowe T, Ettestad PJ, et
     al. Mild hantavirus disease caused by Sin Nombre virus in a
     four-year-old-child. Pediatr Infect Dis J 1995;12:1108-10.
  8. Centers for Disease Control and Prevention. Case definitions for
     infectious conditions under public health surveillance. MMWR Morb
     Mortal Wkly Rep 1997;46:16.
  9. Enria D, Padula P, Segura EL, Pini N, Edelstein A, Riva Posse C,
     Weissenbacher MC. Hantavirus pulmonary syndrome in Argentina.
     Possibility of person-to-person transmission. Medicina (B Aires)
     1996;56:709-11.
 10. Wells RM, Sosa Estani S, Yadon ZE, Enria DA, Padula P, Pini N, et al.
     An unusual hantavirus outbreak in southern Argentina: person-to-person
     transmission? Emerg Infect Dis 1997;2:171-4.
 11. Silberberg L, Rollin PE, Keourani G, Courdrier D. Haemorrhagic fever
     with renal syndrome and pregnancy: a case report. Trans R Soc Trop Med
     Hyg 1993;87:65.
---------------------------------------------------------------------------
Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA
URL: http://www.cdc.gov/ncidod/EID/vol4no1/pini.htm
------------------------------------------------------------------------
Dispatches
Reemergence of Dengue in Cuba: A 1997 Epidemic in Santiago de Cuba
Gustavo Kour,* Mara Guadalupe Guzmn, Luis Valds, Isabel Carbonel,
Delfina del Rosario,
* Susana Vazquez, *Jos Lafert,* Jorge Delgado, and Mara V. Cabrera
*Instituto de Medicina Tropical "Pedro Kour" (IPK), Marianao, Havana,
Cuba; WHO/PAHO Collaborating Center for the Study of Viral Diseases,
Havana, Cuba; Provincial Center for Hygiene, Epidemiology and
Microbiology, Santiago de Cuba, Cuba; and Ministry of Public Health, Cuba
---------------------------------------------------------------------------
Text Version
      After 15 years of absence, dengue reemerged in the municipality
      of Santiago de Cuba because of increasing migration to the area
      by people from disease-endemic regions, a high level of vector
      infestation, and the breakdown of eradication measures. The
      1997 epidemic was detected early through an active surveillance
      system. Of 2,946 laboratory-confirmed cases, 205 were dengue
      hemorrhagic fever, and 12 were fatal. No deaths were reported
      in persons under 16 years of age. Now the epidemic is fully
      controlled.
Cuba had its first dengue epidemic of modern times in 1977; transmission
continued probably until 1981, and more than 500,000 mild cases were
reported. A 1978 serologic survey for flavivirus antibody indicated that
44.6% of the Cuban population had been infected with dengue-1 virus,
whereas before 1977 only 2.6% had antibodies (1,2).
A second dengue epidemic in 1981, caused by dengue-2 virus (2), was
unusually severe and widespread. Of 344,203 cases, 10,312 were clinically
classified as dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), and
158 persons (101 children and 57 adults) died (3). Before 1981, only 60
suspected or confirmed DHF sporadic cases had been reported in the region
(4). Dengue-2 virus isolated during the 1981 epidemic was classified in the
same genotype as New Guinea 1944 (5). Not previously known to circulate in
the Americas, this genotype was not isolated again in the region until 1994
in Venezuela and in 1995 in Mexico (6). Retrospective studies show that
although the 1981 epidemic was detected in May, the first cases occurred in
December 1980. After the epidemic ended on October 10, 1981, a campaign to
improve mosquito control and eradicate _Aedes aegypti_ was immediately
launched. Eradication was not achieved, but most of the 169 Cuban
municipalities were free of the vector.
Passive Surveillance-1981
A passive dengue surveillance system was established at the end of the 1981
epidemic. Of 9,543 paired sera (acute- and convalescent-phase) from all
suspected dengue patients, only 14 showed seroconversion to immunoglobulin
G (IgG) by enzyme-linked immunosorbent assay (ELISA) (7); none developed
IgM antibodies to dengue virus by capture IgM ELISA (8). Dengue virus
infection was excluded on the basis of clinical and epidemiologic
investigation. No _Ae. aegypti_ mosquitoes were found in the residence
localities of these patients. The surveillance system detected cases,
imported from other Latin American countries, that had no evidence of
indigenous transmission. Since 1987, 4,983 samples received through the
surveillance system for measles and rubella, as well as paired sera of
patients with rash, were studied for dengue antibodies [Mara Guzmn, World
Health Organization (WHO)/Pan American Health Organization (PAHO)
Collaborating Center for the Study of Viral Diseases, unpub. info.). No
dengue cases were identified. The low _Ae. aegypti_ premise indexes and the
results of the passive surveillance system indicate no dengue transmission
in Cuba between 1981 and the end of 1996. However, reinfestation has
occurred in some areas; the municipality of Santiago de Cuba was reinfested
in 1992 by _Ae. aegypti_ transported in imported tires (9).
Active Surveillance-1997
In January 1997, the Institute of Tropical Medicine "Pedro Kour" of the
Cuban Ministry of Health (a WHO/PAHO Collaborating Center for the Study of
Viral Diseases) established an active surveillance system for dengue in
Santiago de Cuba municipality. The municipality is located in Santiago de
Cuba province, in the eastern part of the country, and has several risk
factors for the reemergence of dengue: limited water supply, inadequate
eradication efforts, high vector infestation, and increasing migration of
people from Latin American and Caribbean disease-endemic countries to the
municipality. Following the Guidelines for the Prevention and Control of
Dengue and Dengue Hemorrhagic Fever in the Americas (4), this surveillance
system actively searched for febrile patients in the primary health-care
subsystem whose clinical picture was compatible with dengue fever and whose
sera collected 5 to 6 days after onset of the disease contained dengue IgM
antibories. As a result of this system, dengue cases were detected on
January 28, 1997, in one area of the municipality. In three of the first
seven cases, dengue-2 virus was detected by polymerase chain reaction (10)
and was confirmed by viral isolation and identification using C6/36 cell
line and monoclonal antibodies to the four dengue serotypes.
Although retrospective seroepidemiologic studies indicated that the initial
transmission occurred during the second half of December 1996, it is highly
probable that the cases detected on January 28 were the first. Of 60,000
cases reported from the emergency rooms of Santiago de Cuba hospitals from
November 1 to January 28, 592 were clinically compatible with dengue fever.
Home interviews of these 592 patients reduced the figure to 154. Blood
samples from 143 of 154 patients were examined for IgM antibodies, but no
positive cases were detected.
The breakdown of the vector control campaign in this municipality
interfered with our efforts to abort the epidemic, despite the early
detection of the first dengue cases; however, the partial vector control
measures implemented once the outbreak was detected prevented its extension
to the other 30 Cuban municipalities infested with the _Ae. aegypti_
mosquito.
Active surveillance continued from January to July 1997. Serologic
confirmation of cases was carried out by IgM capture ELISA, confirming
recent infection. The serologic diagnosis was decentralized to the
Provincial Laboratory in Santiago de Cuba, which used an ultramicro-ELISA
for dengue IgM detection (11). The Institute of Tropical Medicine served as
the national reference laboratory for serology, viral isolation, and strain
identification and characterization.
During the epidemic, 17,114 febrile patients were initially considered to
have dengue, but serologic testing of 10,024 of these patients confirmed
dengue in only 2,946; 46 dengue-2 isolates from 160 serum samples were
obtained. The nucleotide sequence of the E\NS1 gene junction of the first
isolated strain (12) indicated that it belonged to the Jamaica genotype,
which during recent years is being transmitted extensively throughout Latin
American and Caribbean countries and is associated with DHF/DSS in some
countries (6,13).
Epidemiology
After the end of the 1981 Cuban DHF epidemic, seroepidemiologic studies in
Palmira, Cienfuegos, and Cerro municipalities examined dengue-1 and
dengue-2 seroprevalence in these populations (14,15). Taking into
consideration these data and the total population of the Santiago de Cuba
municipality, we estimated the prevalence of dengue-1 and dengue-2
antibodies. The estimated total population at risk for dengue-2 infection
was 301,986 adults and children susceptible to a primary infection by any
dengue virus serotype (63.5% of the population) and 88,108 adults with
antibodies to dengue-1 virus acquired during the epidemic of 1977 to 1980,
now susceptible to a secondary infection with dengue-2 and at increased
risk for DHF/DSS (18.5% of the population).
The earlier Cuban experience (3) confirms other reports of secondary
infection (dengue-1 and dengue-2) as the main risk factor for DHF/DSS.
During the 1997 dengue outbreak, secondary infection was again confirmed as
a risk factor for DHF/DSS. Of the 2,946 confirmed cases, 205 (including 12
fatal adult cases) were classified as DHF/DSS cases according to the
criteria established by PAHO (4). DHF/DSS was observed mostly in adults,
the only age group in whom secondary infection was possible.
DHF/DSS-compatible symptoms were seen only in one child with primary
infection. Preliminary studies indicated that secondary infection was
present in 100 (98%) of 102 DHF/DSS cases. In fatal cases, secondary
infection could be documented in 11 (92%) of 12 cases. In Thailand the
greatest risk appeared when the secondary infection occurred 6 months to 5
years after the primary one (16). For that reason, an epidemic of DHF/DSS
was not expected in Santiago de Cuba, perhaps only sporadic cases. However,
DHF/DSS in adults who contracted a secondary infection at least 16 years
after the primary infection was not previously reported.
Because in Cuba dengue-1 circulated from 1977 to 1980-81, the youngest
patients expected to contract secondary infection should be older than 16
years of age; the youngest DHF/DSS patient with confirmed secondary
infection was a 17-year-old, which indicates that the "enhancing"
antibodies can circulate and be effective for at least 16 years and maybe
for life.
A significant number of febrile patients with suspected dengue had
respiratory signs and symptoms; therefore, simultaneous circulation of
respiratory or other pathogens was considered. Serologic screening for
respiratory viruses using hemagglutination-inhibition and ELISA confirmed
that 29.3% of 41 nonconfirmed dengue cases were influenza A, influenza B,
or adenovirus infections. Additionally, some children had fever and rash
clinically compatible with herpangina, and some had diarrheal disease with
fever, as is common in Cuba during the summer. These febrile syndromes
contributed to the high number of patients whose infections were
provisionally considered suspect dengue cases. Suspect dengue cases were
broadly defined to maximize sensitivity of detection and retain all
possible dengue cases. This active surveillance excluded other febrile
syndromes but recorded them as suspected cases. In practice, the risk
perception by the population was very high, especially when the epidemic
was officially declared and deaths were noted. Both the patients and the
health providers appeared to think of dengue as the first diagnostic
possibility. For this reason, the figure of 17,114 cases was considered the
magnitude of the epidemic from the clinical management perspective. Since
most cases were tested serologically, the incidence of clinical cases was
probably close to the 2,946 serologically or virologically confirmed cases.
Because asymptomatic and subclinical dengue cases are frequent, especially
in children, the true rate of infection may be higher. In a separate and
limited study on asymptomatic contacts of dengue cases, for every clinical
case, 13.9 asymptomatic or subclinical cases were produced. Serologic
studies of contacts in Santiago de Cuba are planned for a more in-depth
study of this question.
Clinical Management
The health authorities established a liberal policy of hospitalization that
varied with the availability of beds. Hospitalization permitted vector
control of the human reservoir, more precise case classification, and close
clinical surveillance.
When beds were available, all patients with suspected cases were
hospitalized. When the numbers of patients surpassed the availability of
beds, patients were treated at home under the supervision of the family
doctor. The family doctor transferred the patient to the hospital if any
medical complication appeared. Wards with specialized personnel were
established where the patients were protected from vectors, and observation
wards were organized for patients with complications. Intensive and
intermediate care units, as well as an emergency subsystem for the transfer
of patients from one unit to another, were available. As in 1981, some
patients rapidly developed hypovolemic shock and died within hours of
admission to the hospital (17).
An ad hoc task force followed the case definitions for dengue and DHF/DSS
established by PAHO (4) for classifying the cases at the closure of the
medical record. The accumulated experience of the Cuban scientists and
doctors and the increased international knowledge about dengue and DHF/DSS
in the last 15 years permitted a much deeper and more comprehensive study
of this outbreak with more accurate classification and management of cases
than in 1981. Nevertheless, the case-fatality rate was three times higher,
mainly because of a much better classification of DHF/DSS cases. Other
countries in the region with a very accurate case classification, such as
Puerto Rico (13), also have a high case-fatality rate.
Vector Control
The campaign to control the vector started before the beginning of the 1997
dengue outbreak and is well established. Although the campaign required the
mobilization of scarce financial resources and experts from all over the
country, early intervention prevented spread of the outbreak to other
potentially vulnerable municipalities. Of 169 municipalities in Cuba, 30
had _Ae. aegypti_ mosquitoes. The epidemic was limited to the municipality
of Santiago de Cuba; no autochthonous transmission to other municipalities
of the province or country was detected.
An active search for cases detected transmission very early, before "fever
alert" signaled an outbreak. In the Provincial Center for Hygiene,
Epidemiology, and Microbiology of Santiago de Cuba, a special Unit for
Analysis and Trends maintains a permanent fever alert system. For several
years, this system has provided a weekly tabulation of febrile patients for
every population. The tabulation allows us to evaluate fever alert (4) as
applied to an active surveillance system. Because the fever alert did not
appear in the epidemic area until May 1997, after the epidemic was already
occurring, we consider fever alert an indicator with low sensitivity for
the early and timely detection of dengue transmission, at least under the
conditions of this study.
As a result of the 1997 epidemic, an epidemiologic alert was established,
and antivector intervention, as well as active seroepidemiologic
surveillance, was reinforced in the entire country. The epidemiologic
characterization of the outbreak (now fully controlled) is in the final
phase. Although mosquitoes persisted at a low level after the 1981 DHF/DSS
epidemic, the campaign was successful in eradicating dengue from Cuba for
more than 15 years, precisely when the disease was reemerging in nearly all
the other tropical regions of the Americas. According to PAHO, 250,707
cases of dengue fever and 4,440 cases of DHF/DSS were reported in 1996
alone; 29 countries reported dengue in 1996, and 10 of these reported
DHF/DSS. Overall, from 1981 to 1996, 25 countries reported 41,000 cases of
DHF/DSS (F. Pinheiro, pers. comm.).
The 1997 Cuban dengue outbreak demonstrated once again that dengue
reappears where _Ae. aegypti_ control is relaxed. Taking into account these
facts, Cuba maintains its policy of vector eradication and recommends an
exerted effort in the American region to prevent a recurrence of dengue
similar to the one in Southeast Asia, where DHF/DSS is the leading cause of
hospitalization and death among children (18).
References
  1. Cantelar N, Fernndez A, Albert L, Prez E. Circulacin de dengue en
     Cuba 1978-1979. Rev Cubana Med Trop 1981;33:72-8.
  2. Kour G, Mas P, Guzmn MG, Soler M, Goyenechea A, Morier L. Dengue
     hemorrhagic fever in Cuba, 1981: rapid diagnosis of the etiologic
     agent. Bull Pan Am Health Org 1983;17:126-32.
  3. Kour G, Guzmn MG, Bravo J, Triana C. Dengue hemorrhagic fever/dengue
     shock syndrome: lessons from the Cuban epidemic. Bull World Health
     Organ 1989;67:375-80.
  4. Dengue and dengue hemorraghic fever in the Americas: guidelines for
     prevention and control. Washington: Pan American Health Organization;
     1994. Scientific publication No. 548.
  5. Guzmn MG, Deubel V, Pelegrino JL, Rosario D, Sariol C, Kour G.
     Partial nucleotide and amino-acid sequences of the envelope and the
     envelope/nonstructural protein-1 gene junction of four dengue 2 virus
     strains isolated during the 1981 Cuban epidemic. Am J Trop Med Hyg
     1995:52:241-6.
  6. Ricco-Hesse R, Harrison LM, Salas RA, Tovar D, Nisalak A, Ramos C, et
     al. Origins of dengue type 2 viruses associated with increased
     pathogenicity in the Americas. Virology 1997;230:244-51.
  7. Fernndez R, Vzquez S. Serological diagnosis of dengue by an ELISA
     inhibition method (EIM). Mem Inst Oswaldo Cruz 1990;85:347-51.
  8. Vzquez S, Saenz E, Huelva G, Gonzlez A, Kour G, Guzmn MG.
     Deteccin de IgM contra el virus del dengue en sangre entera absorbida
     en papel de filtro. Rev Panamericana de Salud Pblica. In press 1998.
  9. Ministerio de Salud Pblica de Cuba. Dengue en Cuba. Julio 1997.
     Boletn Epidemiolgico Organizacin Panamericana de la Salud
     1997;18:7.
 10. Lanciotti RS, Calisher CH, Gubler DG, Chang G, Vordam V. Rapid
     detection and typing of dengue viruses from clinical samples by using
     reverse transcriptase-polymerase chain reaction. J Clin Microbiol
     1992;30:545-51.
 11. Laferte J, Pelegrino JL, Guzmn MG, Gonzlez G, Vzquez S, Hermida C.
     Rapid diagnosis of dengue virus infection using a novel 10l IgM
     antibody capture ultramicroELISA assay (MAC UMELISA Dengue). Advances
     in Modern Biotechnology 1992;1:19.4.
 12. Rico-Hesse R. Molecular evolution and distribution of dengue viruses
     type 1 and 2 in nature. Virology 1990;174:479-93.
 13. Divisin de Prevencin y Control de Enfermedades, Programa de
     Enfermedades, Programa de Enfermedades Transmisibles, HCP/HCT, OPS.
     Resurgimiento del dengue en las Amricas. Boletn Epidemiolgico.
     Organizacin Panamericana de la Salud 1997;18:1-6.
 14. Guzmn MG, Kour G, Bravo J, Hoz de la F, Soler M, Hernndez B.
     Encuesta seroepidemiolgica retrospectiva a virus dengue en los
     municipios Cienfuegos y Palmira. Rev Cubana Med Trop 1989;41:321-32.
 15. Guzmn MG, Kour G, Bravo J, Soler M, Vzquez S, Morier L. Dengue
     hemorrhagic fever in Cuba, 1981: a retrospective seroepidemiologic
     study. Am J Trop Med Hyg 1990;42:179-84.
 16. Halstead SB. Observations related to pathogenesis of dengue
     hemorrhagic fever. Yale J Biol Med 1970;42:350-60.
 17. Daz A, Kour G, Guzmn MG, Lobaina L, Bravo J, Ruiz A, et al.
     Description of the clinical picture of dengue hemorrhagic fever/dengue
     shock syndrome (DHF/DSS) in adults. Bull Pan Am Health Organ
     1988;22:133-44.
 18. Gubler DJ, Clark GG. Dengue/dengue hemorrhagic fever: the emergence of
     a global health problem. Emerg Infect Dis 1995;1:55-7.
---------------------------------------------------------------------------
Dispatches
Hantavirus Pulmonary Syndrome in a Chilean Patient with Recent Travel in
Bolivia
Ricardo Espinoza,* Pablo Vial,* Luis M. Noriega,* Angela Johnson, Stuart
T. Nichol, Pierre E. Rollin, Rachel Wells, Sherif Zaki, Enrique
Reynolds,* and Thomas G. Ksiazek
*Clnica Alemana de Santiago, Santiago, Chile; Escuela de Medicina,
Universidad Catlica de Chile, Santiago, Chile; Centers for Disease
Control and Prevention, Atlanta, Georgia, USA.
---------------------------------------------------------------------------
      A case of hantavirus pulmonary syndrome (HPS) was serologically
      confirmed in a critically ill patient in Santiago, Chile. The
      patient's clinical course had many similarities to that of
      other HPS patients in North and South America but was
      complicated by acute severe renal failure. The patient's
      history included self-reported urban and probable rural rodent
      exposure during travel in Bolivia. Comparison of a viral
      sequence from an acute-phase serum sample with other known
      hantaviruses showed that the hantavirus nucleic acid sequence
      from the patient was very similar to a virus recently isolated
      from rodents associated with HPS cases in Paraguay.
Since its discovery in 1993 (1) and its association with Sin Nombre virus
in North America (2), hantavirus pulmonary syndrome (HPS) has been
identified in several countries in South America and is associated with
Juquitiba virus in Brazil (3), Andes virus in Argentina and Chile (4-6),
and Laguna Negra virus in Paraguay (7,8). Hantaviruses are rodent-borne,
and each is associated with a specific primary rodent reservoir.
Sigmodontine rodents are the vectors of hantaviruses associated with HPS
(3).
Case Report
A 20-year-old male resident of Santiago, Chile, who had no prior history of
medical problems, became ill after he backpacked (from February 4 to March
9, 1997) as a tourist in Bolivia. His travel itinerary included Barrio,
Oruro, La Paz, Cochabamba, Villa Tunari, Santa Cruz, Vallegrande, Higueras,
and Sucre. The tourist and a fellow traveler stayed in hotel in Santa Cruz
where they saw black rats running through the bathroom they used. While in
Higueras, they stayed in a rustic adobe house with no floor and joined
local villagers in agricultural jobs, primarily harvesting hay. A diagnosis
of HPS was considered.
On March 26, 3 weeks after returning to Chile, the young man became ill
with fever and cough. On March 28, he was admitted to the emergency room of
a private hospital in Santiago with high fever (39C), cough, and chest
pain. Chest X-rays showed interstitial infiltrates in the left lower lobe.
Laboratory results were as follows: hemoglobin 15.3 gm/dl, white blood
cells 5,900, platelets 130,000, erythrocyte sedimentation rate 6, and a
C-reactive protein 2.8 mg/dl. He was sent home on clarithromycin 250 mg,
twice a day. Three days later (March 31), he returned to the emergency room
with persistent high fever, myalgia, and shortness of breath; distal
cyanosis was noted. Vital signs were as follows: blood pressure 115/80,
pulse 120, temperature 38.5C, and respiratory rate 32. Physical
examination found few petechiae on the forearms, diffuse bilateral rales,
regular cardiac rhythm, no murmurs, and no hepatomegaly or splenomegaly.
Chest X-rays showed diffuse bilateral alveolar infiltrates. Oxygen
saturation was 90%. Laboratory values included white blood cells 11,700
with a left shift, platelets 150,000, hemoglobin 19.4, erythrocyte
sedimentation rate 2, C-reactive protein 8.18, international normalized
ratio 1.6, activated partial thromboplastin 43s, blood urea nitrogen 38.9
mg/dl, and liver function tests normal limits.
The patient was transferred to the intensive care unit with a diagnosis of
bilateral pneumonia of unknown etiology and secondary respiratory failure.
In 3 to 4 hours, acute respiratory distress developed; arterial blood gases
(on 50% oxygen) were PaO(sub 2) 61, PaCO(sub 2) 22.7, pH 7.49, and HCO(sub 3) 17.1. The
patient was connected to a ventilator and was started on imipenen,
erythromycin, amantadine, dopamine, dobutamine, fluids, and nitric oxide.
Two 500-mg doses of methylprednisolone were administered, and a Swan-Ganz
catheter was installed. The pulse wedge pressure was 7, and the cardiac
output 6.1. During the first 24 hours, acute renal failure developed, and
hemodialysis was started.
Hypotension was quite refractory to vasoactive drugs (mean BP 30 to 40),
and critical hypoxemia (O(sub 2) saturation 70% to 80%) and hypotension persisted
for 48 hours. Echocardiography showed a mild pericardial effusion. Blood
cultures were negative for bacteria, as well as for influenza, adenovirus,
respiratory syncytial virus, and parainfluenza virus. Serologic results for
_Mycoplasma_, _Legionella_, and HIV were also negative. Blood gases started
to improve on day 3, and the patient's clinical condition also slowly
improved. On day 10, hemolytic anemia (Coombs negative) developed, and a
bone marrow aspirate showed hemophagocytosis. On day 11, an episode of
bleeding from the respiratory tract occurred. However, the patient's
ventilatory function continued to improve. On day 15 after admission, his
chest X-ray was clearing, but he was still on a mechanical ventilator (O(sub 2)
saturation 98% on 40% O(sub 2)). However, he was anuric (BUN 100). On day 17, he
had a second episode of bronchial bleeding, and bronchoscopy showed only a
4-mm tracheal ulcer. Platelets were 72,000, and hematocrit fell to 24%.
Hantavirus serology was reported positive [Centers for Disease Control and
Prevention (CDC), April 18], and intravenous ribavirin was started on April
22. A central venous catheter-related infection with _Pseudomonas_ and
_Staphylococcus aureus_ was documented. The patient's condition
deteriorated progressively, with further bronchial bleeding and markedly
unstable ventilatory function, and required constant administration of
vasoactive drugs; he died of massive pulmonary hemorrhage and shock on
April 28. A postmortem lung sample was taken with a needle.
The patient's serum, collected on April 1, was tested for immunoglobulin G
(IgG) and IgM antibodies to Sin Nombre virus at CDC. Both IgG and IgM
antibodies were found, which suggested recent infection with a hantavirus
associated with Sigmodontine rodents. Subsequent testing of sera collected
on April 21 and April 25 confirmed the initial findings. Hematoxylin and
eosin stained sections of the lung sample showed diffuse alveolar damage
with extensive hyaline membrane formation, proliferation of type II
pneumocytes, and fibroblastic and edematous thickening of alveolar walls.
Abundant fibrin, necrotic debris, and acute inflammatory cellular
infiltrates were also observed in the alveolar spaces. Rare endothelial
cells and macrophages were hantavirus-antigen positive by previously
described immunohistochemical procedures (9). The destructive changes seen
by histopathology and a small amount of antigen found in the patient's
tissues are compatible with the long course of the patient's illness (9).
Viral RNA was extracted from the earliest serum sample, and reverse
transcriptase-polymerase chain reaction amplification with primers designed
specifically for hantaviruses associated with Sigmodontine rodents (8)
yielded polymerase chain reaction fragments for the S and M segments. These
cDNA fragments were sequenced and compared with those of other American
hantaviruses. These comparisons show that the virus is closely related to
other South American hantaviruses, and most closely related to Laguna Negra
virus detected in patients and _Calomys laucha_ rodents (vesper mice) in
the Chaco region of Paraguay (7,8). The nucleotide sequence identity was
84% for the G1 protein encoding fragment and 87% for the N protein encoding
fragment. However, the deduced amino acid sequences for both fragments were
identical to Laguna Negra virus. This shows that the virus associated with
this HPS case is a Laguna Negra virus variant and suggests that the virus
is probably associated with the same or a very closely related species of
rodent host.
_C. laucha_, the apparent primary rodent reservoir for Laguna Negra virus,
is common in savanna and grassland areas as far north as southern Brazil
and Bolivia, throughout much of Paraguay and Uruguay, and in Argentina as
far south as Rio Negro province, but not in Chile (10). A number of the
lowland rural locations that the patient visited in Bolivia are within the
range of _C. laucha_.
The clinical course and travel history of the patient and the laboratory
serology and molecular characterization of the viral RNA are compatible
with infection in Bolivia with a Laguna Negra virus variant. This report
and evolving information concerning hantaviruses associated with clinical
HPS in Argentina and Paraguay strongly suggest that a diagnosis of HPS
should be considered in patients with febrile respiratory distress syndrome
throughout Latin America.
References
  1. Duchin JS, Koster F, Peters CJ, Simpson G, Tempest B, Zaki S, et al.
     Hantavirus pulmonary syndrome: a clinical description of 17 patients
     with a newly recognized disease. N Engl J Med 1994;330:949-55.
  2. Nichol ST, Spiropoulou CF, Morzunov S, Rollin PE, Ksiazek TG, Feldmann
     H, et al. Genetic identification of a hantavirus associated with an
     outbreak of acute respiratory illness. Science 1993;262:914-7.
  3. Nichol ST, Ksiazek TG, Rollin PE, Peters CJ. Hantavirus pulmonary
     syndrome and newly described hantaviruses in the United States. In:
     Elliott RM, editor. The Bunyaviridae. New York: Plenum Press,
     1996:269-80.
  4. Lopez N, Padula P, Rossi C, Lazaro ME, Franze-Fernandez MT. Genetic
     identification of a new hantavirus causing severe pulmonary syndrome
     in Argentina. Virology 1996;220:223-6.
  5. Centers for Disease Control and Prevention. Hantavirus pulmonary
     syndrome in Chile-1997. MMWR Morb Mortal Wkly Rep 1997;46:949-51.
  6. Lopez N, Padula P, Rossi C, Miguel S, Edelstein A, Ramirez E, et al.
     Genetic characterization and phylogeny of Andes virus and variants
     from Argentina and Chile. Virus Research 1997;50:77-84.
  7. Williams RJ, Bryan RT, Mills JN, Palma RE, Vera I, de Velasquez F, et
     al. An outbreak of hantavirus pulmonary syndrome in Western Paraguay.
     Am J Trop Med Hyg 1997;57:274-82.
  8. Johnson AM, Bowen MD, Ksiazek TG, Williams RJ, Bryan RT, Mills JN, et
     al. Laguna Negra virus associated with HPS in western Paraguay and
     Bolivia. Virology. In press 1997.
  9. Zaki S, Greer PW, Coffield LM, Goldsmith CS, Nolte KB, Foucar K, et
     al. Hantavirus pulmonary syndrome: pathogenesis of an emerging
     infectious disease. American Journal of Pathology 1995;146:552-79.
 10. Redford KH, Eisenberg JF. Mammals of the neotropics, the southern
     cone. Chicago: The University of Chicago Press, 1992:279-80.
---------------------------------------------------------------------------
Dispatches
Prevalence of Tick-Borne Pathogens in _Ixodes scapularis_ in a Rural New
Jersey County
Shobha Varde,* John Beckley, and Ira Schwartz*
*New York Medical College, Valhalla, New York, USA; and Department of
Health, County of Hunterdon, Flemington, New Jersey, USA
---------------------------------------------------------------------------
      To assess the potential risk for other tick-borne diseases, we
      collected 100 adult _Ixodes scapularis_ in Hunterdon County, a
      rapidly developing rural county in Lyme disease-endemic western
      New Jersey. We tested the ticks by polymerase chain reaction
      for _Borrelia burgdorferi_, _Babesia microti_, and the
      rickettsial agent of human granulocytic ehrlichiosis (HGE).
      Fifty-five ticks were infected with at least one of the three
      pathogens: 43 with _B. burgdorferi_, five with _B. microti_,
      and 17 with the HGE agent. Ten ticks were coinfected with two
      of the pathogens. The results suggest that county residents are
      at considerable risk for infection by a tick-borne pathogen
      after an _I. scapularis_ bite.
The vector-borne diseases Lyme disease, human babesiosis, and human
granulocytic ehrlichiosis (HGE) are emerging in the Northeast and upper
Midwest regions of the United States (1,2). The etiologic agents for these
diseases (_Borrelia burgdorferi_, _Babesia microti_, and the HGE agent,
respectively) appear to share the same vertebrate reservoir host
(_Peromyscus leucopus_) and tick vector (_Ixodes scapularis_) (3-6).
Immunologic evidence of human coinfection with these pathogens has been
reported (7-10), and a culture-confirmed case of coinfection with _B.
burgdorferi_ and the HGE agent has recently been described (11). Thus, in
Lyme disease-endemic areas, there may be a substantial risk for coinfection
with _B. burgdorferi_ and either _B. microti_ or the HGE-causing
rickettsia.
Hunterdon County (population 118,000) is a rural, but rapidly developing,
county in western New Jersey (Figure), with many homes in wooded settings.
In 1996, the county had the third highest case rate of Lyme disease (524
per 100,000) of all counties in the United States (Centers for Disease
Control and Prevention case reports). The county has also had many
suspected, but no serologically confirmed, cases of HGE.
 [fig]
 Figure. Map of New Jersey showing Hunterdon County. Black dots indicate
 tick collection sites.
The risk of acquiring a tick-borne pathogen in a specific geographic
location depends largely on tick density and the prevalence rate of the
agent in the tick population. Although cultivation of a microbial agent is
considered optimal, molecular detection is more practical since it permits
the rapid assay of large numbers of individual specimens. We report here
the prevalence rate of _B. burgdorferi_, _B. microti_, and the agent of HGE
in _I. scapularis_ in Hunterdon County by species-specific polymerase chain
reaction (PCR).
One hundred adult _I. scapularis_ were collected by drag cloth or from
personal clothing at 10 sites in the county during the fall of 1996
(Figure). Ticks were stored at room temperature in 70% ethanol until
analysis. DNA was isolated from individual ticks with the Isoquick DNA
extraction kit (ORCA Research, Bothell, WA) (12). The final DNA pellets
were resuspended in 50 l of sterile water, and a 10-l aliquot was used
for each PCR test. _B. burgdorferi_-specific PCR used primers IS1 and IS2
(13). The HGE agent was detected by PCR using primers GER3 and GER4, as
described by Munderloh et al. (14), and _B. microti_ DNA was detected
according to methods used by Persing et al. (15). PCR products were
electrophoresed on 2% agarose gels stained with ethidium bromide. For _B.
burgdorferi_ and _B. microti_, hybridization with specific probes was
carried out after transfer to nylon membranes (13). In each PCR experiment
appropriate negative controls were used; these included master mix controls
lacking template and reactions containing _B. burgdorferi_ DNA, HGE agent
DNA, or _Escherichia coli_ DNA as additional controls for HGE agent, _B.
burgdorferi_, or _B. microti_ PCR, respectively. To prevent
cross-contamination, specimen preparation, PCR amplification, and post-PCR
analysis were performed in separate laboratories.
Results of PCR analysis for each of the pathogens on individual ticks are
presented in the Table. At least one of the three pathogens was present in
55% of the ticks. The highest prevalence rate was that for _B. burgdorferi_
(43/100), followed by the agent of HGE (17/100) and _B. microti_ (5/100).
Furthermore, these pathogens were widely distributed. At least one tick at
each site was infected with _B. burgdorferi_, nine of 10 sites had HGE
agent-positive ticks, and _B. microti_-positive ticks were found at five
different collection sites. Ten ticks were coinfected with two of the three
agents; no ticks were infected with all three pathogens. The _B. microti_
PCR amplification products were gel purified and subjected to automated DNA
sequencing. The 238 bp products yielded sequences identical to that
reported for _B. microti_ 18S ribosomal DNA (15).
Table. Prevalence rate of _Borrelia burgdorferi_, human granulocytic
ehrlichiosis (HGE) agent, and _Babesia microti_ in 100 adult _Ixodes
scapularis_
------------------------------------------------------------------
Pathogen                                     No. infected ticks
------------------------------------------------------------------
_B. burgdorferi_                                       43
 HGE agent                                             17
_B. microti_                                            5
_B. burgdorferi_ and HGE agent                          6
_B. burgdorferi_ and _B. microti_                       2
HGE agent and _B. microti_                              2
_B. burgdorferi_, HGE agent, and _B. microti_           0
------------------------------------------------------------------
The prevalence rates for the three pathogens reported here are consistent
with earlier studies at selected sites in the northeastern United States.
Telford et al. reported prevalence rates of 36%, 11%, and 9% for _B.
burgdorferi_, the HGE agent, and _B. microti_, respectively, in adult _I.
scapularis_ on Nantucket Island, Massachusetts; coprevalence of _B.
burgdorferi_ and the HGE agent was 4%, and no simultaneous infection with
_B. burgdorferi_ and _B. microti_ was observed (4). We have reported
prevalence rates of 52% and 53% for _B. burgdorferi_ and the HGE pathogen,
and coprevalence of 26% at a site in Westchester County, New York (5).
Given the simultaneous infection of _I. scapularis_ with these pathogens,
it is not surprising that numerous serosurveys of Lyme disease patients
indicate the coincident presence of antibodies to _B. burgdorferi_ and _B.
microti_ and human granulocytic ehrlichia (7-10). These findings do not
establish concurrent, active infection, nor do they demonstrate
simultaneous transmission by a single tick bite; however, they suggest that
persons living in disease-endemic areas are exposed to these agents,
presumably as a result of tick bites.
Human infection by any of the three tick-borne agents alone generally
results in similar acute manifestations, including fever, headache, and
myalgia (1,10,16). Reported cases of Lyme disease greatly outnumber those
of babesiosis or HGE. This, in conjunction with seroepidemiologic data,
suggests that many infections with _B. microti_ and granulocytic ehrlichia
are subclinical. Dual infection with _B. burgdorferi_ and _B. microti_
often results in more severe illness (10); whether this is also true for
concurrent infection by the agents of Lyme disease and HGE remains to be
established. Human coinfection by multiple tick-borne agents may account
for the variable nature of the clinical manifestations of Lyme disease.
This study demonstrates that the agents of Lyme disease, human babesiosis,
and HGE coexist in the tick population of a previously identified Lyme
disease-endemic region of New Jersey. The study provides further evidence
that these three pathogens may be prevalent throughout the range of _I.
scapularis._ Infection with any of these three tick-borne pathogens should
be considered for residents or visitors of a disease-endemic area who have
flulike symptoms and a history of a tick bite.
Acknowledgments
We thank Gary Wormser and Dennis White for helpful comments, Chris Kolbert
and David Persing for providing advice and positive controls for _B.
microti_ PCR, and the Hunterdon County Tickborne Disease Research Group and
Hunterdon Medical Center for their support. This work was supported in part
by NIH grant AR41511.
References
  1. Walker DH, Barbour AG, Oliver JH, Lane RS, Dumler JS, Dennis DT, et
     al. Emerging bacterial zoonotic and vector-borne diseases: ecological
     and epidemiological factors. JAMA 1996;275:463-9.
  2. Persing DH, Conrad PA. Babesiosis: new insights from phylogenetic
     analysis. Infect Agents Dis 1995;4:182-95.
  3. Anderson J, Mintz E, Gadbaw J, Magnarelli L._ Babesia microti_, human
     babesiosis and _B. burgdorferi_ in Connecticut. J Clin Microbiol
     1991;29:2779-83.
  4. Telford SR III, Dawson JE, Katavolos P, Warner CK, Kolbert CP, Persing
     DH. Perpetuation of the agent of human granulocytic ehrlichiosis in a
     deer tick-rodent cycle. Proc Natl Acad Sci U S A 1996;93:6209-14.
  5. Schwartz I, Fish D, Daniels TJ. Prevalence of the rickettsial agent of
     human granulocytic ehrlichiosis in ticks from a hyperendemic focus of
     Lyme disease [letter]. N Engl J Med 1997;337:49-50.
  6. Piesman J, Mather TN, Telford SR III, Spielman A. Concurrent _Borrelia
     burgdorferi_ and _Babesia microti_ infection in nymphal _Ixodes
     dammini_. J Clin Microbiol 1986;24:446-7.
  7. Benach JL, Coleman JL, Habicht GS, MacDonald A, Grunwaldt E, Giron JA.
     Serological evidence for simultaneous occurrences of Lyme disease and
     babesiosis. J Infect Dis 1985;152:473-7.
  8. Magnarelli LA, Dumler JS, Anderson JF, Johnson RC, Fikrig E.
     Coexistence of antibodies to tick-borne pathogens of babesiosis,
     ehrlichiosis, and Lyme borreliosis in human sera. J Clin Microbiol
     1995;33:3054-7.
  9. Mitchell PD, Reed KD, Hofkes JM. Immunoserologic evidence of
     coinfection with _Borrelia burgdorferi, Babesia microti_, and human
     granulocytic _Ehrlichia_ species in residents of Wisconsin and
     Minnesota. J Clin Microbiol 1996;34:724-7.
 10. Krause PJ, Telford SR III, Spielman A, Sikand V, Ryan R, Christianson
     D, et al. Concurrent Lyme disease and babesiosis. Evidence for
     increased severity and duration of illness. JAMA 1996;275:1657-60.
 11. Nadelman RB, Horowitz HW, Hsieh TC, Wu JM, Aguero-Rosenfeld ME,
     Schwartz I, et al. Simultaneous human granulocytic ehrlichiosis and
     Lyme borreliosis. N Engl J Med 1997;337:27-30.
 12. Schwartz I, Varde S, Nadelman RB, Wormser GP, Fish D. Inhibition of
     efficient polymerase chain reaction amplification of _Borrelia
     burgdorferi_ DNA in blood-fed ticks. Am J Trop Med Hyg 1997;56:339-42.
 13. Schwartz I, Wormser GP, Schwartz JJ, Cooper D, Weissensee P, Gazumyan
     A, et al. Diagnosis of early Lyme disease by polymerase chain reaction
     amplification and culture of skin biopsies from erythema migrans
     lesions. J Clin Microbiol 1992;30:3082-8.
 14. Munderloh UG, Madigan JE, Dumler JS, Goodman JL, Hayes SF, Barlough
     JE, et al. Isolation of the equine granulocytic ehrlichiosis agent,
     _Ehrlichia equi_, in tick cell culture. J Clin Microbiol
     1996;34:664-70.
 15. Persing D, Mathiesen D, Marshall W, Telford S, Spielman A, Thomford J,
     Conrad P. Detection of _Babesia microti_ by polymerase chain reaction.
     J Clin Microbiol 1992;30:2097-103.
 16. Boustani MR, Gelfand JA. Babesiosis. Clin Infect Dis 1996;22:611-5.
---------------------------------------------------------------------------
Dispatches
Plague, a Reemerging Disease in Madagascar
Suzanne Chanteau,* Lala Ratsifasoamanana, Bruno Rasoamanana,* Lila
Rahalison,* Jean Randriambelosoa, Jean Roux,* and Dieudonn Rabeson
*Institut Pasteur, Antananarivo, Madagascar; and Ministre Sant,
Antananarivo, Madagascar
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[download.gif (1218 bytes)]
      Human cases of plague, which had virtually disappeared in
      Madagascar after the 1930s, reappeared in 1990 with more than
      200 confirmed or presumptive cases reported each year since. In
      the port of Mahajanga, plague has been reintroduced, and
      epidemics occur every year. In Antananarivo, the capital, the
      number of new cases has increased, and many rodents are
      infected with Yersinia pestis. Despite surveillance for the
      sensitivity of Y. pestis and fleas to drugs and insecticides
      and control measures to prevent the spread of sporadic cases,
      the elimination of plague has been difficult because the host
      and reservoir of the bacillus, Rattus rattus, is both a
      domestic and a sylvatic rat.
In the last 15 years, Madagascar (population 13 million) has accounted for
45% of the cases of plague in Africa (1).
Epidemiology
Plague was brought to the island of Madagascar in 1898 by steamboats from
India and has never disappeared. As a result of vaccination campaigns,
improved housing and public hygiene, and the discovery of streptomycin and
insecticides, plague was controlled in the 1950s. During the next 30 years,
only 20 to 50 cases per year were reported in the entire country. However,
since 1989, the number of suspected cases has increased steadily (Figure
1). Since 1990, 800 to 1,500 cases of suspected plague have been reported,
of which 150 to 230 were smear-positive (presumptive cases) or confirmed by
the isolation of Y. pestis (2,3). The population exposed to plague in the
plateaus is approximately 5 million; the mean annual rate of known human
cases (except in Mahajanga) is 3 to 4 per 100,000 inhabitants. The mean
annual death rate is 20% of the confirmed or presumptive cases. Bubonic
plague is the main clinical form of the disease (approximately 95% of
cases).
                         [Fig]
Figure 1. Human plague, Madagascar, 1982-1996.
With the exception of the west coast port of Mahajanga, plague is endemic
in areas more than 800 m high. The major focus area is a central triangle,
and the minor focus is a northern diamond (Figure 2). The main plague foci
(in order of importance) are Mahajanga, the district of Ambositra, the town
of Antananarivo, and the districts of Fianarantsoa II, Miarinarivo, Betafo,
and Soavinandriana. In the plateaus, the human plague season is September
to April, while in Mahajanga it is July to November.
                         [Fig]
Figure 2. Plague foci in Madagascar. The plague-endemic zones are in the
gray areas and in the port of Mahajanga.
In 1996, 1,644 suspected plague cases were reported; biologic samples were
available for 1,316. A total of 173 confirmed and 56 presumptive cases were
officially reported. In 1997, 2,127 suspected cases were reported from
January to October, and more than 2,500 total cases were expected by the
end of 1997. So far, 260 are confirmed and 33 are presumptive cases. The
number of confirmed or presumptive plague cases is underestimated because
of underreporting in remote areas and the lack of sensitivity of the
bacteriologic techniques used for diagnosis. Our preliminary results,
obtained by immunodiagnostic tests, such as anti-F1 antibody detection (4)
and F1 antigen tests (tests supplied by the Naval Medical Research
Institute, Bethesda, MD), suggest a number of plague cases two to three
times higher than that obtained by conventional methods (S. Chanteau and J.
Burans, unpub. data). Whether the significant increase between 1996 and
1997 results from better notification of the cases following educational
campaigns or a real increase in disease incidence is unknown.
In Madagascar, the reservoirs of Y. pestis are two species of rats, Rattus
norvegicus and R. rattus. The sylvatic reservoir of Y. pestis is not well
documented. The urban domestic rat R. norvegicus, introduced to Madagascar
during the 20th century, can only be found (in larger numbers than R.
rattus) in coastal cities and Antananarivo. In contrast, R. rattus,
probably imported into Madagascar with the first human migrations, has
invaded every ecosystem of the island. Thus, it is both a domestic and a
wild rodent. It is generally the only type of rodent found in small towns,
villages, rice fields, and grasslands. The reservoirs of Y. pestis during
the interseason period are the R. rattus and R. norvegicus that have
resisted infection; therefore, it is almost impossible to eliminate plague
from the island (2).
Two species of rat fleas are vectors of plague in Madagascar. The most
effective are the classic oriental rat flea, Xenopsylla cheopis, and the
endemic flea, Synopsyllus fonquerniei (2); both are found on R. rattus,
although X. cheopis is almost exclusively on urban and indoor rodents,
while S. fonquerniei is found on outdoor and wild rodents (S. Laventure and
J.M. Duplantier, unpub. data). Ongoing research is examining the
interrelationships between the two species of rats and the two species of
fleas in the epidemiologic cycle of plague in Madagascar.
The reemergence of plague in Madagascar probably reflects the general
breakdown of traditional measures of plague control. Prolonged maintenance
of the domestic rat-to-flea cycle that accounted for the historic plague
pandemics will almost surely provide an opportunity for selection of new
traits that could make the organism even more virulent. In fact, three new
variants of Y. pestis have recently emerged in the region of Ambositra and
Ambohimahasoa, one of the most active plague foci of the island. These new
ribotypes tend to spread to new geographic areas; whether or not they have
acquired selective advantages is being explored (5). Furthermore, the
opportunity for gene exchange with enteric bacteria is greatly enhanced. It
may be important that the first naturally occurring antibiotic-resistant
strain of Y. pestis was recently isolated in Madagascar (6,7).
Plague in Mahajanga
In Mahajanga (population 150,000), after two epidemics in the beginning of
the 20th century, plague remained under control between 1928 and 1990 (2).
In July 1991, the disease suddenly reappeared in a shantytown near the
marketplace of Marolaka; of the 170 suspected cases, 41 were confirmed or
presumptive. After 3 years without reported cases, three successive
outbreaks occurred: July 1995 (8,9), July 1996, and July 1997. During the
1995 and 1996 outbreaks, a total of 1,058 suspected cases were reported;
only 109 cases were confirmed, and 30 cases were smear-positive. However,
serologic testing, mainly F1 antibody detection by enzyme-linked
immunosorbent assay (ELISA) (4), allowed the diagnosis of 93 additional
cases (10). Thus, the mean annual rate of known human cases is 77 per
100,000 inhabitants. During the ongoing epidemic, from July to October
1997, 376 suspected cases (120 bacteriologically confirmed) have been
reported and tended to spread in new quarters of the town.
Every human outbreak has been preceded by a high number of rat deaths.
During and after epidemics, all shrews captured were infected with many X.
cheopis. Ongoing studies are examining the role of the shrew Suncus murinus
in the maintenance of plague between epidemics.
Plague in Antananarivo
Since plague was introduced in the highlands in 1921, it has never
disappeared from remote villages; however, in Antananarivo, it has been
controlled; no human cases were reported between 1953 and 1978. In 1979,
the first confirmed case was found in one of the ancient foci of the town
(11). In the 1990s, a growing number of cases have been reported; 10 to 25
each year have been confirmed or are presumptive (Figure 3). In 1996,
confirmed plague cases were found in 17 quarters of the capital. The mean
annual rate of human cases is 1.4 per 100,000 inhabitants in 1995 and 1996.
                          [Fig]
Figure 3. Human plague, Antananarivo, 1976-1996.
In 1995, 10% of 625 rats trapped near a marketplace were infected with Y.
pestis; 80% were anti-F1 seropositive (S. Chanteau, J.A. Drominy, and B.
Rasoamanana, unpub. data). The monthly flea index (mean number of X.
cheopis per rat) in this quarter was more than 4 year-round. Such a high
index represents a serious threat, especially since most of these fleas are
resistant to deltamethrin (12). Other families of insecticides, such as the
carbamates, can be proposed. Since 1996, the Ministry of Health and local
government have made serious efforts to educate the population and improve
public hygiene.
A preliminary study of rats trapped in eight other quarters of the capital
in 1997 showed a mean seroprevalence of 14% by ELISA (J.A. Drominy and S.
Chanteau, unpub. data). The circulation of Y. pestis in the rat population
was significantly larger than it was in 1965 (4.5%) (2).
National Plague Control Program
The national plague control program is financially supported by the World
Bank and the French Ministry of Cooperation. The surveillance system used
is based on immediate compulsory notification of every suspected case of
plague and its biologic confirmation by the Central Laboratory. All
patients with suspected cases are treated with streptomycin, and their
contacts are treated with sulfonamides to prevent disease spread.
Insecticides are used for flea control. Every strain of Y. pestis isolated
from humans, rats, and fleas is tested for drug sensitivity, and the
susceptibility of fleas to insecticides is determined. The isolation of the
first multidrug-resistant strain of Y. pestis in 1995 (6,7) and the
increasing resistance of fleas to insecticides have caused much concern
(12).
This national program, implemented in Madagascar for several decades, has
been hampered by economic and operational difficulties and urgently needs
to be strengthened.
References
  1. World Health Organization. Human plague in 1995. Wkly Epidemiol Rec
     1997;46:344-8.
  2. Brygoo ER. Epidemiologie de la peste  Madagascar. Arch Inst Pasteur
     Madagascar 1966;35:7-147.
  3. Blanchy S, Ranaivoson G, Rakotonjanabelo A. Epidmiologie clinique de
     la peste  Madagascar. Arch Inst Pasteur Madagascar 1993;60:27-34.
  4. Rasoamanana B, Leroy F, Boisier P, Rasolomaharo M, Buchy P, Carniel E,
     Chanteau S. Field evaluation of an IgG anti-F1 ELISA test for the
     serodiagnosis of human plague in Madagascar. Clin Diagn Lab Immunol
     1997;4:587-91.
  5. Guiyoule A, Rasoamanana B, Buchreiser C, Michel P, Chanteau S, Carniel
     E. Recent emergence of new variants of Yersinia pestis in Madagascar.
     J Clin Microbiol 1997;35:2826-33.
  6. Rasoamanana B, Leroy F, Raharimanana C, Chanteau S. Surveillance de la
     sensibilit aux antibiotiques des souches de Y. pestis  Madagascar de
     1989  1995. Arch Inst Pasteur Madagascar 1995;62:108-10.
  7. Galimand M, Guiyoule A, Gerbaud G, Rasoamanana B, Chanteau S, Carniel
     E, Courvalin P. Multiple antibiotic resistance in Yersinia pestis
     mediated by a self-transferable plasmid. N Engl J Med 1997;337:677-80.
  8. Rasolomaharo M, Rasoamanana B, Andrianirina Z, Buchy P,
     Rakotoarimanana N, Chanteau S. Plague in Mahajanga, Madagascar. Lancet
     1995;346:1234.
  9. Boisier P, Rasolomaharo M, Ranaivoson G, Rasoamanana B, Rakoto L,
     Andriamahefazafy B, Chanteau S. Urban epidemic of bubonic plague in
     Mahajanga, Madagascar. Epidemiological aspects. Trop Med Int Health
     1997;5:422-7.
 10. Chanteau S, Rasoamanana B, Rasolomaharo M, Leroy F, Rahalison L, Buchy
     P, et al. Apport de la dtection des anticorps anti-F1 et de
     l'antignmie F1 dans l'analyse des pidmies de peste de la ville de
     Mahajanga. Proceedings of Colloque Scientifique de la Runion du
     Conseil des Directeurs des Instituts Pasteurs et Instituts Associs;
     1997; Paris.
 11. Coulanges P. Situation de la peste  Tananarive, de son apparition en
     1921  sa rsurgence en 1979. Arch Inst Pasteur Madagascar
     1989;56:9-35.
 12. Laventure S, Ratovonjato J, Rajaonarivelo E, Rasoamanana B, Rabarison
     P, Chanteau S, Roux J. Rsistance aux insecticides des puces
     pestignes  Madagascar et implications pour la lutte antivectorielle.
     Proceedings of the 5me Congrs International de Mdecine Tropicale de
     Langue Franaise; 1996; Mauritius.
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Dispatches
Bayou Virus-Associated Hantavirus Pulmonary Syndrome in Eastern Texas:
Identification of the Rice Rat, _Oryzomys palustris_, as Reservoir Host
Norah Torrez-Martinez,* Mausumi Bharadwaj,* Diane Goade,* John Delury,
Peggy Moran,* Bradley Hicks, Beverlee Nix, James L. Davis, and Brian
Hjelle*
*University of New Mexico School of Medicine, Albuquerque, New Mexico, USA;
Texas Department of Health, Austin, Texas, USA; Texas Department of
Health, Houston, Texas, USA; and St. Elizabeth's Hospital, Beaumont,
Texas, USA.
---------------------------------------------------------------------------
      We describe the third known case of hantavirus pulmonary
      syndrome (HPS) due to Bayou virus, from Jefferson County,
      Texas. By using molecular epidemiologic methods, we show that
      rice rats (_Oryzomys palustris_) are frequently infected with
      Bayou virus and that viral RNA sequences from HPS patients are
      similar to those from nearby rice rats. Bayou virus is
      associated with _O. palustris_; this rodent appears to be its
      predominant reservoir host.
The 1993 discovery of a clinically distinct form of hantavirus disease now
known as hantavirus pulmonary syndrome (HPS) highlighted the existence of a
previously unrecognized complex of New World hantaviruses, each of which is
associated with a specific rodent of the subfamily Sigmodontinae, family
Muridae. The prototype of this complex is the Sin Nombre (SN) virus of the
deer mouse, _Peromyscus maniculatus_. SN virus, which occurs most
frequently in the western United States and Canada, is responsible for more
than 95% of cases of HPS in North America. The case-fatality rate of HPS is
nearly 50% (1).
Three viruses other than SN have been associated with HPS in North America.
All cases associated with viruses other than SN have occurred outside the
range of the deer mouse. Two cases have been linked to New York virus in
the northeastern United States; the white-footed mouse, _P. leucopus_, was
the only hantavirus carrier in the area where the infections were
contracted. The cotton rat (_Sigmodon hispidus_)-associated Black Creek
Canal virus is believed to have caused a single case in southern Florida,
albeit without molecular confirmation. Bayou (BAY) virus has been
identified from cDNA sequences amplified from the necropsied tissues or the
blood of patients from Louisiana and eastern Texas (1-6).
Evidence implicates the rice rat, _Oryzomys palustris_, as the carrier of
BAY virus. In a survey of archived rodent tissue samples obtained from
species indigenous to Louisiana, only _O. palustris_ samples were positive
for hantavirus antibodies, and cDNAs of a BAY-like virus were amplified
from two of those samples (7). A few additional BAY virus-RNA-positive rice
rat samples have appeared in subsequent studies (6). However, no
substantial rodent collections have been conducted in association with BAY
virus HPS cases, and no direct molecular association has been found between
human BAY virus genomic sequences and those obtained from samples from
rodents trapped at possible sites of human infection. The purpose of this
study was to identify the carrier rodents associated with two human cases
of BAY virus infections and to further characterize the clinical
consequences of human infection with BAY virus.
Diagnostic Studies
Initial serologic investigations to detect antibodies to hantavirus used a
sandwich -capture enzyme-linked immunosorbent assay (ELISA) as well as an
immunoglobulin (Ig) G ELISA, with recombinant SN virus nucleocapsid (N)
protein as the target antigen (8). Confirmatory testing with a larger array
of recombinant-expressed viral N antigens was conducted by using strip
immunoblot and Western blot formats (9-11). The strip immunoblot assay
incorporates five membrane-bound antigens (recombinant-expressed SN virus N
and G1, recombinant-expressed Seoul virus N, and synthetic peptides of SN N
and G1)(11). Antibodies reactive to these antigens are detected with an
anti-human immunoglobulin heavy+light chain conjugate, and the assay thus
has some sensitivity for IgM but greater sensitivity for IgG. The Western
blot uses both anti-IgG and anti-IgM conjugates in separate assays (9).
Western blot studies used a panel of affinity-purified, full-length N
antigens produced in _Escherichia coli_ in fusion with the phage T7 gene 10
protein in the pET23b vector (10). Recombinant N antigens were purified
over metal chelation affinity columns through a polyhistidine tag on the
C-terminus of each protein. The recombinant proteins were produced in an
isogenic background and loaded at 500ng/lane before sodium dodecyl
sulfate-polyacrylamide gel electrophoresis separation and electrophoretic
transfer. The antigens were derived from the following hantaviruses: SN
(3H226); BAY (OP-LA-475); Rio Mamor (OM-556), Muleshoe (SH-Tx-339),
Puumala (P360), and Seoul (80/39).
Rodent Collection and Processing
Since the two BAY virus-HPS cases from Texas occurred in neighboring
cities, this study includes rodent samples collected in the investigation
of both case P/Tx (6) and the case described here, T/Tx. Sherman live-traps
were used to collect rodents in Jefferson County and neighboring Orange
County. Heart blood samples were screened by a recombinant SN virus N
antigen ELISA (8).
Viral Sequencing and Phylogenetic Analyses
Human peripheral blood mononuclear cells (2 x 106) and rodent lung, kidney,
and spleen samples (~200 mg total) were used to make RNA (6,10). A standard
set of partially nested primers in the viral S segment was used in reverse
transcription-polymerase chain reaction (RT-PCR) analyses of the RNA
(6,10). This procedure produces a 442-nucleotide (nt) amplification
product; 397 nt of that sequence is internal to the primers. The PCR
products were subjected to direct DNA sequencing with an ABI 377 automatic
sequencer. Phylogenetic trees were constructed from the 397 nt of
informative sequence by using maximum parsimony with PAUP 3.1 software
(12).
A Third Case of HPS Due to BAY Virus
Patient T/Tx is a 54-year-old African-American man from Jefferson County,
Texas. A heavy tobacco user, he has a more than 100 pack-year smoking
history and chronic shortness of breath. Approximately 2 weeks before
admission, his spouse noted that he had increased fatigue and somnolence.
By August 20, 1996, his symptoms worsened, and he sought medical care. He
visited a local emergency room with complaints of increasing shortness of
breath and low-grade fever for 2 days. Hospital personnel believed that he
had probable chronic obstructive pulmonary disease and chronic bronchitis.
Results of a physical examination of the patient at that time were
unremarkable, and a chest radiograph was interpreted as hyperinflated but
without infiltrates (Figure 1A). He was afebrile (37C) in the emergency
room. Laboratory analysis showed a white blood cell count of 4,080/l and a
platelet count of 122,000/l. He was given amoxicillin and bronchodilators
and was discharged.
 [Fig]
 Figure 1. Chest radiographs of patient T/Tx at hospital admission on August
 20, 1996 (Panel A), and during a period of increasing respiratory distress
 on August 22, 1996 (Panel B). Note the diffuse interstitial infiltrate and
 peribronchial cuffing that developed over that interval.
On August 21, the patient returned to the emergency room with persistent
and worsening dyspnea. He also had flulike symptoms, including myalgias,
and had had a temperature of 39.4C. Physical examination revealed
bilateral rhonchi. He had a blood pressure of 90/60, pulse of 96/min,
respiratory rate of 16/min, and an oral temperature of 37C. Laboratory
analysis noted an increase in his white blood cell count to 7,700/l with a
left shift and a decrease in platelet count to 65,000/l. The patient was
admitted to the hospital with a preliminary diagnosis of chronic
obstructive pulmonary disease exacerbation and possible pneumonia. He was
given broad-spectrum antibiotics, methylprednisolone, aminophylline, and
ipatronium nebulizer treatments.
The patient's condition deteriorated rapidly over the next 24 hours, and he
was severely short of breath by the second hospital day. He was also
febrile and was transferred to the intensive care unit to manage his
worsening shortness of breath. His oxygen demand increased, and on the
third hospital day, the patient required endotracheal intubation. A
physical examination foundbilateral expiratory wheezing and bibasilar
rales. A chest radiograph showed increasing bilateral interstitial and
alveolar infiltrates. By the fourth hospital day, the patient required 100%
oxygen and positive end-expiratory pressure support. A chest radiograph
again showed interstitial and alveolar infiltrates with peribronchial
cuffing and pleural effusion. His fluid intake was restricted to manage his
pulmonary edema; he briefly required dopamine for hypotension. Blood and
urine cultures remained negative. Sputum Gram stain showed a moderate
number of white blood cells and few budding yeast; culture showed no
pathogens. Serologic tests for mycoplasma and _Legionella_ were negative.
By the fifth hospital day, the patient's condition had begun to improve.
Over the next 5 days the fever diminished, the pulmonary edema improved,
and requirement for supplemental oxygen decreased. The endotracheal tube
was removed on the 10th day of hospitalization; by the 14th day, the
patient was discharged-ambulatory, afebrile, and no longer requiring
supplemental oxygen.
The hematologic laboratory findings were generally most abnormal when the
patient was most acutely ill clinically; the peak serum chemistry
abnormalities trailed the clinical illness by several days. The maximum
white blood cell count was 31,700/l on August 23, with a differential
count of 7% lymphocytes (including the characteristic plasmacytoid forms;
[13]), 44% mature neutrophils, 35% band neutrophils, 3% metamyelocytes, and
2% myelocytes. The platelet count reached a nadir of 19,000/l on that day,
and the hemoglobin reached a maximum of 17.2 g/dL on August 22. The serum
creatinine was 0.6 mg/dL on August 24 but peaked at 1.9 mg/dL on August 25.
This mild azotemia was associated with proteinuria (300 mg/dL in a spot
sample obtained on August 22). The serum enzymes creatine kinase and lactic
dehydrogenase peaked on August 27 at 917 units/L and 933 units/L,
respectively. Fractionation of the creatine kinase and lactic dehydrogenase
isoenzyme forms were not supportive of myocardial injury as a cause for
their elevation. Serum levels of the liver transaminases were modestly
elevated.
Environmental Assessment and Rodent Collection
The patient worked as a laborer at a railroad construction company. For 2
months before his illness, he replaced old rail lines at three industrial
plants (Beaumont, Neches River, and Orange) in Orange and Jefferson
Counties and cleaned the yard and garage at work headquarters. He manually
removed and replaced tracks and ties serving warehouses, transport pipes,
and scrap metal piles.
The Beaumont and Orange work sites were chemical plants. All rail work
occurred within the confines of the mowed plant complex. Some of the track
sites were next to drainage ditches and canals; others coursed by
warehouses reported by plant employees to have rats. The patient had not
seen any rodents in the months before becoming ill, but he remembered
rodent droppings at a wooden crew trailer at the Beaumont plant. All
rodents trapped at these plants tested negative for hantavirus antibodies.
The Neches River work site was a recycling plant situated for commerce by
rail and ship. The rails were within several meters of permanent bayous and
swamps, piles of scrap metal, and the Neches River. The vegetation near the
water was dense with trees and undergrowth. Truck and train traffic at the
plant created a substantial dust problem. The site contained stray dogs and
cats as well as a variety of wildlife, including snakes and alligators.
Although trapped heavily, few rodents were collected from near the tracks
and adjacent swamps. All rodents trapped at this facility were negative for
hantavirus antibodies.
Although the patient lived in an older pier-and-beam home, he and his wife
saw no evidence of rodent infestation. A Texas Department of Health
investigator verified the absence of rodents and rodent excreta. Although
traps were set, no rodents were collected at the residence.
During the 2 months before he became ill, the patient visited a casino boat
in Louisiana and fished three times from the Pleasure Island jetty in
southern Jefferson County. The jetty had large rock boulders at the water's
edge and an asphalt road along its length. Sand and dense 6-foot high
grasses covered the remainder of the jetty. Household trash and fishing
debris were scattered throughout the area. Most rodents collected by the
Texas Department of Health investigation team during this investigation
were from this jetty; and most were _O. palustris_, including three
seropositive specimens (Area 1, Figure 2).
 [Fig]
 Figure 2. Location of rodents trapped during investigations related to patient P/Tx
 (March 1996; [6]) and patient T/Tx (October 1996; this report). The locations in which
 rodents were collected are broadly classified into four areas, designated 1 through 4.
Other sites were chosen between the patient's work sites and home because
their habitats were likely to support rodents. Rice rat habitats (permanent
water and grasses) were among those targeted, because the first Jefferson
County hantavirus investigation documented the Bayou strain of hantavirus
in _O. palustris_ (6). The Table and Figure 2 show the location and species
of each rodent collected and the number of seropositive specimens in each
of four targeted areas. Four hantavirus antibody-positive rodents were
trapped at these miscellaneous locations during the investigation of the
current HPS case. Two of the seropositive samples were from Area 1 of
Figure 2 (not the jetty), and two were in Area 2. Area 2 yielded two
seropositive rodents during the first Texas BAY virus case investigation
(6).
Table. Rodents collected in association with two cases of Bayou
virus-hantavirus pulmonary syndrome in Jefferson and Orange Counties,
Texas, March and October 1996
---------------------------------------------------------------------------
                                   No. specimens collected
                                      (no. seropositive)
                             ----------------------------------- No. PCR (sup b)
                             Area   Area  Area    Area  Total      tested
Species                      1(sup a) 2     3       4              (no. pos.)
---------------------------------------------------------------------------
_Baiomys taylori_            0      0     1(0)    3 (0) 4 (0)        0
_Mus musculus_               30 (0) 21 (0)12 (1)  26 (0)88 (1)     1 (0)
_Ochrotomys nuttali_         0      0     0       1 (0) 1 (0)        0
_Oryzomys palustris_         45 (7) 20 (5)3 (0)   8 (3) 76 (15)   15 (14)
_Peromyscus leucopus_        0      3 (0) 0       0     3 (0)        0
_Rattus norvegicus_          1 (0)  1 (0) 0       1 (0) 3 (0)        0
_Rattus rattus_              21 (0) 8 (0) 0       1 (0) 30 (0)       0
_Reithrodontomys fulvescens
_                            0      7 (0) 0       7 (0) 14 (0)       0
_Reithrodontomys humulis_    1(0)   0     0       2 (0) 3 (0)        0
_Sigmodon hispidus_          95 (0) 5 (0) 11 (0)  4 (1) 114 (1)    1 (1)
---------------------------------------------------------------------------
(sup a) Refer to Figure 2 for map areas.
(sup b) PCR=polymerase chain reaction.
---------------------------------------------------------------------------
Diagnostic Studies
The IgG ELISA assay for SN virus antibodies in patient T/Tx's serum was
negative, but the IgM test was positive at a 1:6400 dilution (8). The strip
immunoblot assay show
ed antibody reactivities typically seen with HPS
caused by a hantavirus other than SN virus, with 2+ reactivity to the
immunodominant SN virus N peptide and 4+ reactivity to the full-length
recombinant N protein (11). There was no reactivity to the SN virus G1
antigen, either in peptide or recombinant form, or to Seoul virus
recombinant N protein (data not shown). Antibody reactivity to the SN virus
G1 antigen is specific for infection with SN virus.
Western blot analysis of patient T/Tx's serum showed IgG antibodies to
hantavirus N proteins (Figure 3, Panel A). Although the most intense
staining was observed with the homologous (BAY virus) antigen, varying
cross-reactivity was present against the N antigens of Muleshoe, SN, Rio
Mamor, and Puumala viruses but not of Seoul virus. A similar pattern was
observed when another blot membrane was probed with an anti-human IgM
conjugate, except that reactivities were somewhat more intense (data not
shown). Serum samples of seropositive rice rats and deer mice showed
similar cross-reactivities, although the intensity of reactivity to a
particular N antigen was related to the sequence similarity between the
membrane-bound antigen and the virus against which the antibodies were
directed (Figure 3, Panels B and C).
 [Fig]
 Figure 3. Western blot assay for detecting IgG antibodies in patient and
 rodent blood samples. A 1:500 dilution of serum was used to probe Western
 blots containing equimolar amounts of recombinant-expressed N antigens of
 various hantaviruses: 1) Bayou; 2) Muleshoe; 3) Puumala; 4) Rio Mamor;
 5) Seoul; and 6) Sin Nombre. Serum samples are directed against specific
 hantaviruses as verified by reverse transcription-PCR and sequence
 analyses: (A) patient T/Tx, Bayou virus; (B) Bayou virus-seropositive
 _Oryzomys palustris_ specimen #505, collected in Jefferson County, Texas;
 (C) a Sin Nombre-seropositive deer mouse, collected in California; (D), a
 negative control (hantavirus-seronegative) deer mouse. Antibodies bound
 to the solid-phase antigens were detected with alkaline
 phosphatase-conjugated anti-human IgG (A) or with anti-_Peromyscus
 leucopus_ IgG (B-D). The arrow indicates the migration of the full-length
 T7-viral N antigen, ~55kDa.
RT-PCR analyses and sequencing of the cDNA product of the patient's blood
sample identified definitively BAY virus as the etiologic agent. When the
442 nt viral S segment amplification product from patient T/Tx was compared
with the homologous sequence of other hantaviruses, it was closely similar
to previously described BAY viruses from Louisiana and Texas (Figure 4).
Nine viral sequences from seropositive _O. palustris_ and one from a
seropositive _S. hispidus_ from Jefferson County, Texas, and Cameron and
Terrebonne Parishes, Louisiana, were compared with those of patients T/Tx
and P/Tx and with that of a 1993 case-patient from northern Louisiana
(Case-LA-93, Figure 4).
 [Fig]
 Figure 4. Unweighted maximum parsimony tree produced by PAUP 3.1 software
 comparing a 397 nt portion of the hantavirus S genomic segments (residues
 207 to 603) of patients and rodents infected with Bayou virus. A
 selection of other prototypical hantavirus sequences, each of which was
 compared antigenically in Figure 2, was included for comparison. Other
 hantaviruses are abbreviated as follows: RM=Rio Mamor; MULE=Muleshoe;
 SN= Sin Nombre; PUU=Puumala; and SEO=Seoul. Human-derived Bayou virus
 sequences are indicated by Case, and rodent sequences by OP (_Oryzomys
 palustris_) or SH (_Sigmodon hispidus_). TX=Texas, LA=Louisiana.
The sequences of all the Jefferson County rodents and patient T/Tx were
closely related to each other, differing by 1 to 10 nt (0.25% to 2.5% of
397 evaluable residues) in pairwise comparisons. Four rodent-derived viral
sequences differed from that of the patient T/Tx virus by 4 nt (1%), and
all were from rodents collected in Jefferson County.
Except for a brief visit to a Louisiana casino, patient T/Tx reported that
he had not traveled outside east Texas in the 2 months before his illness.
The generally close genetic similarity among the viral sequences from east
Texas supports the hypothesis that patient T/Tx became infected in that
region (14). Potential exposure sites, as defined by his travel history,
were too numerous to allow us to identify a more precise site of infection.
No rodent sequence was completely identical to that of patient T/Tx.
The sequence obtained from patient P/Tx (6), who was thought to have been
infected in Jefferson County, was not closely aligned with any eastern
Texas rodent sequence or with that of patient T/Tx. The closest of the 11
eastern Texas viral sequences differed from that of patient P/Tx at 13
residues (3.3%). One sequence available from previous studies was far
closer to that of patient P/Tx, differing at only four residues (1%). This
sequence was obtained from a rice rat (OP-LA-5) collected in Cameron
Parish, Louisiana, approximately 30 km from the former residence of patient
P/Tx. Although patient P/Tx initially reported no travel back to his former
residence (where his mother still lived) in the 6 weeks before his illness
(6), he later was not certain about his travel history in the weeks before
his illness, stating that he might have visited his mother in Louisiana in
the weeks before his illness.
In previous studies, _O. palustris_ was tentatively implicated as the
predominant rodent reservoir for BAY virus, but the sample sizes were
small, and specific cases of HPS were not linked to the occurrence of BAY
virus-infected rodents at the presumed sites of infection (6,7). For this
study, analysis of a substantial collection of rodents collected in the
vicinity of sites of human infection showed that _O. palustris_ is the
species with the highest hantavirus seroprevalence. Furthermore, the
hantavirus genotypes in circulation in the eastern Texas/western Louisiana
area were related to those of two human case-patients from the area, and
all but one of those rodent-derived sequences came from _O. palustris_.
Rice rats are found in wetlands and marshes from Texas throughout the
southeastern United States, extending north as far as New Jersey on the
eastern seaboard. Like all carriers of viruses associated with HPS, _O.
palustris_ is a member of the subfamily Sigmodontinae, family Muridae.
Although only a single species exists in the United States, the tribe
Oryzomyini has scores of species in Latin America, where HPS is
increasingly recognized as an important zoonotic disease. Viruses similar
to BAY virus have been recognized recently in members of the Oryzomyini
from Bolivia and Argentina, and in some cases these rodent-borne viruses
have been linked to HPS in humans (1,15).
The possibility that the genetically distinct clade of viruses associated
with oryzomine and _Sigmodon_ rodents produces a disease that is
qualitatively different from SN virus-associated HPS (16) has been raised
repeatedly as new cases have been identified (5,6,17). Although patient
T/Tx had a relatively mild course of HPS, his serum creatinine, urine
protein concentration, and creatine kinase were nevertheless slightly
elevated. Both the elevation of creatine kinase and chemical evidence of
myositis have been observed in HPS caused by viruses of the oryzomine and
_Sigmodon_ clade, but much less commonly in SN virus infection (1). Further
studies are needed to determine the basis for the variant clinical symptoms
of HPS in these patients.
Acknowledgments
We thank G. Haigh, McNeese State College, for donating the rice rat
specimen OP-LA-5; J. Griffith for technical assistance with sequencing; and
W. George, St. Elizabeth Hospital, for first recognizing HPS as a possible
cause of patient T/Tx's illness. This study was supported by DHHS grant RO1
AI36336 (to B.H.).
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Dispatches
Laboratory Survey of Drug-Resistant _Streptococcus pneumoniae_ in New York
City, 1993-1995
Richard Heffernan, Kelly Henning, Anne Labowitz, Annette Hjelte, and
Marcelle Layton
New York City Department of Health, New York, New York, USA
---------------------------------------------------------------------------
      Wide geographic variation in the prevalence of drug-resistant
      _Streptococcus pneumoniae_ demonstrates the importance of
      tracking antimicrobial resistance locally. This survey of
      hospital microbiology laboratories in New York City found that
      penicillin resistance (MIC >/= 2.0 mg/ml) increased from 1.5% of
      _S. pneumoniae_ isolates in 1993 to 6.3% in 1995 and that in
      1995, one-third of isolates nonsusceptible to penicillin (MIC >/=
      0.1 g/ml) were also nonsusceptible to an extended-spectrum
      cephalosporin (MIC >/= 1 g/ml).
The emergence of drug-resistant _Streptococcus pneumoniae_ underscores the
need for timely, local, population-based surveillance of antimicrobial
resistance. The prevalence of resistance in U.S. communities varies widely,
with 2% to 53% of _S. pneumoniae_ isolates found to have reduced
susceptibility to penicillin (1-4). The Centers for Disease Control and
Prevention recommends that empiric antibiotic therapy for pneumococcal
infections be based upon local susceptibility patterns (2,5). However, few
communities track drug-resistant _S. pneumoniae_.
The Survey
To estimate the prevalence of drug-resistant _S. pneumoniae_ in New York
City, we surveyed hospital-based clinical microbiology laboratories from
1993 to 1995. A standardized questionnaire was mailed annually to each
laboratory, and those that did not respond were contacted by telephone or
were visited. To evaluate compliance with _S. pneumoniae_ penicillin
susceptibility testing guidelines established by the National Committee for
Clinical Laboratory Standards (NCCLS) (6), we asked about criteria for
selecting specimens and techniques for oxacillin disk diffusion screening
and determination of penicillin MICs.
To determine the prevalence of penicillin resistance, we asked for the
number of _S. pneumoniae_ isolates identified during the year, the number
tested for susceptibility to penicillin, and the number found to be
possibly resistant by the oxacillin disk diffusion test and
penicillin-intermediate or -resistant by MIC testing. We also asked that
information be provided separately for isolates from normally sterile sites
(e.g., blood, cerebrospinal fluid) and from nonsterile sites (e.g., sputum,
nasopharyngeal swab). In 1995, we added questions regarding the MIC test
results for extended-spectrum cephalosporins (ESCs), including the number
of penicillin-nonsusceptible isolates that were also nonsusceptible to an
ESC. No individual patient information was obtained. A report summarizing
the results of the survey and describing NCCLS guidelines was mailed
annually to microbiology laboratories, hospital infection control
departments, and local infectious disease physicians and pediatricians.
Analysis
Of 67 hospital-based clinical microbiology laboratories in New York City,
100% completed the survey in 1993, 98% in 1994, and 100% in 1995. Overall,
more than 5,000 _S. pneumoniae_ isolates were reported annually.
Data were analyzed by using EpiInfo Version 6.0 (CDC, Atlanta, GA, USA).
Drug-susceptibility results are presented for laboratories that conformed
with NCCLS guidelines and provided complete data on all _S. pneumoniae_
isolates identified (Table 1).
Table 1. Penicillin resistance among _Streptococcus pneumoniae_ isolates at
New York City hospital laboratories
---------------------------------------------------------------------------
No. of isolates        1993          1994            1995
                       No. (%)       No. (%)         No. (%)
------------------------------------------------------------------
Screened with
oxacillin disk (sup    3,227         4,133           4,912
a)
zone size </= 19 mm
(sup b)                273 (9)       549 (13)        995 (20)
Screened and
confirmed with         1,229         2,491           3,535
approved MIC (sup c)
zone size </=19 mm     154 (13)      350 (14)        704 (20)
I (sup d)              70 (6)        209 (8)         310 (9)
R (sup d)              19 (2)        115 (5)         222 (6)
Sterile-site isolates
screened and
confirmed with MIC     462           649             1,321
(sup e)
I                      16 (3)        66 (10)         83 (6)
R                      5 (1)         20 (3)          65 (5)
Nonsterile isolates
screened and
confirmed with MIC     456           662             1,596
(sup f)
I                      12 (3)        83 (13)         200 (13)
R                      5 (1)         31 (5)          110 (7)
------------------------------------------------------------------
(sup a)Number of laboratories reporting oxacillin disk diffusion
test results in 1993, 1994, 1995 was 33, 40, 51, respectively.
(sup b)Oxacillin disk diffusion test zone size.
(sup c)Approved penicillin MIC tests include antibiotic gradient
strips and broth dilution or micro-dilution using Mueller-Hinton
broth supplemented with 2-5% lysed horse blood. Number of
laboratories reporting MIC test results in 1993, 1994, 1995 was
10, 22, 35, respectively.
(sup d)I=Penicillin-intermediate (MIC >/= 0.1 and </= 1.0 g/ml);
R=Penicillin-resistant (MIC >/= 2.0 g/ml).
(sup e)Number of laboratories reporting sterile site isolate
results in 1993, 1994, 1995 was 8, 12, 32, respectively.
(sup f)Number of laboratories reporting nonsterile site isolate
results in 1993, 1994, 1995 was 5, 9, 28, respectively.
---------------------------------------------------------------------------
Susceptibility Criteria
The NCCLS recommends routine screening by the oxacillin disk diffusion test
of clinically important _S. pneumoniae_ isolates for susceptibility to
penicillin. Isolates with a zone size </= 19 mm, or any isolate from the
blood or cerebrospinal fluid, should be tested with an approved MIC method
such as broth dilution or antibiotic gradient strips (e.g., E-test).
Isolates whose penicillin MICs are either intermediate (MIC >/= 0.1 and </= 1
g/ml) or resistant (MIC >/= 2 g/ml) should also have MICs determined for
susceptibility to an ESC such as cefotaxime or ceftriaxone
(ESC-intermediate MIC = 1 g/ml; ESC-resistant MIC >/= 2 g/ml) (6). We will
use the term "nonsusceptible" to refer to both intermediate and resistant
isolates.
Findings
The proportion of laboratories conforming with NCCLS guidelines for
penicillin susceptibility testing of _S. pneumoniae_ increased from 22% in
1993 to 69% in 1995. This was due to an increase in the number of
laboratories that screened all isolates, a sharp decrease in the use of
automated MIC tests, and a fourfold rise in the use of antibiotic gradient
strips for determining MICs (Table 2). Overall, the proportion of isolates
with oxacillin disk diffusion test zone size </=19 mm, suggesting possible
resistance, increased from 8.5% of isolates in 1993 to 20.2% in 1995 (Table
1). MIC test results showed that 5.7% of isolates were
penicillin-intermediate and 1.5% penicillin-resistant in 1993, compared
with 8.8% and 6.3%, respectively, in 1995. The prevalence of resistant
organisms increased among isolates from both sterile and nonsterile sites
and was somewhat higher among nonsterile-site isolates than among
sterile-site isolates in 1995 (6.9% vs. 4.9%, Chi-square p = 0.03).
Seven of the laboratories followed NCCLS-recommended methods and provided
complete penicillin susceptibility results for both 1993 and 1995. These
seven reported that among 779 isolates in 1993, 25 (3.2%) were intermediate
and six (0.8%) were resistant to penicillin. Among 896 isolates in 1995, 83
(9.2%) were intermediate, and 47 (5.2%) were resistant.
Table 2. Penicillin susceptibility testing protocols for _Strepococcus
pneumoniae_ in New York City hospital laboratories
---------------------------------------------------------------------------
No. of laboratories   1993            1994               1995
(sup a)               No. (%)         No. (%)            No. (%)
---------------------------------------------------------------------------
Screened with
oxacillin disk        55 (82)         63 (97)            65 (97)
Screened all
isolates              38 (57)         56 (86)            59 (88)
Screened only
sterile               9 (13)          6 (9)              6 (9)
Other                 8 (12)          1 (2)              0
Performed penicillin
MIC test              39 (58)         51 (78)            56 (84)
MIC-tested
All isolates          10 (15)         9 (14)             7 (10)
Oxacillin-resistant
only                  18 (27)         39 (60)            42 (63)
Sterile isolates
only                  5 (7)           1 (2)              0 (0)
By request only       4 (6)           1 (2)              5 (7)
Other                 2 (3)           1 (2)              2 (3)
Penicillin MIC
technique (sup b)
Antibiotic gradient   11(16)          41 (63)            50 (75)
strip (sup c)
Automated tests (sup
c)                    21 (31)         9 (14)             2 (3)
Broth dilution        8 (12)          11 (17)            5 (7)
Unknown               2 (3)           3 (5)              0
Conformed with NCCLS
guidelines (sup d)    15 (22)         38 (58)            46 (69)
---------------------------------------------------------------------------
(sup a)Total number of laboratories responding to survey in 1993, 1994,
1995 was 67, 65, 67, respectively.
(sup b)13 laboratories used more than one MIC technique.
(sup c)Antibiotic gradient strip: e.g., E-test. Automated tests: e.g.,
Microscan, Vitek.
(sup d)NCCLS=National Committee for Clinical Laboratory Standards.
Screened all _S. pneumoniae_ isolates with oxacillin disk diffusion test
and confirmed resistance with approved penicillin MIC test.
---------------------------------------------------------------------------
The prevalence of penicillin resistance varied between laboratories and by
geographic area. At 35 laboratories in 1995, 0% to 80% (median 13.1%) of
isolates were nonsusceptible to penicillin. Laboratories in the New York
City borough of Manhattan reported that 18% of isolates were nonsusceptible
to penicillin, compared with 16% in Queens, 14% in the Bronx, and 10% in
Brooklyn (MIC data from Staten Island were not available).
In 1995, 18 laboratories reported that of 275 _S. pneumoniae_ isolates
nonsusceptible to penicillin, 90 (33%) were also nonsusceptible to an ESC.
Limitations
The survey design had several limitations. First, levels of expertise
varied among laboratories regarding antibiotic susceptibility testing of
pneumococci. Laboratory variation may partly account for the wide range in
the proportion of isolates nonsusceptible to penicillin observed at
individual laboratories, which could in turn influence citywide estimates.
We did not collect and test isolates to confirm laboratory results. Second,
the number of laboratories included in our results increased each year as
more laboratories adopted NCCLS methods and provided complete data. This
improved the accuracy of the survey but made interpreting trends difficult.
Third, citywide estimates inevitably mask important differences in the risk
for drug-resistant _S. pneumoniae_ infections in specific subpopulations;
for example, day-care attendance and prior antibiotic use have been
associated with drug-resistant pneumococcal infections in children (1,7).
Finally, since information was collected on isolates, rather than
individual patients or infections, actual disease incidence could not be
calculated.
Conclusions and Recommendations
Our results document a marked improvement in penicillin susceptibility
testing protocols for _S. pneumoniae_ during this period. Because of an
increase in the proportion of isolates tested and widespread adoption of
antibiotic gradient strips for MIC testing, more than two-thirds of
laboratories conformed with NCCLS guidelines in 1995, compared with fewer
than one-fourth in 1993.
The survey demonstrates that drug-resistant pneumococci are prevalent and
may be increasing in New York City. Penicillin resistance increased
fourfold between 1993 and 1995, and data from 1996 indicate that it has
remained at this level or continued to increase. Nine percent of blood
isolates in 1996 were intermediate and 6% resistant to penicillin (New York
City Department of Health, unpub. data), compared with 6% and 5%,
respectively, among sterile-site isolates in our survey for 1995. The
proportion of _S. pneumoniae_ isolates resistant to penicillin in New York
City was similar to U.S. national averages during this period (2-4). Our
finding that 33% of penicillin-nonsusceptible _S. pneumoniae_ isolates were
resistant to ESCs is also similar to the 29% (4) observed nationally.
On the basis of these results, we recommended that New York City clinicians
consider carefully the possibility of penicillin and ESC resistance when
treating suspected _S. pneumoniae_ infections. Although the response to
therapy may vary by route of administration, site of infection, and age and
immune status of the patient, treatment failure is increasingly common and
can have serious consequences (8,9). Educational efforts are needed to
promote appropriate use of antibiotics and encourage use of the 23-valent
pneumococcal vaccine in populations at increased risk for pneumococcal
disease (10).
This laboratory survey was a relatively low-cost method of estimating the
prevalence of drug-resistant pneumococci in New York City. It may serve as
a model in areas where clinical laboratories routinely perform
drug-susceptibility testing but the resources to collect or test isolates
centrally for surveillance are limited. Despite the survey's limitations,
the estimates provided may be sufficient to guide clinicians in selecting
appropriate empiric therapy for suspected pneumococcal infections.
Considerable effort was spent each year disseminating these data to
laboratories, hospital infection control departments, infectious disease
physicians, pediatricians, and other primary care physicians.
Our ability to expand this survey approach beyond _S. pneumoniae_,
penicillin, and ESCs is limited by constraints on hospital laboratory
staff. As laboratories computerize and standard formats are developed for
the electronic transmission of laboratory test results, collection and
dissemination of susceptibility data will be possible on a wider range of
pathogens and antimicrobial drugs.
Acknowledgments
The authors thank Alexandra Dow for her contributions to the 1993 survey
and laboratory staff throughout New York City, whose time and effort made
this project possible.
References
  1. Hoffman J, Cetron MS, Farley MM, Baugham WS, Facklam RR, Elliott JA,
     et al. The prevalence of drug-resistant _Streptococcus pneumoniae_ in
     Atlanta. N Engl J Med 1995;333:481-6.
  2. Breiman RF, Butler JC, Tenover FC, Elliott JA, Facklam RR. Emergence
     of drug-resistant pneumococcal infections in the United States. JAMA
     1994;271:1831-5.
  3. Doern GV, Brueggemann A, Holley HP, Rauch AM. Antimicrobial resistance
     of _Streptococcus pneumoniae_ recovered from outpatients in the United
     States during the winter months of 1994 to 1995: results of a
     30-center national surveillance study. Antimicrob Agents Chemother
     1996;40:1208-13.
  4. Butler JC, Hoffmann J, Cetron MS, Elliott JA, Facklam RR, Breiman RF,
     et al. The continued emergence of drug-resistant _Streptococcus
     pneumoniae_ in the United States: an update from the Centers for
     Disease Control and Prevention's pneumococcal sentinel surveillance
     system. J Infect Dis 1996:174:986-93.
  5. Cetron MS, Jernigan DB, Breiman RF, The DRSP Working Group. Action
     plan for drug-resistant _Streptococcus pneumoniae_. Emerg Infect Di
s
     1995;1:64-5.
  6. National Committee for Clinical Laboratory Standards. Methods for
     dilution antimicrobial susceptibility tests for bacteria that grow
     aerobically. Approved standard. M7-A4. Villanova (PA): The Committee;
     1997.
  7. Arnold KE, Leggiadro RJ, Breiman RF, Lipman HB, Schwartz B, Appleton
     MA, et al. Risk factors for carriage of drug-resistant _Streptococcus
     pneumoniae_ among children in Memphis, Tennessee. J Pediatr
     1996;128:757-64.
  8. Appelbaum PC. Epidemiology and in vitro susceptibility of
     drug-resistant _Streptococcus pneumoniae_. Pediatr Infect Dis J
     1996;15:932-9.
  9. Chesney PJ, Wilimas JA, Presbury G, Abbasi S, Leggiadro RJ, Davis Y,
     et al. Penicillin- and cephalosporin-resistant strains of
     _Streptococcus pneumoniae_ causing sepsis and meningitis in children
     with sickle cell disease. J Pediatr 1995;127:526-32.
 10. Munford RS, Murphy TB. Antimicrobial resistance in _Streptococcus
     pneumoniae_: can immunization prevent its spread? J Investig Med
     1994;42:613-21.
---------------------------------------------------------------------------
Dispatches
B-virus from Pet Macaque Monkeys: An Emerging Threat in the United States?
Stephanie R. Ostrowski,* Mira J. Leslie, Terri Parrott, Susan Abelt, and
Patrick E. Piercy
*Centers for Disease Control and Prevention, Atlanta, Georgia, USA;
Arizona Department of Health Services, Phoenix, Arizona, USA; Pembroke
Park Animal Hospital, Pembroke Park, Florida, USA; Lake Superior
Zoological Gardens, Duluth, Minnesota, USA; and Illinois Department of
Public Health, Springfield, Illinois, USA
---------------------------------------------------------------------------
      Of primary concern when evaluating macaque bites are bacterial
      and B-virus infections. B-virus infection is highly prevalent
      (80% to 90%) in adult macaques and may cause a potentially
      fatal meningoencephalitis in humans. We examined seven
      nonoccupational exposure incidents involving 24 persons and
      eight macaques. Six macaques were tested for herpes B; four
      (67%) were seropositive. A common observation was that children
      were more than three times as likely to be bitten than adults.
      The virus must be assumed to be a potential health hazard in
      macaque bite wounds; this risk makes macaques unsuitable as
      pets.
B-Virus in Nonhuman Primates
_Cercopithecine herpesvirus_ 1 (_Herpesvirus simiae_ or B-virus) frequently
infects Old World primates of the genus _Macaca_. Of at least 19 species of
macaques, rhesus, Japanese, cynomolgus, pig-tailed, and stump-tailed
macaques are the species most commonly used in biomedical research (1).
Seroprevalence of neutralizing antibodies to B-virus in captive adult
macaque populations is 73% to 100% (1-3). Like _Herpesvirus simplex_ virus
infection in humans, B-virus infection in monkeys is characterized by
lifelong infection with intermittent reactivation and shedding of the virus
in saliva or genital secretions, particularly during periods of stress or
immunosuppression (4). B-virus infection is transmitted among free-ranging
or group-housed animals, primarily through sexual activity and bites. In
captivity, as well as in the wild, mature macaques are more likely than
immature animals to have been infected with, and shed, the virus. Antibody
titer to B-virus indicates infection but can neither confirm nor eliminate
actual viral shedding at the time of the bite (4).
B-Virus in Humans
B-virus disease in humans usually results from macaque bites or scratches
(4). Incubation periods may be as short as 2 days, but more commonly are 2
to 5 weeks (1,3,5-7; Centers for Disease Control and Prevention [CDC],
unpub. data). Most documented infections have occurred among biomedical
research employees who had occupational exposure to macaques, although
transmission has also been documented among laboratory workers handling
infected central nervous system and kidney tissues (1,5).
From 1990 to 1992, 28 U.S. residents reported nonoccupational macaque bites
to CDC (L. Chapman, pers. comm.). Since 1993, additional nonoccupational
exposure cases have been reported, seven of which (involving 24 persons and
eight macaques) are listed in Table 1. Of the six macaques for which herpes
B serologic results were available, four (67%) were positive. Two owners
refused requests for testing. Four (44%) of nine exposed children were
bitten, versus only three (20%) of 12 adults. Children were 3.2 times more
likely to be bitten than adults; although a common observation, this
association is not statistically significant for this case series.
Table 1. Selected pet macaque bite cases (sup a,b)
---------------------------------------------------------------------------
Location   Primate species, age,     Nature of exposure Comments
           B-virus status
---------------------------------------------------------------------------
Illinois   Rhesus, 20+ yrs, B-virus  Household contact  Bought at auction,
           positive Cynomolgus, 2-4  (2 adults/3        wife bitten
           yrs, B-virus negative     children), bites,  multiple sites,
                                     scratches (2       children hand-fed
                                     adults)            monkey
Florida    Cynomolgus, 2 yrs,        Household contact  Kissed on lips, ate
           B-virus positive          (1 adult), bite (1 off owner's  plate,
                                     child)             shared bed
Arizona    Cynomolgus, 2 yrs,        Bites on toe and   Unprovoked attack
           B-virus negative          buttock (child)    on neighbor,
                                                        declared vicious
                                                        animal by   judge,
                                                        no. of household
                                                        contacts (owner)
                                                        unreported
           Cynomolgus, 7 weeks,      Household contact  Diapered, shared
           B-virus positive          (6 adults), bite   chewed gum, oral
                                     on face (1 adult)  ulcers noted by
                                                        veterinarian, bite
                                                        incident  at
                                                        neighborhood bar
           Macaque, (species         Bite on thigh (1   Unprovoked attack
           undetermined), 2 yrs,     child)             (climbed fence to
           B-virus status unknown                       bite child)
           Macaque, (species and age Severe bite (1     Injured child
           undetermined), B-virus    child)             attended an
           status unknown                               unlicensed day-care
                                                        facility run by
                                                        monkey owner, 7
                                                        other monkeys on
                                                        premises
Minnesota  Rhesus, 2 yrs, B-virus    Household          Acquired as
           positive                  contact,  owners'  "child-substitute"
                                     friend bitten      (full-time
                                                        baby-sitters hired)
---------------------------------------------------------------------------
(sup a)Cases referred to Centers for Disease Control and Prevention since
1993.
(sup b)As of November 1997, no confirmed transmission of B-virus in these
persons has been documented.
---------------------------------------------------------------------------
Most free-ranging monkey populations are thought to be part of the exotic
fauna of distant tourist destinations and wild animal parks; however,
macaque species have established free-ranging feral populations in Texas
and Florida. In such settings, contact between humans and macaques cannot
be safely controlled (8-10), and workers and visitors are at risk.
Guidelines for B-virus prevention and diagnosis have recently been
published (9-12).
Symptomatic human infection with B-virus is rare; fewer than 40 cases were
reported from 1933 to 1994 (1,4-7,13-15; CDC, unpub. data). However, the
consequences of symptomatic infection may be severe. Viral infection
rapidly progresses to central loci in the spinal cord and, eventually, the
brain. Of 24 known symptomatic patients whose cases were reviewed in 1992,
19 (79%) died (CDC; unpub. data).
Before 1987, most surviving human patients had moderate to severe
neurologic impairment, sometimes requiring lifelong institutionalization
(1). Recently, acyclovir has prevented progression of the disease in a
limited number of patients. In at least three patients, this treatment
reversed the neurologic symptoms and was life-saving (7,14,15). Rapid
diagnosis and initiation of therapy are of paramount importance in
preventing death or permanent disability in surviving patients.
Human and Macaque Interactions
Most owners form an emotional bond with infant primates. This bond is
probably strengthened by the neonatal monkey's physical and behavioral
resemblance to a human infant. Although physically and emotionally
dependent on their mothers (or human substitutes) for up to 2 years of age,
most macaques exhibit unpredictable behavior as they mature. Males tend to
become aggressive, and both male and female macaques bite to defend
themselves and to establish dominance. Dominance within the social
hierarchy of macaques is established by aggression toward other monkeys,
generally the younger and smaller members of the group. Both veterinary
specialists and breeders of nonhuman primates agree that as a rule, all
these animals bite (16,17). Biting incidents eventually bring the animals
to the attention of animal control authorities. Most state health
departments can require that any biting nondomestic animal be euthanized
and the brain be submitted for rabies testing.
Regulations, Guidelines, and Policies Regarding Nonhuman Primates
Table 2 lists the principal federal regulations affecting the possession,
distribution, and uses of nonhuman primates. The United States is obligated
under the Convention in International Trade in Endangered Species (CITES)
to restrict and control trafficking in exotic and endangered species.
Table 2. Federal regulations regarding nonhuman primates
---------------------------------------------------------------------------
Agency             Statute           Regulation         Subjects
---------------------------------------------------------------------------
Departments of     Public Health     42 CFR 71.53       Importation,
Health, Centers    Services Act, 42                     distribution, bona
for Disease        USCS 201                             fide uses in the
Control and                                             U.S., breeding
Prevention                                              colony
                                                        requirements,
                                                        pet/avocationist
                                                        uses
Department of      Animal Welfare    9 CFR Subchapter A Licenses
Agriculture        Act, 7 USCS                          (breeders,
                   2131-2159                            dealers,
                                                        laboratories,
                                                        exhibitors,
                                                        auctions),
                                                        interstate health
                                                        certificates,
                                                        humane care and
                                                        transport
Department of the  Endangered        50 CFR 10, 11, 13, Endangered
Interior, U.S.     Species Act, 16   14, 16             species,
Fish  and Wildlife USCS 1540 Lacey                      smuggling,
Service            Act, 18 USCS 42                      interstate sales
---------------------------------------------------------------------------
Since October 10, 1975, U.S. Public Health regulation 42 CFR 71.53(c) has
prohibited the importation of nonhuman primates into the United States as
pets, and neither nonhuman primates imported since that date nor their
offspring may be legally bred or distributed for any uses other than bona
fide science, university-level educational programs, or full-time zoologic
exhibition. Furthermore, the regulation states, "the maintenance of
nonhuman primates as pets, hobby, or an avocation with occasional display
to others is not a permissible use" (18).
All states require their citizens to comply with applicable federal
regulations. Many state officials, however, may be unaware of regulatory
restrictions on the uses and distribution of nonhuman primates and may be
confused by the distinctions among federal agencies regarding regulatory
restrictions on captive-bred animals. State wildlife authorities may not
know that a federal public health regulation prohibits the keeping
("maintenance") of nonhuman primates imported after October 10, 1975, as
pets, for a hobby, or as an avocation; likewise, many do not know the
compelling public health and safety reasons for enforcement.
Captive-bred offspring of animals purported to have been imported before
October 10, 1975, are frequently offered for sale. Without documentation it
is very difficult to determine whether this is the case. Depending on the
specific circumstances, it is possible for undocumented animals to be
considered deliberately misclassified (i.e., intentionally mislabeled), a
violation under the Lacey Act (18 USC 42) and under 16 USC 3373 (19).
In 1987 and 1988, occupational safety guidelines were published based on
evidence that all macaque species are inherently dangerous to humans
because of the risk for B-virus transmission, as well as the likelihood of
serious physical injury from bite wounds (9-12,14,15). Several recent
reviews of monkey-bite injuries worldwide indicate that severe lacerations,
wound infections, and permanent sequelae (e.g., flexure contractures,
osteomyelitis) were present in 33% of cases (20,21).
In 1990, the American Veterinary Medical Association issued a general
policy statement opposing the keeping of wild animals (especially those
inherently dangerous to humans) as pets and advising veterinarians to exert
their influence to discourage this practice (22). In 1995, updated
guidelines for the prevention and treatment of B-virus infections in
exposed persons were published (12). Despite these continuing public health
educational efforts, nonhuman primates (including macaques) continue to be
marketed and kept as pets in many states (16,17,23).
The Frequency of Exposure Resulting in Infection
Much remains to be learned about the pathogenesis of B-virus infection in
humans. In this very limited case series (Table 1), one family (two adults
and two of three children) exposed to a B-virus positive macaque had
flulike symptoms. One of the adults had additional symptoms related to the
injury site, which suggested B-virus infection. In the other six cases, no
suspect clinical symptoms were noted, and disease-specific antiviral
postexposure prophylaxis was not given. B-virus is still rare, and
diagnostic evaluation of clinical cases of aseptic meningitis does not
routinely include B-virus testing.
Owners of pet macaques are often reluctant to report bite injuries from
their pets, even to their medical care providers, and may fail to
appreciate that the premonitory headache and flulike symptoms (which may
lead them to seek medical attention) could be associated with healed, often
minor, bite wounds dating back more than a month (23). The Southwest
Foundation for Biomedical Research, which is the designated National
Institutes of Health B-virus resource laboratory, reports processing 2,000
to 3,000 human diagnostic specimens per year between 1990 and 1994, or
approximately 200 per month, most of which reflect occupational exposure
(8).
Some Public Health Consequences of the Nonhuman Primate Pet Trade
The pet trade in a variety of nonhuman primate species, and particularly
the apparent increase in macaque species as part of this trade, may
constitute an emerging infectious disease threat in the United States.
Although the U.S. Fish and Wildlife Service indicates that illegal traffic
in nonhuman primates is a significant aspect of the estimated $3 billion
worth of wildlife illegally traded in the United States annually, more data
are needed on the actual number of macaques in the private sector and on
trends in the population (24; U.S. Fish and Wildlife special agents, pers.
comm.).
The public resources deployed when a monkey-bite case is referred to public
health authorities are similar to those required for rabies investigations
(M. Lesl
ie and T. Parrott, unpub. obs.). Persons bitten by pet and feral
macaques are more likely than persons bitten in the workplace to require
public resources, delay seeking medical care, and have an initial medical
evaluation by care givers who are largely unfamiliar with the potentially
serious consequences of B-virus exposure (23). In contrast, occupational
exposure generally occurs within highly structured workplace settings,
where health professionals are prepared to provide prompt, appropriate, and
specific care at no public cost.
Ongoing efforts to establish B-virus-free macaque colonies illustrate the
difficulties of ascertaining B-virus-negative status, even with a battery
of sophisticated laboratory tests and extended longitudinal follow-up of
individual macaques (25). The high percentage of death in known cases of
human B-virus disease underscores the potential seriousness of all bite or
scratch exposures from macaques.
The extremely high prevalence of B-virus along with their behavioral
characteristics make the macaque species unsuitable as pets.
Acknowledgments
The authors thank Drs. D. Manning, S. O' Marro, R. Montrey, T. Burke, D.
Morton, and J. Thulin; Mr. J. Francis, Mr. C. Langkop; Drs. R. Martin, J.
Hilliard, D. Watkins; Capt. J. Thompson; Special Agents P. Bosco, D.
Burleson, J. English, S. Hamilton, T. Karabinoff, D. Kirkby, D. Manera, G.
Phillips, G. Phocas, T. Santelle, J. Sommers, C. Tabor; Dr. J. Cheek; and
the Division of Viral and Rickettsial Diseases, Animal Resources Branch,
and the Division of Quarantine, CDC.
References
  1. Palmer AE. B virus, _Herpesvirus simiae_: historical perspective. J Med
     Primatol 1987;16:99-130.
  2. Shah KV, Morrison JA. Comparison of three rhesus groups for antibody
     patterns to some viruses: absence of active simian virus 40
     transmission in the free-ranging rhesus of Cayo Santiago. Am J
     Epidemiol 1972;99:308-15.
  3. Orcutt RP, Pucak GJ, Foster HL, Kilcourse JY. Multiple testing for the
     detection of B virus antibody in specially handled rhesus monkeys
     after capture from virgin trapping grounds. Lab Anim Sci 1976;26:70-4.
  4. Weigler BJ. Biology of B Virus in macaque and human hosts: review.
     Clin Infect Dis 1992;14:555-67.
  5. Hummeler K, Davidson WL, Henle W, LaBoccetta AC, Ruch HG.
     Encephalomyelitis due to infection with _Herpesvirus simiae_ (Herpes B
     virus): report of two fatal laboratory acquired cases. N Engl J Med
     1959;261:64-8.
  6. Centers for Disease Control. B-virus infections in humans-Michigan.
     MMWR Morb Mortal Wkly Rep 1989;38:453-4.
  7. Holmes GP, Hilliard JK, Klontz KC, Rupert AH, Schindler CM, Parrish E,
     et al. B-virus (_Herpesvirus simiae_) infection in humans: epidemiologic
     investigations of a cluster. Ann Intern Med 1990;112:833-9.
  8. Centers for Disease Control. Guidelines for prevention of _Herpesvirus
     simiae_ (B-virus) infection in monkey handlers. MMWR Morb Mortal Wkly
     Rep 1987;36:681-2, 687-9.
  9. The B Virus working group. Guidelines from prevention of _Herpesvirus
     simiae_ (B virus) infection in monkey handlers. J Med Primatol
     1988;17:77-83.
 10. Hilliard JK. 1990-1994 yearly comparisons; B-virus Resource
     Laboratory. San Antonio (TX): Southwest Foundation for Biomedical
     Research; 1995.
 11. Wells DL, Lipper SL, Hilliard JK, Stewart JA, Holmes GP, Herrmann KL,
     et al. _Herpesvirus simiae_ contamination of primary rhesus monkey
     kidney cell cultures. CDC recommendations to minimize risks to
     laboratory personnel. Diagn Microbiol Infect Dis 1989;12:333-6.
 12. Holmes GP, Chapman LE, Stewart JA, Straus SE, Hilliard JK, Davenport
     DS, et al. Guidelines for the prevention and treatment of B-virus
     infections in exposed persons. Clin Infect Dis 1995;20:421-39.
 13. Artenstein AW, Hicks CB, Goodwin BS, Hilliard JK. Human infection with
     B virus following a needlestick injury. Reviews of Infectious Diseases
     1991;13:288-91.
 14. Centers for Disease Control. B-virus infection in humans-Pensacola,
     Florida. MMWR Morb Mortal Wkly Rep 1987;36:289-90, 295-6.
 15. Centers for Disease Control. B-virus infection in humans-Michigan.
     MMWR Morb Mortal Wkly Rep 1989;38:453-4.
 16. Hoctor P. Short hints on raising exotics. Animal Finders Guide 1995
     Sep 1;12:44-5.
 17. Johnson-Delaney CA. The pet monkey: health care and husbandry
     guidelines. Journal of Small Exotic Animal Medicine 1991;1:32-7.
 18. Centers for Disease Control. Import restrictions on nonhuman primates.
     Atlanta (GA): Centers for Disease Control; 1982 Nov 17. DQ-CPS
     Advisory Memorandum No. 65.
 19. Anderson RS. Lacey Act. Case law update and current issues in federal
     wildlife prosecution. Washington: U.S. Department of Justice,
     Environment and Natural Resources Division; 1993.
 20. Goldstein EJC, Pryor EP, Citron DM. Simian bites and bacterial
     infection. Clin Infect Dis 1995;20:1551-2.
 21. Janda DH, Ringler DH, Hilliard JK, Hankin RC, Hankin FM. Nonhuman
     primate bites. J Orthop Res 1990;8:146-50.
 22. American Veterinary Medical Association. Policy Statement on Wild
     Animals as Pets. In: 1995 AVMA Directory. Schaumburg (IL): The
     Association; 1995.
 23. Paulette J. "Yes, monkeys will bite!" The Simian 1996 Feb;6-8.
 24. Webster D. The looting and smuggling and fencing and hoarding of
     impossibly precious, feathered and scaly wild things: inside the $10
     billion black market in endangered animals. The New York Times
     Magazine 1997 Feb 16: 27-33, 48-50.
 25. Hilliard J. Managing macaques and herpes B. Presented at 4th National
     Symposium on Biosafety: Working Safely with Research Animals; 1997 Jan
     27-31; Atlanta, Georgia.
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[Emerging Infectious Diseases * Volume 4 * Number 1 * January - March 1998]
Letters
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Infectious Diseases and Mental Illness: Is There a Link?
To the Editor: The report by Hatalski et al. (1) on Borna virus as a
probable human pathogen provides yet another example of an infectious agent
being tentatively associated with neuropsychiatric disorders. Earlier this
year, researchers at Rockefeller University and the National Institute of
Mental Health suggested that after streptococcal infection, some children
may be at increased risk for obsessive-compulsive disorders and Tourette
syndrome (2). The human B-cell antigen D8/17, believed to be a marker for
increased susceptibility to poststreptococcal rheumatic heart disease, has
been tentatively linked to this increased risk for psychiatric illness in
children. Other reports of patients with complicated Lyme borreliosis,
including some whose infections have progressed to encephalopathies,
describe persistent verbal and memory deficits among these patients (3). In
a few Lyme disease patients, the only overt symptoms of disease at the time
of initial diagnosis and treatment were classified as mental confusion (4).
Two newly emergent infectious diseases in the United States, leptospirosis
and neurocysticercosis, have been found among inner city residents and poor
immigrants, respectively. Occasionally leptospirosis has been associated
with a variety of postinfectious psychiatric symptoms, including
depression, dementia, and psychosis (5). Neurocysticercosis, a tropical
parasitic infection, is increasingly associated with emergency room
admissions for seizures and epilepsy (6). Still other infectious diseases
are being examined for links with cognitive symptoms and emotional
disorders.
The primary cause of many common psychiatric disorders, including
depression, manic depression, anxiety, and schizophrenia, remains a
mystery. The World Health Organization estimates that 1.5 billion people
worldwide suffer from a neuropsychiatric disorder. Of the 10 leading causes
of disability in 1990, four were psychiatric disorders: unipolar
depression, manic depression, schizophrenia, and obsessive-compulsive
disorders (7). The National Institute of Mental Health recently estimated
that as many as 20% of young Americans ages 7 to 14 approximately 10
million children have mental health problems severe enough to compromise
their ability to function (8). Infectious agents may play a role in some of
these diseases to some unknown degree. A better understanding of the role
of infection may speed treatment and prevention efforts and reduce the
degree of disability and stigma associated with mental illness.
Vaccines and antimicrobial agents might enhance current therapeutic options
for mental illnesses. Even if infectious diseases were a primary factor in
only 1% of neuropsychiatric illnesses, some 10 million persons might
benefit from antimicrobial therapies. Identifying those susceptible to
neuropsychiatric illnesses (because of environmental factors or genetic
predisposition) may also permit vaccination or antimicrobial prophylaxis
and a subsequent lowering of disease incidence.
Physicians and federal agencies addressing the problems of emerging
infectious diseases should examine the possibility of infection as a cause
of mental illness. Better communication among infectious disease and mental
health experts, as well as additional training, will be needed to shed
light on the growing phenomenon of infectious diseases manifesting
themselves as neuropsychiatric disorders.
Edward McSweegan
National Institute of Allergy and Infectious Diseases, Bethesda, Maryland,
USA
References
  1. Hatalski CG, Lewis AJ, Lipkin WI. Borna disease. Emerg Infect Dis
     1997;3:129-35.
  2. Swedo SE, Leonard HL, Mittleman BB, Allen AJ, Rapoport JL, Dow SP, et
     al. Identification of children with pediatric autoimmune
     neuropsychiatric disorders associated with streptococcal infections by
     a marker associated with rheumatic fever. Am J Psychiatry
     1997;154:110-2.
  3. Benke T, Gasse T, Hittmair-Delazer M, Schmutzhard E. Lyme
     encephalopathy: long-term neuropsychological deficits years after
     acute neuroborreliosis. Acta Neurol Scand 1995;91:353-7.
  4. Fallon BA, Nields JA. Lyme disease: a neuropsychiatric illness. Am J
     Psychiatry 1994;151:1571-83.
  5. Mumford C, Dudley N, Terry H. Leptospirosis presenting as a flaccid
     paraplegia. Postgrad Med J 1990;66:218-20.
  6. Garcia H, Tsang V, Gonzalez A, Gilman R. Cysticercosis in the U.S. and
     Peru. Presented at the 6th Annual National Institutes of Health
     International Centers for Tropical Disease Research Meeting; 1997 May
     5-7; Bethesda, Maryland.
  7. World Health Organization. The global burden of disease [cited 1998
     Jan 5]; [2 screens]. Available from URL:
     http://www.who.ch/programmes/mnh/msamain.htm
  8. Children's mental health research severely neglected, says National
     Institute of Mental Health Director. Washington Fax [serial online]
     1997 Apr 8 [cited 1998 Jan 5]. Available from URL:
     http://www.washington-fax.com
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Mycobacterium nonchromogenicum Bacteremia in an AIDS Patient
To the Editor: Mycobacterium avium complex is the most common
nontuberculous mycobacterium that causes disseminated infection in
HIV-positive patients (1). Other less common nontuberculous mycobacteria
responsible for disseminated disease in these patients are M. fortuitum
(2), M. genavense (3), M. gordonae (4), M. haemophilum (5), M. kansasii
(6), M. malmoense (7), M. marinum (8), M. scrofulaceum (9), M. simiae (10),
M. szulgai (11), M. terrae (9), and M. xenopi (2,9). Although only M.
genavense, M. kansasii, and M. xenopi are significantly more frequent in
these patients (2,3,9), HIV infection is likely a predisposing condition
for all nontuberculous mycobacterial infections. We report the first case
of disseminated infection caused by M. nonchromogenicum in an HIV-infected
patient.
A 28-year-old man with HIV infection acquired by sharing injection tools
was seen in our outpatient clinic because of intermittent fever, drenching
nocturnal sweats, and cough with purulent sputum of 4 months' duration. He
also reported a weight loss of 10 kg in the previous 2 months. He had been
treated for bronchial infection with an unknown antibiotic in another
hospital. After this treatment, respiratory symptoms had improved somewhat,
but fever and constitutional symptoms continued. His only previous
opportunistic infection had been recurrent oral and esophageal candidiasis.
The last CD4-cell count had been 16/L 1 year earlier, and he was receiving
didanosine and prophylactic therapy with cotrimoxazole and fluconazole. On
physical examination the patient appeared ill; he was febrile, cachectic,
and had thrush and oral hairy leukoplakia. Neither lymphadenopathy nor
abnormal cardiopulmonary symptoms were found. The liver, which had enlarged
since the last examination, was palpated 6 cm below the right costal
margin. Abnormal laboratory values included aspartate aminotransferase 61
U/L, gamma-glutamyl transferase 209 U/L, lactate dehydrogenase 516 U/L,
hemoglobin 12.3 g/dL, leukocyte count 4,300/L (66% neutrophils, 19% band
forms, 1% metamyelocytes, 3% lymphocytes, 11% monocytes), platelet count
130,000/L, and erythrocyte sedimentation rate 72 mm/h. Chest X-rays were
unremarkable, and a set of blood cultures was sterile. A sputum culture
yielded Haemophilus influenzae sensitive to ampicillin, and smears and
cultures for mycobacteria in one stool and three sputum samples were
negative.
The patient was treated with oral amoxicillin for 2 weeks without
improvement. Empirical therapy against M. avium complex with
clarithromycin, ciprofloxacin, and ethambutol was started; the patient's
condition improved dramatically within the next few days, and the fever and
diaphoresis disappeared, although cough and sputum production remained
unchanged. Three weeks later, a slow-growing nonphotochromogenic
mycobacterium, identified as M. nonchromogenicum in a reference laboratory
(Centro Nacional de Microbiologa, Majadahonda, Madrid, Spain) by
biochemical tests and confirmed by polymerase chain reaction-restriction
enzyme pattern analysis, was isolated from a blood sample obtained on
admission. This microorganism was sensitive to the three drugs
administered, and the treatment was continued. Two months later the patient
had gained 10 kg, hemoglobin had increased to 13.8 g/dL, the erythrocyte
sedimentation rate had decreased to 52 mm/h, and the differential leukocyte
count had returned to normal. Antimycobacterial drugs were withheld after 1
year of treatment. Twenty-two months after the diagnosis, the patient is
doing well. He is receiving combination antiretroviral therapy, and his
CD4-cell count is 128/L.
M. nonchromogenicum, a slow-growing nonpigmented (Runyon's group III)
mycobacterium, belongs to the M. terrae complex, together with M. triviale;
it is traditionally considered nonpathogenic. However, it has been involved
in a few cases of pulmonary infection (12) and chronic tenosynovitis
secondary to puncture wounds (13), like the related organism M. terrae. In
fact, some authors think that M. nonchromogenicum is the true pathogen in
the M. terrae complex (13), and it is possible that some reports have
misidentified this organism. This complex was first isolated in soil
washings from radishes, but it has been found to be ubiquitous in the
aquatic environment, including a hospital potable water supply (14).
Unlike osteoarticular infections, which commonly occur in previously
healthy people, the scanty reports on pulmonary and disseminated infection
by M. terrae complex suggest that either immunosuppression or local
predisposing conditions (e.g., tuberculous cavities) are necessary
pathogenetic cofactors (15). To our knowledge, M. nonchromogenicum
bacteremia has never been reported before.
No specific DNA probes exist for M. terrae complex, but false-positive
reactions with M. tuberculosis complex DNA probes have been described (16).
Isolates are usually resistant to most antituberculosis drugs, with the
exception of ethambutol and streptomycin, and susceptible to erythromycin,
ciprofloxacin, and sulfonamides.
Only one case of disseminated infection by M. terrae has been described in
a patient with advanced HIV infection and positive cultures in blood and
bronchoalveolar lavage fluid, but no additional data were provided (9).
Although the isolate we recovered might represent a laboratory contaminant,
several pieces of evidence make this possibility very unlikely: lack of
alternative explanation for a persistent and progressive clinical picture
of 4 months' duration, absence of response to standard antibiotic therapy,
negative results in the search for other pathogens, rapid and sustained
clinical and laboratory response to drugs active against this strain, clear
improvement despite the lack of treatment for other conditions, and absence
of other isolates of this pathogen in our hospital despite the large number
of samples examined for mycobacteria.
Acknowledgments
We are indebted to Mara Soledad Jimnez, Mycobacterial Laboratory of the
Centro Nacional de Microbiologa, Majadahonda, Madrid, for providing the
microbiologic data.
Jos Mayo, Julio Collazos, and Eduardo Martnez
Hospital de Galdakao, Vizcaya, Spain
References
  1. Nightingale SD, Byrd LT, Southern PM, Jockusch JD, Cal SX, Wynne BA.
     Incidence of Mycobacterium avium-intracellulare complex bacteremia in
     human immunodeficiency virus-positive patients. J Infect Dis
     1992;165:1082-5.
  2. Raszka WV Jr, Skillman LP, McEvoy PL, Robb ML. Isolation of
     nontuberculous, non-avium mycobacteria from patients infected with
     human immunodeficiency virus. Clin Infect Dis 1995;20:73-6.
  3. Bessesen MT, Shlay J, Stone-Venohr B, Cohn DL, Reves RR. Disseminated
     Mycobacterium genavense infection: clinical and microbiological
     features and response to therapy. AIDS 1993;7:1357-61.
  4. Lessnau KD, Milanese S, Talavera W. Mycobacterium gordonae: a
     treatable disease in HIV-positive patients. Chest 1993;104:1779-85.
  5. Straus WL, Ostroff SM, Jernigan DB, Kiehn TE, Sordillo EM, Armstrong
     D, et al. Clinical and epidemiologic characteristics of Mycobacterium
     haemophilum, an emerging pathogen in immunocompromised patients. Ann
     Intern Med 1994;120:118-25.
  6. Parenti DM, Symington JS, Keiser J, Simon GL. Mycobacterium kansasii
     bacteremia in patients infected with human immunodeficiency virus.
     Clin Infect Dis 1995;21:1001-3.
  7. Chocarro A, Gonzlez-Lpez A, Breznes MF, Canut A, Rodrguez J, Diego
     JM. Disseminated infection due to Mycobacterium malmoense in a patient
     infected with human immunodeficiency virus. Clin Infect Dis
     1994;19:203-4.
  8. Tchornobay AM, Claudy AL, Perrot JL, Levigne V, Denis M. Fatal
     disseminated Mycobacterium marinum infection. Int J Dermatol
     1992;31:286-7.
  9. Schafer RW, Sierra MF. Mycobacterium xenopi, Mycobacterium fortuitum,
     Mycobacterium kansasii, and other nontuberculous mycobacteria in an
     area of endemicity for AIDS. Clin Infect Dis 1992;15:161-2.
 10. Valero G, Peters J, Jorgensen JH, Graybill JR. Clinical isolates of
     Mycobacterium simiae in San Antonio, Texas: an 11-yr review. Am J
     Respir Crit Care Med 1995;152:1555-7.
 11. Roig P, Nieto A, Navarro V, Bernacer B, Borras R. Micobacteriosis por
     Mycobacterium szulgai en paciente con infeccin por el virus de la
     inmunodeficiencia humana. An Med Interna 1993;10:182-4.
 12. Tsukamura M, Kita N, Otsuka W, Shimoide H. A study of the taxonomy of
     the Mycobacterium nonchromogenicum complex and report of six cases of
     lung infection due to Mycobacterium nonchromogenicum. Microbiol
     Immunol 1983;27:219-36.
 13. Ridderhof JC, Wallace RJ Jr, Kilburn JO, Butler WR, Warren NG,
     Tsukamura M, et al. Chronic tenosynovitis of the hand due to
     Mycobacterium nonchromogenicum: use of high-performance liquid
     chromatography for identification of isolates. Rev Infect Dis
     1991;13:857-64.
 14. Lockwood WW, Friedman C, Bus N, Pierson C, Gaynes R. An outbreak of
     Mycobacterium terrae in clinical specimens associated with a hospital
     potable water supply. American Review of Respiratory Diseases
     1989;140:1614-7.
 15. Peters EJ, Morice R. Miliary pulmonary infection caused by
     Mycobacterium terrae in an autologous bone marrow transplant patient.
     Chest 1991;100:1449-50.
 16. Ford EG, Snead SJ, Todd J, Warren NG. Strains of Mycobacterium terrae
     complex which react with DNA probes for M. tuberculosis complex. J
     Clin Microbiol 1993;31:2805-6.
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Escherichia coli O157:H7 Infection in Colombia
To the Editor: The prevalence of Escherichia coli O157:H7 in Colombia is
not known; we conducted a study to determine its prevalence in children
with infectious diarrhea, in adult cattle, and in ground beef.
Between March 1996 and March 1997, we examined 538 children under 5 years
of age with infectious diarrhea who had been admitted to either of the two
children's hospitals in Bogot. Diarrhea was defined as three or more loose
stools within the previous 24 hours. One hundred and sixty-one children
under 10 years of age admitted to the hospital for a medical reason other
than infectious diarrhea served as controls.
Stool samples from children with and without diarrhea were placed in Stuart
transport medium and sent to the laboratory within 24 hours; the samples
were injected into sorbitol MacConkey agar (Oxoid Basingstoke, United
Kingdom). After 24 hours of incubation at 37C, sorbitol nonfermenting
colonies were tested with 4-methylumbelliferyl--glucuronide (MUG); all
typical colonies of E. coli O157:H7 that were sorbitol-negative were
confirmed as E. coli by biochemical tests (1,2) and were tested for
agglutination with a latex test kit (Oxoid Basingstoke, United Kingdom) for
detecting E. coli O157 and E. coli H antiserum H7 (Difco, Detroit, MI,
USA). All human isolates were confirmed as Shiga-toxin producers by latex
agglutination (Oxoid Basingstoke, United Kingdom). We used as a control
strain E. coli O157:H7 provided by M. Karmali.
Rectal swabs from 307 healthy adult cattle from farms in Cundinamarca and
Meta Departments were placed in Stuart transport medium, stored at 4C, and
transported to the laboratory within 6 hours. Swabs were injected into
sorbitol MacConkey agar, and colonies that did not ferment sorbitol were
characterized by standard techniques (3).
One hundred and fifty beef patties (31 cooked, 119 raw), collected in
Bogot, were examined by direct plating and enrichment culturing. Samples
(1.0 g each) were serially diluted (1:10) in 0.85% NaCl solution, and 0.1
ml portions were plated in duplicate onto sorbitol MacConkey agar.
Serologic and biochemical confirmation was done as mentioned above.
E. coli O157:H7 prevalence among children was 7.2%, with an age range of
0-60 months (average 21 months); diarrheal illness lasted an
average of 2.5 days. Of 39 patients, eight were 6 months old or younger,
five were 6 to 12 months old, 17 were 12 to 24 months old, and nine were
older than 24 months. Renal failure associated with hemolytic uremic
syndrome (HUS) developed in three (7.7%). Epidemiologic data were not
collected regarding contaminated foods as a possible source of E. coli
O157:H7 infection in the patients. E. coli O157:H7 was isolated from five
(3.1%) of the 161 controls; the prevalence of E. coli O157:H7 was
substantially higher in patients with infantile diarrhea than in controls.
All 39 strains from human cases were sorbitol-negative; five did not
display MUG activity. Overall, 39 strains agglutinated strongly with
antiserum O157:H7. Antimicrobial susceptibility tests were performed by the
Bauer method (4). All E. coli O157:H7 isolated were susceptible to
ciprofloxacin; 92% were resistant to ampicillin; 76% were resistant to
furazolidone; and 76% were resistant to trimethoprim-sulfamethoxazole
(TMP-SMZ).
E. coli O157:H7 was isolated from 20 (6.5%) of 307 rectal swabs from
cattle. The strains isolated were sorbitol-negative and agglutinated
strongly with antisera; five did not present activity. All strains were
susceptible to ciprofloxacin; 90% were resistant to ampicillin; and 26%
were resistant to TMP-SMZ. E. coli O157:H7 was isolated from 13 (87%) of
150 beef patties, six from raw beef, and seven from cooked beef.
Stool cultures of all patients with acute bloody diarrhea should be tested
for E. coli O157:H7 to identify those at risk for HUS (5); however,
serotyping, cytotoxicity assays, or DNA probing for E. coli O157:H7 are not
routinely performed in Colombia.
This preliminary report suggests that E. coli O157:H7 is emerging as an
important cause of endemic childhood diarrhea in Colombia and that the
chain of contamination is present. The incidence is greatly underestimated
because of limited surveillance and reporting. Further studies are needed
to identify the pathogenic mechanisms of these E. coli O157:H7 strains and
to determine the fecal carriage rate in healthy children. Data obtained
will help elucidate the role of E. coli O157:H7 in childhood diarrhea. In
addition, molecular analysis should be performed to establish the
connection between the strains isolated from different sources in Colombia.
Our findings suggest that the risk for E. coli O157:H7 infection in
Colombia is high; therefore,
more active screening and surveillance would enhance case detection,
epidemiologic understanding of E. coli O157:H7 infection and HUS, and could
lead to more specific therapeutic interventions.
Salim Mattar and Elizabeth Vsquez
Facultad de Ciencias, Universidad Javeriana, Bogot, Colombia
References
  1. Bockemuhl J, Aleksic S, Karch H. Serological and biochemical
     properties of Shiga-like toxin (verocytotoxin)-producing strains of
     Escherichia coli, other than O-group 157, from patients in Germany.
     Zentralbl Bakteriol 1992;276:189-95.
  2. Gunzer F, Bohm H, Rossmann H, Bitzan M, Aleksic S, Karch H. Molecular
     detection of sorbitol-fermenting Escherichia coli O157 in patients
     with hemolytic-uremic syndrome. J Clin Microbiol 1992;30:1807-10.
  3. Chapman PA, Siddons CA, Wright DJ, Norman P, Fox J, Crick E. Cattle as
     a possible source of verocytotoxin-producing Escherichia coli O157
     infections in man. Epidemiol Infect 1993;439:447.
  4. Bauer AW, Kirby WMM, Sherris JC, Turk M. Antibiotic susceptibility
     testing by a standardized single disc method. Am J Clin Pathol
     1966;45:493-6.
  5. Wells JG, Davis BR, Wachsmuth IK, Riley LW, Remis RS, Sokolow R,
     Morris GK. Laboratory investigation of hemorrhagic colitis outbreaks
     associated with a rare Escherichia serotype. J Clin Microbiol
     1983;18:512-20.
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Autofluorescence and the Detection of Cyclospora Oocysts
To the Editor: From May through July 1997, we searched for the seasonally
occurring Cyclospora cayetanensis, along with other coccidia and
microsporidia, in fecal samples from 385 patients. The samples, in 10%
formalin for evaluation of coccidia and microsporidia, were initially
processed by a routine formalin-ethyl acetate concentration method; the
parasite was detected in 18 patients (1,2). The resulting sediment was
examined as follows. A drop of sediment was placed on a slide,
cover-slipped, and examined microscopically as a wet mount at 200x and 400x
magnification and subsequently at 200x magnification by epifluorescence
with a 330 to 380 nm UV filter. Four smears were also prepared and stained
by routine trichrome (2), modified trichrome (3), auramine-rhodamine (4),
and Kinyoun acid-fast (5) procedures. All wet mount and stained
preparations were evaluated by at least two trained persons.
Of the 385 fecal samples examined, 18 were positive for C. cayetanensis.
The positive samples were from eight states, which encompassed northeastern
(Rhode Island, New York, Massachusetts, Pennsylvania), midwestern
(Wisconsin), western (Oregon, California), and southern (Florida) sections
of the United States.
In 12 of 18 patients, the organisms were detected without much difficulty
in wet mounts as round or partially collapsed nonrefractile bodies;
however, in the other six, repeated wet preparations were needed to detect
the organisms. When the same wet mounts were examined with epifluorescence
microscopy, oocysts were easily discerned in all samples, even the six in
which repeated wet preparations and stains were needed. While the trichrome
procedures were ineffective, the auramine-rhodamine and Kinyoun stains gave
varied results. The autofluorescence technique, however, was distinctly
superior to the wet mount and staining procedures.
Extensive outbreaks of diarrhea caused by C. cayetanensis were reported in
1997 from different parts of the United States (6-8), and several
procedures have been used to confirm the diagnosis in clinical samples.
While the organisms are large enough to be seen in direct wet mounts, they
are frequently caught up in mucus or covered by debris, so they are
difficult to detect. Autofluorescence in C. cayetanensis oocysts makes them
easily visible in clinical samples (1,9) with the use of a 330 to 380 nm UV
filter; this feature enhanced their detection at least twofold over the
direct wet mount, especially when the wet mount and stained slides
contained few oocysts. (The same wet mount preparation can be used for the
epifluorescence procedure.)
The 18 patients with cyclosporiasis were ages 2 to 71 years, which
indicates that the infection was not specific to any age group. Twelve of
the 18 cases were in women. Massachusetts had 11, the largest number of C.
cayetanenis-positive patients. Of the 18, 16 were adults; the other two
were children with a coexisting parasite (Dientamoeba fragilis). In one
instance, three members of the same family were infected, the parents with
only C. cayetanensis, the son with D. fragilis and Blastocystis hominis.
Because C. cayetanensis is a seasonal diarrheal agent, fecal samples from
persons with persistent unexplained explosive diarrhea during the summer
should be carefully evaluated for this infection. Stool specimens should be
fixed in 10% formalin and examined with autofluorescence microscopy for
enhanced detection.
O.G.W. Berlin,* J.B. Peter, C. Gagne, C.N. Conteas, and L.R. Ash*
*School of Public Health, University of California, Los Angeles,
California, USA; Specialty Laboratories, Santa Monica, California, USA;
and Kaiser Permanente Medical Center, Los Angeles, California, USA
References
  1. Berlin OGW, Novak SM, Porschen RK, Long EG, Stelma GN, Schaeffer FW.
     Recovery of Cyclospora organisms from patients with prolonged
     diarrhea. Clin Infect Dis 1994;18:606-9.
  2. Ash LR, Orihel TC. Collection and preservation of feces. Parasites: a
     guide to laboratory procedures and identification. Chicago: ASCP
     Press; 1991. p. 3-53.
  3. Weber R, Bryan RT, Owen RL, Wilcox CM, Gorelkin L, Visvesvara GS.
     Improved light microscopical detection of microsporidia spores in
     stool and duodenal samples. N Engl J Med 1992;326:161-6.
  4. Berlin OGW. Mycobacteria. In: Baron EJ, Finegold SM, editors.
     Diagnostic microbiology. 8th ed. St. Louis: C.V. Mosby; 1990. p.
     597-640.
  5. Ash LR, Orihel TC. Atlas of human parasitology. 4th ed. Chicago: ASCP
     Press; 1997.
  6. Centers for Disease Control and Prevention. Update: outbreaks of
     cyclosporiasisUnited States, 1997. MMWR Morb Mortal Wkly Rep
     1997;46:451-2.
  7. Centers for Disease Control and Prevention. Update: outbreaks of
     cyclosporiasisUnited States and Canada, 1997. MMWR Morb Mortal Wkly
     Rep 1997;46:521-3.
  8. Colley DG. Widespread food-borne cyclosporiasis outbreaks present
     major challenges. Emerg Infect Dis 1996;2:354-6.
  9. Eberhard ML, Pienazek NJ, Arrowood MJ. Laboratory diagnosis of
     Cyclospora infections. Arch Pathol Lab Med 1997;121:792-7.
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Partnerships for Detecting Emerging Infectious Diseases: Nepal and Global
Influenza Surveillance
To the Editor:With new influenza strains emerging each year, identification
of circulating strains by coordinated global surveillance is crucial to
vaccine development for the coming year (1-3). Approximately 110
laboratories in 80 countries voluntarily participate in the World Health
Organization (WHO) influenza surveillance network (4). Comprehensive
surveillance is especially important in Asia, since new influenza strains
often originate there. To participate in influenza global surveillance,
countries need not rely on their own laboratory capability. Clinical
specimens from patients thought to have influenza can be sent to designated
laboratories around the world for analysis. A unique partnership has led to
the expansion of the WHO global influenza surveillance network to Nepal.
The U.S. Army Medical Component-Armed Forces Research Institute for Medical
Sciences (AFRIMS) (5) in Bangkok, Thailand, is well situated to assist with
surveillance in Asia. Scientists at AFRIMS have conducted medical research
in collaboration with Nepali colleagues for more than 20 years. Several
studies have been conducted in collaboration with the CIWEC Clinic Travel
Medicine Center (a travel medicine clinic that serves the diplomatic, aid,
and tourist communities in Nepal). The clinic has approximately 5,000
patient visits per year, of which half are drawn from the 2,500 expatriates
in Nepal and half from the 200,000 non-Indian tourists who visit Nepal
annually.
A protocol was developed for a pilot influenza surveillance program. The
staff of the CIWEC Clinic was responsible for volunteer recruitment,
clinical evaluation, and specimen collection. Febrile upper respiratory
infections were defined as temperature > 100F (37.8C, oral or equivalent)
and cough or sore throat of 72 hours duration. Other symptoms, such as
streptococcal pharyngitis, were excluded. No age or gender restrictions
were included. Volunteers had to have been in Nepal for the 5 days
preceding illness. Only the first patient in any single household with
similar symptoms within days of other household members was asked to
participate.
The AFRIMS field station in Kathmandu (locally known as the Walter
Reed/AFRIMS Research Unit-Nepal or WARUN) was responsible for shipping
specimens collected by the CIWEC Clinic to AFRIMS, Thailand. Since dry ice
was not available in Kathmandu, dry ice and shipping containers were sent
by AFRIMS, Thailand for use by WARUN. Shipments from WARUN were then sent
back to AFRIMS, where specimens were repacked in dry ice and sent for
testing at the central laboratory of the U.S. Air Force's Project Gargle
(6) in San Antonio, Texas. Project Gargle has been testing viral
respiratory specimens from distant Air Force installations for more than 20
years. Each specimen was tested for influenza A and B; parainfluenza virus
1, 2, and 3; adenovirus, enterovirus; and herpesvirus. Characterization of
selected influenza A and B isolates by hemagglutination-inhibition testing
was performed by the Centers for Disease Control and Prevention (CDC).
Between December 1996 and February 1997, the CIWEC staff collected
specimens from 18 patients. Samples were collected from 11 (61%) residents
and seven (39%) tourists, who were evenly distributed by gender and had a
median age of 35 years. Influenza B/Beijing/184/93-like viruses were
isolated from five (28%) of the 18 specimens. All patients from whom
influenza viruses were obtained had mild illnesses with fever and upper
respiratory syndromes. Herpes virus type 1 and adenovirus type 6 were each
identified in one other specimen. No respiratory viruses were identified in
the remaining 11 specimens.
Because of the importance of China in the emergence of new strains of
influenza, CDC's WHO Collaborating Center for Surveillance, Epidemiology,
and Control of Influenza has worked with colleagues in China to establish a
national Chinese network of influenza surveillance sites. Analysis of
viruses isolated in China between 1988 and 1997 in comparison with other
viruses obtained through WHO's global influenza surveillance network has
shown that influenza variants are frequently identified in China before
becoming prevalent in other regions of the world. Nepal is another
especially valuable surveillance site, given its location between China and
India (at the crossroads between northern and southern Asia) and its
historic importance as a trans-Himalayan trade route.
Especially relevant are data from China demonstrating that the two
antigenically and genetically distinct lineages of influenza B viruses
represented by B/Victoria/02/87 and B/Yamagata/16/88 (7) have continued to
circulate and evolve in China, while only viruses related to B/Yamagata
have been detected elsewhere in the world and are represented in the
current trivalent vaccine by the B/Bejing/184/93-like component. Virologic
surveillance in surrounding countries (8) such as Nepal is necessary to
detect geographic spread of B/Victoria-like virus in the region. Our data
suggest that these viruses have not yet spread to Kathmandu.
Our unique international partnership between several civilian and military
organizations (e.g., CIWEC Clinic, CDC, U.S. Air Force, and U.S. Army)
demonstrates the feasibility of such partnerships as well as the usefulness
of influenza surveillance data at both the local and global levels. Despite
the small number of isolates obtained during this study, we were able to
determine that the influenza B component of the trivalent vaccine prepared
for the 1996-1997 influenza season would likely have offered protection for
travelers and the local population against the influenza B strains isolated
in Kathmandu. Ongoing surveillance data will establish geographic and
temporal patterns of circulation of influenza viruses and thus provide
valuable information for guiding public health policies for influenza
vaccination. On a global level, these data are useful for annual vaccine
strain selection.
Advances in communication, laboratory, and specimen transport technologies
contributed greatly to the identification of viral pathogens from a new
sentinel surveillance site in Nepal. In evaluating future collaborative
sites, prior surveillance experience and reliable specimen shipping should
be prime considerations. Approaches that use existing resources might
foster greater international cooperation toward improved global detection
and reporting of infectious diseases.
Acknowledgment
Financial support was given by the United States Department of Defense
Emerging Infectious Disease Program.
Jeffrey M. Gambel,* David R. Shlim, Linda C. Canas, Nancy J. Cox, Helen
L. Regnery, Robert M. Scott, David W. Vaughn, Charles H. Hoke Jr.,* and
Patrick W. Kelley*
*Walter Reed Army Institute of Research, Washington, D.C., USA; CIWEC
Clinic, Kathmandu, Nepal; Armstrong Laboratory - Diagnostic Virology,
Brooks Air Force Base, Texas, USA; Centers for Disease Control &
Prevention, Atlanta, Georgia, USA; and Armed Forces Research Institute of
Medical Sciences, Bangkok, Thailand
References
  1. Gross PA. Preparing for the next influenza pandemic: a reemerging
     infection. Ann Internal Med 1996;124:682-5.
  2. Glezen WP. Emerging infections: pandemic influenza. Epidemiol Rev
     1996;18:64-76.
  3. Patriarca PA, Cox NJ. Influenza pandemic preparedness plan for the
     United States. J Infect Dis 1997;176(Suppl 1):S4-S7.
  4. Hampson AW. Surveillance for pandemic influenza. J Infect Dis
     1997;176(Suppl 1):S8-S13.
  5. Gambel JM, Hibbs RG. U.S. military overseas medical research
     laboratories. Mil Med 1996;161:638-45.
  6. Williams RJ, Cox NJ, Regnery HL, Noah DL, Khan AS, Miller JM, ET AL.
     Meeting the challenge of emerging pathogens: the role of the United
     States Air Force in global influenza surveillance. Mil Med
     1997;162:82-6.
  7. Rota PA, Hemphill ML, Whistler T, Regnery HL, Kendal AP. Antigenic and
     genetic characterization of the hemagglutinins of recent circulating
     strains on influenza type B virus. J Gen Virol 1992:73:2737-42.
  8. Centers for Disease Control and Prevention. Update influenza
     activity-United States and worldwide, 1996-97 season, and composition
     of the 1997-98 influenza vaccine. MMWR Morb Mortal Wkly Rep
     1997;46:325-30.
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HIV-2 Infection and HIV-1/HIV-2 Dual Reactivity in Patients With and
Without AIDS-Related Symptoms in Gabon
To the Editor: Between 1996 and 1997, we evaluated the incidence of HIV-2
infection at the Fondation Jeanne Ebori, the second largest hospital in
Libreville, capital of Gabon; we found an unexpected high prevalence of
HIV-2-infected or HIV-1/HIV-2-dually reactive patients.
During a 10-month period, 147 (14.3%) of 1,029 sera from inpatients and
outpatients were found HIV-positive by the type III method recommended by
the World Health Organization (two enzyme-linked immunosorbent assays are
used to screen anti-HIV antibodies) (1). Further discrimination between
HIV-1 and HIV-2 infections was assessed by using synthetic peptides
specific for the gp41 and the gp120 of HIV-1 and the gp36 of HIV-2
(ImmunoComb II, PBS Orgenics, Illkirch, France). Of the 147 HIV-positive
sera, 141 (96.0%) were exclusively HIV-1-positive; four were exclusively
HIV-2-positive; and two were both HIV-1- and HIV-2-positive. Of the six
sera with anti-gp36/HIV-2 reactivities, two (from patients A and B) were
positive on HIV-2 Western blot, with marked anti-gag HIV-1 cross-reactivity
and a discrimination assay positive only for HIV-2; two (from patients D
and E) were positive on HIV-2 Western blot, with anti-gag and pol
reactivities markedly lower than anti-env reactivities and a discrimination
assay positive only for HIV-2; the two remaining sera (from patients C and
F) showed typical dual reactivities for HIV-1 and HIV-2 infections, with
positive patterns of HIV-1 and HIV-2 Western blots and a discrimination
test positive for both viruses. As a whole, six (4.1%) of 147 HIV-positive
sera showed either HIV-2 infection alone (n = 4) or dual reactivity. Of
those, four were from Gabonese patients B, C, D, and E, and two were from
immigrants from West Africa (patient A from Mali and patient F from
Nigeria); two were female patients B and E. Among Gabonese patients, only
one (patient E) had traveled to West Africa; the remaining three had never
visited any neighboring country. However, one Gabonese man (patient C)
lived in Port-Gentil, which has many West African immigrants. For all
patients, the most likely risk factor for HIV was a heterosexual
relationship with an unknown HIV-infected person. In three asymptomatic
patients (A, B, and C) the HIV-2-serostatus was unexpected; in contrast,
the three other patients had AIDS-related symptoms. Patients D and E had an
HIV-2 Western blot pattern showing a marked decrease in anti-gag and pol
reactivities compatible with their advanced stage of HIV-2 disease.
The case of a 55-year-old exclusively heterosexual asymptomatic woman
(patient B) suggests the possibility of a specific variant of HIV-2 in
Central Africa (2). The high frequency in primates in Gabon of natural
infection with simian immunodeficiency retroviruses, which show a high
degree of genetic relatedness to HIV-2 (3), could support such a
hypothesis.
Two patients had typical dual reactivities to HIV-1 and HIV-2 antigens. To
our knowledge, such dual reactivities have never been reported in Gabon
(4). In the patient from Nigeria (patient F), the serologic pattern was
typical of that usually observed in West Africa (5). Dual reactivity can
result from genuine mixed infections and from serologic cross-reactivity in
HIV-1 and HIV-2 infection alone; theoretically, it could also represent
infection with a different, cross-reacting recombinant strain (5).
HIV-2 infection in Gabon is epidemiologically related to West Africa,
because of cultural and, above all, economic ties. However, HIV-2 is not
limited to immigrant populations from West Africa or to Gabonese citizens
traveling in this area; it has also reached the indigenous Gabonese
population. The possibility of rare cases of HIV-1 and HIV-2 coinfections,
recombinant HIV-1 and HIV-2 strains, and also peculiar HIV-2 variants from
Central Africa, should be considered in Gabon. A possible entry of HIV-2
infection into Central Africa from Gabon in the near future could have
major public health implications.
Acknowledgments
We thank Dr. Jean Wickings for reviewing the manuscript. CIRMF is funded by
the Gabonese government, ELF Gabon, and the French Ministry of Cooperation.
Carol Tevi-Benissan,* Madeleine Okome, Maria Makuwa,* Moise Ndong Nkoume,
Joseph Lansoud-Soukate,* Alain Georges,* Marie-Claude Georges-Courbot,* and
Laurent Belec
*Centre International de Recherches Medicales, Franceville, Gabon;
Fondation Jeanne Ebori, Libreville, Gabon; and Centre
Hospitalo-Universitaire Broussais, Paris, France.
References
  1. World Health Organization. Global programme on AIDS. Recommendations
     for the selection and use of HIV antibody tests. Wkly Epidemiol Rec
     1992;67:145-9.
  2. Belec L, Martin PMV, Georges-Courbot MC, Brogan T, Gresenguet G,
     Mathiot CC, et al. Dementia as the primary manifestation of HIV-2
     infection in a Central African patient. Ann Inst Pasteur Virol
     1988;139:291-4.
  3. Georges-Courbot MC, Moisson P, Leroy E, Pingard AM, Nerrienet E,
     Dubreuil G, et al. Occurrence and frequency of transmission of
     naturally occurring simian retroviral infections (SIV, STLV, and SRV)
     at the CIRMF Primate Center, Gabon. J Med Primatol 1996;25:313-26.
  4. Delaporte E, Janssens W, Peeters M, Buve A, Dibanga G, Perret J-L, et
     al. Epidemiological and molecular characteristics of HIV infection in
     Gabon, 1986-1994. AIDS 1996;10:903-10.
  5. Peeters M, Gershy-Damet G-M, Fransen K, Koffi K, Coulibaly M,
     Delaporte E, et al. Virological and polymerase chain reactions studies
     of HIV-1/HIV-2 dual infection in Cte d'Ivoire. Lancet
     1992;340:339-40.
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Q Fever in French Guiana: New Trends
To the Editor: Q fever, the endemic disease caused by the rickettsial
organism Coxiella burnetii, was first described in French Guiana in 1955
(1). Only sporadic cases were reported until 1996 when three patients were
hospitalized in the intensive care unit of the Cayenne Hospital for acute
respiratory distress syndrome. One of the patients died. Many cases of Q
fever were diagnosed in the general population at the same time. A
seroepidemiologic study was performed to determine whether the increase in
cases was due to an increase in incidence or to an improvement in
diagnosis. All paired samples of sera (acute-phase and convalescent-phase)
from patients sent to the arbovirus laboratory for diagnosis of dengue
infection from January 1, 1992, to December 31, 1996, were tested for
antibodies to C. burnetii by immunofluorescence. All positive samples were
also tested for immunoglobulin (IgM) by the same method; the IgG and IgM
titers were determined by using a serial twofold dilution. A diagnosis of Q
fever was made when there was a seroconversion from negative to positive or
a twofold increase in IgG titer associated with the presence of IgM in the
second sample.
One hundred and fifty-one of 426 paired sera collected between 1992 and
1996 were from patients recently infected with dengue fever. Twenty-five
(9.1%) of 275 remaining sera were from Q fever patients. Significant
differences were observed in the rates of Q fever in different years (p <
0.01); one (1.9%) of 53 was positive in 1992, five (9.1%) of 55 in 1993,
five (8.6%) of 58 in 1994, three (4.8%) of 63 in 1995; a large increase was
observed in 1996 (11 [23.9%] of 46). Differences by residence were also
assessed. Rates of infection were higher in Cayenne (21 [13.0%] of 161)
than in rural areas (4 [3.5%] of 114) (p < 0.01).
This study shows that cases of Q fever have occurred in French Guiana in
recent years and that a significant increase in the incidence rate occurred
in 1996. The reasons for this increase are unclear, and further studies of
the epidemiology of Q fever in French Guiana are necessary. The
epidemiology of Q fever is unusual in French Guiana because the rates of
infection are much higher in Cayenne, the capital city, than in rural
areas. No link with classical sources of infection (cattle, sheep, or goat
birth products, or work in a slaughterhouse) was found. Indeed, Cayenne,
with 80,000 inhabitants, is located near the Atlantic Ocean, and the
prevailing winds blow from the sea. Airborne contamination from rural areas
is therefore impossible. Furthermore, no large farm is in the immediate
vicinity of the city. For identical reasons, contamination from the
abattoirs is not likely; they are located on the west side of the city,
near the Cayenne River, and the winds blow from the east. In our study,
cases were almost equally distributed throughout the city, although many
patients came from the same area.
A seroepidemiologic study to determine possible new sources of infection
(e.g., dogs, cats) and estimate rates of seropositivity in cattle and sheep
and a case-control study on new cases are being conducted.
F. Pfaff,* A. Franois,* D. Hommel, I. Jeanne,* J. Margery, G. Guillot,
Y. Couratte-Arnaude, A. Hulin, and A. Talarmin*
*Pasteur Institute of French Guiana, Cayenne, French Guiana; Cayenne
Hospital, Cayenne, French Guiana; and Army Health Care Service, Cayenne,
French Guiana
Reference
  1. Floch H. La pathologie vtrinaire en Guyane franaise (les affections
     des porcins, des caprins et des ovins). Revue Elev Md Vt Pays Trop
     1955;8:11-3.
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Ixodes dammini: A Junior Synonym for Ixodes scapularis
To the Editor: The authors of "A new tick-borne encephalitis-like virus
infecting New England deer ticks, Ixodes dammini" (1) provide useful
information regarding a possibly new tick-borne encephalitis-like virus.
However, the use of the name Ixodes dammini is not accurate for describing
this species. I. dammini (Spielman, Clifford, Piesman, and Corwin) was
synonymized with Ixodes scapularis (Say) in 1993 by Oliver et al. (2) and
was redescribed in 1996 (3) to reduce confusion regarding identification.
Keirans and colleagues summarize a wide array of rigorous studies involving
hybridization, assortative mating, isozymes, and morphometrics, all of
which provide evidence supporting the synonymization of the two tick
species (3).
The synonymization of I. dammini with I. scapularis has been widely
accepted. "I. scapularis (= I. dammini)" is still often used, but the use
of I. scapularis as the sole nomen for this species is becoming more common
(4). Oliver et al. (2) have established I. dammini as a junior subjective
synonym of I. scapularis. If scientifically rigorous evidence exists
justifying the reestablishment of the species name I. dammini, it must be
published according to proper procedure. The proper nomenclature of any
species, let alone one of such widespread notoriety and public health
importance, is too important to be relegated to a footnote. Until such
evidence is presented, the continued misuse of I. dammini serves only to
confuse health-care providers, public health professionals, and lay
persons.
On a secondary matter, on page 167 of the dispatch, the authors state that
"I. (Pholeoixodes) cookei is a one-host tick that is only distantly related
to I. dammini and only rarely feeds on humans or mice" (1). I. cookei is a
three-host tick (D.E. Sonenshine, pers. comm.), as are all the members of
the genus Ixodes.
Martin Sanders
Maryland Department of Health and Mental Hygiene, Baltimore, Maryland, USA
References
  1. Telford SR III, Armstrong PM, Katavolos P, Foppa I, Garcia ASO, Wilson
     ML, Spielman A. A new tick-borne encephalitis-like virus infecting New
     England deer ticks, Ixodes dammini. Emerg Infect Dis 1997;3:165-70.
  2. Oliver JH, Owsley MR, Hutcheson AM, James C, Chen W, Irby S, et al.
     Conspecificity of the ticks Ixodes scapularis and Ixodes dammini
     (Acari: Ixodidae). J Med Entomol 1993;30:54-63.
  3. Keirans JE, Hutcheson HJ, Durden LA, Klompen JSH. Ixodes scapularis
     (Acari: Ixodidae): redescription of all active stages, distribution,
     hosts, geographical variation, and medical and veterinary importance.
     J Med Entomol 1996;33:297-318.
  4. Centers for Disease Control and Prevention. Lyme disease-United
     States, 1996. MMWR Morb Mortal Wkly Rep 1997;46:531-5.
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The Name Ixodes dammini Epidemiologically Justified
To the Editor: Although a large body of evidence has been interpreted as
supporting conspecificity of the deer tick (Ixodes dammini) and the
blacklegged tick (Ixodes scapularis), according to Chapter VI, Article 23 L
of the International Code of Zoological Nomenclature (1), "A name that has
been treated as a junior synonym may be used as the valid name of a taxon
by an author who considers the synonymy to be erroneous...."
Current use of I. scapularis to refer to the vector of Lyme disease
obscures important epidemiologic issues. One of the reasons for "sinking"
I. dammini was to make it easier to diagnose Lyme disease in areas where
the disease was thought to be nonendemic: "The belief that I. dammini does
not occur south of Maryland and that I. scapularis is a separate and
distinct species yet unproven as a natural vector of Lyme disease has
caused delays in Lyme disease surveillance in the South. The general
attitude among physicians and veterinarians has been that Lyme disease is
not a problem in that area, although patients present clinical symptoms of
it" (2). Recognizing and reporting Lyme disease in southern and
southcentral states should not, however, depend on whether the two ticks
are conspecific. Only peer-reviewed descriptions of human cases of Lyme
disease, with appropriate documentation of the diagnoses, should be
accepted as evidence. Few such reports exist, and the evidence does not
convincingly support a conclusion that Lyme disease exists as an epidemic
zoonosis in southern states (3). This is not to say that residents outside
the well-established eastern United States zoonotic sites (the Northeast
and upper Midwest) do not have symptoms that fit one or more aspects of the
current Centers for Disease Control and Prevention/Council of State and
Territorial Epidemiologists/Association of State and Territorial Public
Health Laboratory Directors surveillance definition for Lyme disease. Lyme
disease-like infections, mainly manifesting as erythema migrans and
strongly associated with Lone Star tick (Amblyomma americanum) bites are
commonly seen in southern and southcentral states, but Borrelia burgdorferi
does not seem to be the etiologic agent (4).
Enzootic transmission of Lyme disease spirochetes among rodents and ticks
had been documented in southern and southcentral states by the late 1980s
(5-7). The question, however, is whether there is frequent zoonotic
transmission. There are widespread southern U.S. enzootic cycles of
Trypanosoma cruzi, but few autochthonous human Chagas disease cases seem to
occur because the vectors (such as Triatoma sanguisuga) have behavioral
traits that reduce their capacity to serve as zoonotic vectors (8). Nymphal
I. scapularis apparently do not frequently bite humans (7,9), although
adult ticks do. The major feature of Lyme disease epidemiology in the
Northeast and in the upper Midwest, however, is transmission by nymphal I.
dammini (10).
Whether the predilection of nymphal I. dammini to feed on humans is
environmentally determined or is a heritable trait with undescribed genetic
markers remains unexplored. Particular mitochondrial DNA haplotypes seem to
be more characteristic of I. dammini (11,12), and the use of such typing
methods may enhance future analyses of the vectorial capacity of these
ticks. For example, one might test the hypothesis that nymphal ticks
removed from residents of sites in coastal North Carolina through Georgia,
where both kinds of ticks have been collected, represent only I. dammini.
But, if it is "widely accepted" that no differences exist between the two
ticks, such studies may never be done. Similarly, many may wrongly assume
that Lyme disease, human babesiosis, and human granulocytic ehrlichiosis
are, or will become, epidemic throughout virtually all of the eastern
United States. An equally likely scenario is that these zoonoses may never
become public health problems for more southerly states. For the moment,
then, distinguishing tick populations that frequently bite humans from
those that rarely do seems to be a rational use of nomenclature,
particularly for public health officials.
Dr. Sanders is correct in pointing out that all Ixodes spp. are
"three-host" ticks, although my intent in using the term "one-host" was to
indicate that all stages of I. cookei tend to feed on the same kind of
animal (sometimes a single animal, within burrows), usually woodchucks,
skunks, or raccoons. I regret the confusion from my use of the acarologic
term in a descriptive context.
Sam R. Telford III
Harvard School of Public Health, Boston, Massachusetts, USA
References
  1. International code of zoological nomenclature. 3rd ed. London:
     International Trust for Zoological Nomenclature; 1985.
  2. Oliver JH Jr, Owsley MR, Hutcheson HJ, James AM, Chen C, Irby WS, et
     al. Conspecificity of the ticks Ixodes scapularis and I. dammini
     (Acari: Ixodidae). J Med Entomol 1993;30:54-63.
  3. Barbour AG. Does Lyme disease occur in the South? A survey of emerging
     tickborne infections in the region. Am J Med Sci 1996;311:34-40.
  4. Campbell GL, Paul WS, Schriefer ME, Craven RB, Robbins KE, Dennis DT,
     et al. Epidemiologic and diagnostic studies of patients with suspected
     early Lyme disease, Missouri, 1990-1993. J Infect Dis 1995;172:470-80.
  5. Levine JF, Apperson CS, Nicholson WL. The occurrence of spirochetes in
     ixodid ticks in North Carolina. Journal of Entomological
     Science1989;24:594-602.
  6. Kocan AA, Mukolwe SW, Murphy GL, Barker RW, Kocan KM. Isolation of
     Borrelia burgdorferi (Spirochaetales: Spirochaetaceae) from Ixodes
     scapularis and Dermacentor albopictus ticks (Acari: Ixodidae) in
     Oklahoma. J Med Entomol 1992;29:630-3.
  7. Luckhart S, Mullen GR, Wright JC. Etiologic agent of Lyme disease,
     Borrelia burgdorferi, detected in ticks (Acari: Ixodidae) collected at
     a focus in Alabama. J Med Entomol 1991;28:652-7.
  8. Pung OJ, Banks CW, Jones DN, Krissinger MW. Trypanosoma cruzi in wild
     raccoons, opossums, and triatomine bugs in southeast Georgia, USA. J
     Parasitol 1995;81:324-6.
  9. Lavender DR, Oliver JH Jr. Ticks (Acari:Ixodidae) in Bulloch County,
     Georgia. J Med Entomol 1996;33:224-31.
 10. Spielman A, Wilson ML, Levine JF, Piesman J. Ecology of Ixodes dammini
     borne human babesiosis and Lyme disease. Annu Rev Entomol
     1985;30:439-60.
 11. Rich SM, Caporale DA, Telford SR III, Kocher TD, Hartl DL, Spielman A,
     et al. Distribution of the Ixodes ricinus-like ticks of eastern North
     America. Proc Natl Acad Sci U S A 1995;92:6284-8.
 12. Norris DE, Klompen JSH, Keirans JE, Black WC IV. Population genetics
     of Ixodes scapularis (Acari:Ixodidae) based on mitochondrial 16S and
     12S genes. J Med Entomol 1996;33:78-89. 996;33:78-89.
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Ebola/Athens Revisited
To the Editor: After our hypothesis that the plague of Athens (430 B.C.-
425 B.C.) could have been caused by Ebola virus was published in this
journal (1996;2:155-6), it was brought to our attention that this
hypothesis had been previously entertained.
Gayle D. Scarrow had published a paper entitled "The Athenian Plague: A
Possible Diagnosis" in The Ancient History Bulletin 2.1 (1988).
Unfortunately, this had not come to our attention in our literature search,
and therefore we assumed that we were the first to recognize the
possibility. Clearly, Ms. Scarrow deserves credit for suggesting this
first. Her arguments are compelling, even without the support of more
recently available information and the observations advanced in our
publication.
We believe an evolving knowledge base (e.g., the information about the Cte
d'Ivoire outbreak where a protracted epidemic has been meticulously
documented) will serve to enhance the credibility of the Ebola/Athens
hypothesis.
P.E. Olson,* A.S. Benenson, and E.N. Genovese
*U.S. Navy Balboa Hospital, San Diego, California, USA; San Diego State
University,
San Diego, California, USA
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[EID Home Page]
Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA
URL: http://www.cdc.gov/ncidod/EID/vol4no1/letters.htm
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[Emerging Infectious Diseases * Volume 4 * Number 1 * January - March 1998]
News and Notes
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Meeting Summaries
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The Hot Zone--1997: Conference on Emerging Infectious Diseases
On June 27-28, 1997, the University of Kentucky College of Medicine,
University of Cincinnati College of Medicine, and Kentucky AIDS Consortium
held a conference for clinicians and researchers in Lexington, Kentucky.
Participants presented the latest findings from worldwide epidemiologic
studies, basic science, and clinical research on emerging infectious
diseases. The findings indicate that the war against infectious diseases is
far from over. In the United States, between 1980 and 1992, deaths from
infectious diseases increased by 58%, making serious infections the third
leading cause of death; HIV infection is the leading cause of death among
25- to 44-year-olds; and antibiotic resistance costs the health-care system
an estimated $100 million to $30 billion each year.
With the global population growing from 2.5 to 5.8 billion over the last 25
years, large urban centers throughout the developing world are overcrowded
and have inadequate sanitation, ideal for the emergence of infectious
diseases. By 2025, the global population will reach 8.6 billion. In
developing countries, this represents an 84% increase, which will intensify
overcrowding in these areas. In industrialized countries, an aging
population base, the advent of immunosuppressive medications, and the
emergence of HIV are combining to increase the risk for opportunistic
infection. Moreover, with increased travel, clinicians see increasing
numbers of patients with exotic diseases acquired abroad. Recent migration
of epidemic diphtheria from the former Soviet Union to Europe and the
emergence of multidrug-resistant tuberculosis (TB) in the United States and
elsewhere are but two examples of infections resulting from international
travel; in addition, nearly 70% of the fruits and vegetables consumed in
the United States originate in developing countries; disease outbreaks
related to imported food frequently go unreported.
Extensive cross-species contact among humans and certain domestic animals
can dictate antigenic shifts in influenza viruses. The likelihood of the
emergence of a new influenza virus in the near future increases with the
growth of the hog population in China. The emergence of new viruses, such
as HIV and filoviruses, indicates the virtually unlimited capacity of
pathogenic organisms to mutate and rapidly adapt to environmental changes
and selective pressures.
HIV/AIDS Research
Recent data from long-term survivors support the concept that HIV
replication occurs when the number of CD4+ cells drops below the minimum
level required to maintain CD8+ cell control of HIV. CD4+ cell production
of IL-2 is needed for strong cell-mediated immunity. Without CD8+ cell
responses, a more virulent, highly cytotoxic viral strain emerges, killing
greater numbers of CD4+ cells and leading to AIDS. Because CD8+ cell loss
appears to be related to a shift from a TH-1- to a TH-2-type cytokine
response, therapeutic approaches that maintain TH-1 cell response, or
enhance CD8+ cell anti-HIV activity through factors yet to be fully
defined, are being actively investigated. Vaccines relying exclusively on
antibody responses will almost certainly prove to be of limited value,
while those using CD8+ cell antiviral activities hold substantially greater
promise.
A basic science research forum described the use of a murine model of
immunodeficiency induced by a type C retrovirus. Because models of this
type reproduce a number of the clinical pathophysiologic manifestations
associated with human AIDS, they can significantly enhance our
understanding of retrovirus-induced immunodeficiency.
The results of recent research involving host immune responses to
Pneumocystis carinii infection indicated that, compared with healthy
controls, HIV patients with less than 200 CD4+ cells have similar IL-4
levels but significantly lower peripheral blood mononuclear cell
proliferative responses and IFN-[gamma] levels to P. carinii major surface
class glycoprotein (MSG). Centers for Disease Control and Prevention (CDC)
Class 3 patients with previous P. carinii pneumonia (PcP) have
significantly higher IL-4 (but not IFN-[gamma]) levels than Class 3
patients with no history of PcP. HIV+ patients who have recovered from PcP
have sufficient memory cells to recognize MSG, but demonstrate a shift from
a TH-1- to a TH-2-type antigen recall response.
HIV Therapy
The current state of CD4 and viral load testing and the 11 drugs available
for HIV treatment were reviewed. Although combination drug therapy has
consistently proved more effective than monotherapy in maintaining reduced
viral load, the results of a recently completed follow-up study confirmed
the effectiveness of zidovudine (AZT) in preventing neonatal HIV
transmission; in a large cohort of pregnant women, AZT plus high titer
immunoglobulin G was no more effective than AZT plus placebo, with both
regimens producing results comparable with those achieved with AZT alone. A
number of issues in HIV treatment remain unresolved, including when
treatment should begin, how to manage inadequate response to therapy,
whether to use HIV resistance genotyping to direct therapy, and how to best
deal with prophylaxis for opportunistic infections in patients showing
dramatic reductions in viral load.
Dengue and Dengue Hemorrhagic Fever (DHF)
Tens of millions of cases of dengue and hundreds of thousands of cases of
DHF are reported annually, and more than 2.5 billion people are at risk for
infection. Factors contributing to the emergence of dengue include
unplanned and uncontrolled population growth associated with urbanization
in tropical regions, lack of effective mosquito control, deteriorating
water systems that increase densities of Aedes aegypti, and viral migration
among tropical urban centers due to increasing international air travel.
More than 50% of all air travel from the United States is to tropical
destinations, and from 1977 to 1994, 2,248 suspected cases of imported
dengue were reported in the United States. Because their clinical symptoms
are initially nonspecific, dengue and other arboviral infections can be
difficult to distinguish from other viral, bacterial, and parasitic
infections. Correct diagnosis requires a detailed clinical summary,
thorough epidemiologic information (including recent travel history), and a
diagnostic laboratory test.
The severe hemorrhagic form, DHF/dengue shock syndrome (DSS), has an
average incubation period of 4 to 6 days before sudden onset of fever and
nonspecific signs and symptoms. Because the major pathophysiologic
abnormality observed in DHF/DSS is increased vascular permeability and
leakage of plasma from the vascular compartment, early fluid replacement is
effective. The geographic distribution of DHF/DSS has been expanding and
now can be found in tropical areas of Asia, the Pacific, and the Americas,
including Central America and Mexico. This expansion is associated with
increased movement of dengue viruses by airplane travelers and the
development of hyperendemicity in the Pacific Region and the Americas. A
similar scenario, generated largely from human encroachment into new
environments, may also emerge for other Aedes-transmitted illnesses such as
yellow fever. The most cost-effective approach to control dengue and DHF is
larval source reduction in disease-endemic areas. Programs should use both
government and community resources to integrate environmental sanitation
with the use of insecticides and biologic controls, targeted to breeding
grounds such as tire dumps.
Other Viral Diseases
The exponential increase in ecologic change, both environmental and
behavioral, was cited as the major driving force for the increasing human
risk for viral infection. Microbial variability can play a causal role in
disease emergence, but it more often enables viruses to adapt to new
circumstances. Travel of infected humans and international transport of
microbes and vectors help provide the maximum possible microbial
evolutionary opportunities in the minimum amount of time.
Viruses have emerged in the past, with measles providing a good example of
the worrisome potential for future emerging RNA viruses. The emergence of
cities in the Mesopotamian basin, resulting largely from the advent of
irrigated agriculture, provided a populated substrate for interhuman
transmission of short-incubation, nonlatent viruses. Domestication of
livestock likely brought measles progenitors into close proximity with
humans; a precursor of rinderpest/peste de petit ruminants then made an
interspecies leap; much later measles spread to Europe, and, in the
post-Columbian interchange, to the Americas.
Viral hemorrhagic fevers include those caused by Ebola filovirus and
hantaviruses. Ebola hemorrhagic fever is characterized by extensive and
disseminated infection and necrosis in major organs, and lymphoid
depletion. Aerosol transmission of Ebola virus has occurred between
nonhuman primates and guinea pigs, but no evidence exists for interhuman
transmission by airborne infection. Barrier nursing precautions generally
prevent the spread to humans, but in areas having inadequate medical care
facilities, the virus can amplify in humans and cause epidemics.
Hantaviruses belong to a single genus in the family Bunyaviridae, and each
virus infects a limited or unique rodent species with no apparent disease.
Although hemorrhagic fever with renal syndrome has rarely been diagnosed in
the Americas, hantaviruses from sigmodontine rodents cause hantavirus
pulmonary syndrome, characterized by large bilateral pleural effusions and
heavy, edematous lungs, interstitial pneumonitis, and extensive infection
of endothelial cells in the pulmonary microvasculature. The first
documented interhuman transmission of a hantavirus was an outbreak of 20
cases in Patagonia, with evidence overwhelmingly indicating spread between
patients and physicians. The reasons for, and mechanism of, the spread are
unknown, but the registry of U.S. cases was revised to ensure that this
phenomenon was adequately monitored. Although early diagnosis and
supportive care are potentially lifesaving in cases of hantavirus pulmonary
syndrome, such efforts are of limited value in Ebola hemorrhagic fever.
Prion Illnesses
In prion illnesses such as Creutzfeldt-Jakob disease (CJD), risk factors
(family history of CJD or dementia, history of poliomyelitis, exposure to
sheep or cows) and iatrogenic factors (dura or corneal grafts and exposure
to pooled human growth hormone) are important. At onset, CJD is commonly
misdiagnosed as psychotic illness, Alzheimer's dementia, paraneoplastic
syndrome, vascular brain disease, parkinsonism, or even drug-induced
delirium. A variant of CJD, caused by a prion with an altered protein
configuration, is bovine spongiform encephalopathy (BSE or mad cow
disease). Although the precise mechanism of infection originally reported
in U.K. cattle is unclear, BSE has been exported to other countries by
feeding cattle inadequately processed bone meal. The potential for the
emergence of BSE in the United States exists because similar reservoirs of
infection are present. Casual handling of beef and beef products in
processing plants and uncontrolled disposal of sick cattle may increase
that risk. Several cases of CJD have been reported in Kentucky patients who
consumed squirrel brains; however, a causal link has not been established.
The most recently identified prion illness is a new variant of CJD reported
in England that, unlike sporadic CJD, occurs in much younger patients (16
to 50 years of age), can last longer than 1 year, and is characterized by
the presence of psychiatric and sensory symptoms and the absence of kuru
plaques and electroencephalogram periodic complexes. The illness is thought
to be linked to BSE. Mathematical modeling suggests that 75,000 to 80,000
cases will occur in the foreseeable future.
Tick-Borne Diseases
As with many other emerging infectious diseases, concern about tick-borne
zoonotic diseases in the United States has increased because of
environmental changes brought on by human settlement and socioeconomic
factors that place humans at greater risk for tick exposure, such as the
development of suburban housing in disturbed natural settings. Current
conditions appear favorable for continued increases in vector tick
populations and their geographic expansion and for increasing interaction
between ticks and humans. In the United States, the prevalence of Rocky
Mountain spotted fever may continue to increase as the urbanization of the
western and southern regions expands opportunities for human exposure to
tick-borne pathogens.
The epidemiology of Lyme disease and tularemia was reviewed, and the
concept of a southern (U.S.) tick-associated rash illness (STARI), the
epidemiology of which is still being defined, was introduced. STARI is
characterized by an expanding erythematous rash resembling that of Lyme
disease, mild or absent constitutional symptoms, and no well-described
sequelae. The rash responds well to antibiotic treatment. STARI is seen in
the range of the human-biting Lone Star tick (Amblyomma americanum) and is
frequently associated with a Lone Star tick bite. Studies indicate that
this infection is not caused by Borrelia burgdorferi or other known
tick-borne agents. Amplified segments of the genome of this spirochete have
recently been described, and some investigators have proposed that it
represents a new species, B. lonestari. It has been suggested that STARI
may be closely related or identical to a commensal spirochete of deer, B.
theileri.
The need for early diagnosis and treatment of Rocky Mountain spotted fever
was stressed by presenting data from 94 infected patients in North
Carolina. Death rates were significantly lower (6.5% vs. 22.9%, p < 0.03)
in patients receiving appropriate treatment within 5 days of onset of
illness than in patients who received delayed or no treatment. Predictors
of death included renal failure; elevated serum creatinine, aspartate
transaminase, and bilirubin; decreased serum sodium; thrombocytopenia;
neurologic involvement; and male gender.
Parasitic Diseases
The role of the cell surface glycoconjugate lipophosphoglycan (LPG) in the
survival of Leishmania parasites whose life cycle alternates between
intracellular parasitism and extracellular life in sand fly vectors was
explored. Data indicating that LPG is a multifactorial molecule may lead to
a biochemical rationale for LPG-targeted chemotherapeutic regimens.
Isolation of a full-length genomic clone of the acid [alpha]-mannosidase
from an epimastigote genomic library of Trypanosoma cruzi was reported.
Sequence analysis showed a single open reading frame encoding the
[alpha]-mannosidase gene. Because the size of this frame is consistent with
that of lysosomal mannosidases in humans, these results could lead to the
exploration of new chemotherapeutic options in Chagas disease.
A bank of 5,200 insertion mutants has been used to characterize the
parasitic mechanisms used by Legionella pneumophila. Results from
transmission electronmicroscopy indicate that L. pneumophila has acquired
genetic loci specific for survival and replication within mammalian cells,
allowing evolution from a protozoan parasite into its present
disease-causing agent. Alternatively, ecologic coevolution of L.
pneumophila to parasite protozoa has led to the development of multiple
redundant mechanisms, some of which do not function within mammalian
macrophages.
TB
Poverty, changing immigration patterns, and the emergence of HIV disease
were cited as factors contributing to an attenuated rate of decrease of TB
incidence in the United States since the late 1980s and its increase as a
major global problem. Poorly managed TB control programs, suboptimal access
to health care, an inadequate physician knowledge base, and poor patient
compliance have combined to increase the incidence of TB and, especially,
multidrug-resistant TB; sensitivity testing is critical in the management
of resistant TB. Updated CDC guidelines for interpreting the purified
protein derivative skin test call for responses to be considered positive
if induration is greater than 5 mm in those with HIV or who have recent TB
contact, and greater than 10 mm in foreign-born patients, intravenous drug
users, the homeless, and immunocompromised patients. Because of the
"boosting" phenomenon, two-step purified protein derivative skin testing is
now recommended for health-care workers and nursing home patients who are
retested periodically. Treatment of active TB should use multiple drugs,
avoid adding single agents, and include compliance monitoring, preferably
directly observed therapy. Workplace prevention measures should be driven
by a consistently high index of suspicion and should include appropriate
isolation of suspected cases, work-ups following exposure, rigorous
reporting to health departments, and locating recalcitrant patients.
Effective control of TB will require social, political, and cultural
changes, as well as medical innovation.
Plague
The epidemiology, pathophysiology, and treatment of plague, as well as the
improvement of diagnostic techniques for infections caused by Yersinia
pestis, were reviewed. Such new tests will permit the public health
laboratory to quickly identify a plague outbreak and apply the appropriate
control measures to limit its spread.
The pathogenesis of plague was explored, and two separate, but essential,
iron transport systems were identified in Y. pestis. The first, the
yersinabactin (Ybt) system, enables the organism to proliferate from the
site of an open wound or bite, while the second, identified as Yfe, is used
by Ybt mutants to obtain iron during infection of internal organs. The Ybt
iron transport system appears to be essential for growth in the early
stages of bubonic plague infection, while the Yfe system functions to allow
growth during, or after, infection of internal organs. Both systems are
required for Y. pestis to be fully virulent.
A novel mechanism by which a Y. pestis virulence protein is sequestered in,
and transported within, host cells was described. When yersiniae contact a
eukaryotic cell, a signaling event activates the expression and secretion
of a set of four toxins (Yops) and causes their vectorial translocation
into the cell cytoplasm. Three of the Yops derange cell signaling and
cytoskeletal functions by kinase, tyrosine phosphatase, and actin
depolymerization activities. Although no activity or intracellular target
has been identified for the fourth known translocated Yop, YopM, immunoblot
analysis and laser scanning confocal microscopy demonstrated that most YopM
is vectorially translocated into HeLa cells or the macrophagelike cell line
J774 by adherent Y. pestis and travels to the eukaryotic cell nucleus.
Because a growing number of important human pathogens (e.g., Salmonella,
Shigella, and Pseudomonas) have similar, but less well-studied,
secretion/translocation mechanisms and putative secreted toxins, these
findings will facilitate studies that ultimately could lead to novel
therapies for these agents.
Antibiotic Resistance
The current development of staphylococci resistant to methicillin or
fluoroquinolones and gram-negative bacilli resistant to extended-spectrum
beta-lactams are but the most recently recognized patterns of antibiotic
resistance. General approaches for modifying these trends include 1) source
control, particularly handwashing, and the need to wear gloves during
contacts with all patients, 2) improved antibiotic use and control, 3)
improved infection control devices, and 4) better use of pathophysiology
and immunologic modulation.
The growing problem of vancomycin-resistant enterococci (VRE), with an
incidence of 20% to 40% in some groups of U.S. hospitalized patients,
necessitates maximal use of all these approaches. Skin proliferation of VRE
produces extensive environmental contamination that may require universal
use of gloves to control outbreaks or hyperendemic disease. In addition,
vancomycin should be limited to treatment of beta-lactam resistant
gram-positive bacteria (such as methicillin-resistant Staphylococcus aureus
and gram-positive bacteria in beta-lactam allergic patients) and
Clostridium difficile (only after metronidazole failure) and to
endocarditis prophylaxis. Vancomycin should be avoided in routine surgical
prophylaxis, empiric treatment of febrile neutropenia with negative
cultures, and pneumonia prophylaxis in the intensive care unit. A number of
experimental peptides and other agents (such as quinupristin-dalfopristin)
under investigation as treatments for VRE infections were identified.
Future trends in resistance may include further spread of
vancomycin-resistant staphylococci (already reported in Japan), quinolone-
or carbapenem-resistant gram-negative bacilli, and treatment-resistant
viruses. Seventeen isolates of methicillin-resistant S. aureus with unique
genotypes were studied to determine rates of resistance to the
fluoroquinolones ciprofloxacin and levofloxacin. The mean single-step
resistance to 4 x MIC ciprofloxacin was 1.05 x 10(sup-5) and to levofloxacin was
4.03 x 10(sup -6). When serially passaged in increasing antibiotic
concentrations, the geometric mean MICs for ciprofloxacin and levofloxacin
increased 3.0 +/- 1.5 times and 1.8 +/- 1.4 times, respectively (p < 0.0005).
Only four strains became resistant to levofloxacin, but eight became
resistant to ciprofloxacin, indicating that ciprofloxacin selects
methicillin-resistant S. aureus more frequently than levofloxacin.
Other Topics
The laboratory evidence supporting the role of Chlamydia pneumoniae in the
development of atherosclerosis was reviewed. Recent data indicate that
infection of vascular endothelial cells with C. pneumoniae is associated
with the production of chemokines and adhesion molecules that promote
transendothelial migration of neutrophils and monocytes. These findings
suggest that immunopathogenic responses to C. pneumoniae infection may
contribute to the development of clogging deposits.
Data were presented demonstrating the proliferation of human CD4+ T cells
from unexposed persons in response to in vitro exposure to Toxoplasma
gondii. Further studies showed that this proliferative response depends on
HLA-DR molecules and requires processing of Tg antigens. In contrast to
typical exogenous superantigens, analysis of TCR V[beta] expression after
stimulation with Tg did not show a pattern of preferential increase of a
specific TCR V[beta]-bearing subpopulation. [alpha][beta]T cells secreted
significant amounts of IFN-[gamma] after incubation with Tg-infected
monocytes. This process may play an important role in the early events of
the immune response to T. gondii.
Richard N. Greenberg,* Judith E. Feinberg, and Claire Pomeroy*
*University of Kentucky Medical Center, Lexington, Kentucky, USA;
University of Cincinnati College of Medicine, Cincinnati, Ohio, USA
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International Meeting on Borreliosis, Prague, Czech Republic
Approximately 150 participants from 10 countries gathered in Prague, the
Czech Republic, August 27-29, 1997, to discuss research topics related to
the theme of the meeting, "Lyme Borreliosis-Basic Science and Clinical
Approaches." The meeting was organized by the National Institute of Public
Health (Centre of Epidemiology and Microbiology); the World Health
Organization Collaborating Center for Reference and Research on
Borreliosis; the Second School of Medicine, Charles University (Prague);
and the Czech Medical Association J.E. Purkyn;. Meeting sessions focused
on topics including epidemiology, clinical treatment, dermatology,
diagnosis and treatment, neurology, and laboratory diagnosis.
The session on epidemiology presented surveillance data on the incidence of
Lyme borreliosis (LB) in the Czech Republic (incidence rates were
61.8/100,000 in 1995 and 41.2/100,000 in 1996) and in Slovakia, Austria,
and Slovenia. Data underscored the high risk for transmission of LB in
central and eastern Europe. The results of vaccine trials using the
recombinant outer surface protein (Osp)A antigen of Borrelia burgdorferi
were also presented; more detailed studies are needed to examine
intraspecies variability of OspA antigens in Europe.
The session on clinical approaches and treatment reviewed research
conducted in the United States and discussed the diagnostic importance of
organism-specific biologic markers, e.g., Borrelia-specific antigens or
DNA, as well as pleocytosis in cerebrospinal or synovial fluid. Experience
with the diagnosis and treatment of LB in the hyperendemic-disease regions
of west Bohemia underscored the importance of accurate diagnosis in
avoiding overtreatment.
The use of nonhuman primates as models for studying neuroborreliosis was
examined in the session on neurology. Problems related to the diagnosis and
treatment of chronic disease, and their economic consequences, were
identified. Several methods to assist clinicians in making a correct
diagnosis were presented and discussed. The persistence of B. burgdorferi
DNA in patients with Lyme arthritis was considered in the rheumatology
session. Ultrastructural evidence for the intracellular location of B.
burgdorferi in synovium also was presented.
The session on laboratory diagnosis focused on the genomic sequence of the
linear chromosome of B. burgdorferi (B31 strain) and the crystal structure
of OspA; both apply to the laboratory diagnosis of LB. Other studies
affirmed the importance of standardizing diagnostic methods to ensure
reproducibility and uniformity of the results from different laboratories.
The influence of certain in vivo-expressed antigens (virulence antigens) on
invasiveness and the ability of B. burgdorferi to adapt to the host
environment were noted. Other topics were the sensitivity and
reproducibility of polymerase chain reaction and the importance of the
primers selected for the assay.
The studies presented in the poster session addressed a wide array of
themes: among them, epidemiology and population awareness, reactivity of B.
burgdorferi antigens in immunoblot procedures when specimens derived from
humans or animals are used, and incidence of ticks and their association
with disease in different regions.
The importance of apoptosis in the morphology of LB, the role of Langerhans
cells in the skin reactions, and the role of integrin CR3 in the
interaction of B. burgdorferi with host cells were discussed. The
sensitivity and the selection of the primers used for polymerase chain
reaction to detect B. burgdorferi in ticks were considered. Aspects of
vector biology and ecology were investigated (e.g., habitats, the tick as
LB's major vector, vector capacity). Other diseases transmitted by Ixodes
ricinus ticks in Europe (e.g., tick-borne encephalitis, babesiosis,
ehrlichiosis) as well as human ehrlichiosis in Europe were reviewed.
Dagmar Hulnsk and Jir Bata
National Institute of Public Health, Prague, Czech Republic
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Workshop on Climate Change and Vector-Borne and Other Infectious Diseases
Climate changes may affect human health through a myriad of pathways; of
particular interest are pathways affecting the geographic ranges and
incidence of vector- and water-borne diseases. As society chooses how to
deal with projections of long-term climate change, decisions must be based
on scientific knowledge. A 2-day workshop(ft1) was convened in September 1997
to discuss what is known about the relationship between projected climate
changes and the incidence of water-borne diseases (e.g., cholera) and
vector-borne diseases, including those typically considered tropical
(malaria, dengue fever, yellow fever, and schistosomiasis), plus
subtropical or temperate-zone diseases whose vectors are likely to be
affected by projected climate changes.
The workshop participants discussed the systems involved in potential
climate changes, from the global ocean-atmosphere-landmass system that
drives climate to the regional ecologic and human socioeconomic systems
where disease dynamics occur. These systems are extremely complex, as are
the interactions among them, which underscores the need for more research
before accurate projections can be made. Major research gaps were
identified, and an agenda was framed for a sound scientific basis for
public policy debates and decisions. The proposed agenda included the
following items: climate modeling; ecosystem and habitat dynamics; disease
surveillance; technologies for disease prevention and mitigation; disease
transmission dynamics; data sets for empirical studies; integrated
assessments; and detecting, understanding, and responding to unexpected
events. Further discussion and implementation of this research agenda is
encouraged. A summary of the workshop is available from the Electric Power
Research Institute, TR-109516, EPRI Distribution Center, 207 Coggins Drive,
P.O. Box 23205, Pleasant Hill, CA 94523; Telephone: 510-934-4212.
(sup1)The workshop was commissioned by the Electric Power Research Institute,
with additional sponsorship from the Department of Energy, the National
Institute of Allergy and Infectious Disease, the National Institute of
Environmental Health Sciences, and the National Aeronautics and Space
Administration. The workshop was organized and conducted by the Washington
Advisory Group. The 28 participants included representatives from agencies
and institutions that conduct or fund research and experts in the fields of
climatology and global climate modeling, public health, and the biology and
ecology of vectors, pathogens, and the ecosystems they inhabit.
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The Fourth International Conference on HFRS and Hantaviruses
Atlanta, Georgia, USA, March 5-7, 1998
The Centers for Disease Control and Prevention and other cosponsors will
host the Fourth International Conference on HFRS and Hantaviruses to
facilitate the exchange of scientific information in the following areas:
1) clinical aspects, 2) laboratory diagnostics, 3) pathogenesis and immune
response, 4) hantavirus ecology, 5) hantavirus epidemiology, 6) molecular
biology and cell interactions, 7) health education and prevention, and 8)
antiviral and vaccine development. The meeting will offer plenary sessions
with invited speakers, as well as oral and poster sessions based on
accepted abstracts.
For further information, contact Amy Corneli, Centers for Disease Control
and Prevention, 1600 Clifton Road, MS A26, Atlanta, GA 30333, USA; fax:
404-639-1509; e-mail: akc8@cdc.gov; URL:
http://www.cdc.gov/ncidod/diseases/hanta/hantconf.htm.
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Third International Congress on Tropical Neurology
November 30-December 2, 1998
Organized by the Groupe Francophone d'Etude et de Recherche en Neurologie
Tropicale, the Third International Congress on Tropical Neurology will
convene in Fort de France, Martinique, from November 30 to December 2,
1998. The four main themes of the congress are central nervous system
inflammatory, neurodegenerative, epileptic, and cerebrovascular disorders
in tropical environments; however, presentations on other themes are
welcome.
A symposium on epilepsy in tropical zones will be held during the congress.
For additional information, contact Professor M. Dumas (phone:
33-5-55-43-58-20, fax: 33-5-55-43-58-21) or Professor J.C. Vernant (phone:
33-5-96-55-22-61, fax: 33-5-96-75-45-90).
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International Conference on Emerging Infectious Diseases
March 8-11, 1998
Atlanta Marriott Marquis Hotel
Late-breaker abstract submission deadline: January 30, 1998
Information on abstract submission, conference registration, and exhibits
can be obtained at www.asmusa.org, by sending an e-mail message to
meetinginfo@asmusa.org, or by calling 202-942-9248. Proceedings of the
conference will be published in the journal Emerging Infectious Diseases.
Registration: limited to 2,500 - register NOW!
Preliminary program information is available at
http://www.cdc.gov/ncidod/EID/98conf.htm.
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Emerging Infectious Diseases
National Center for Infectious Diseases
Centers for Disease Control and Prevention
Atlanta, GA
URL: http://www.cdc.gov/ncidod/EID/vol4no1/ascii/newsnote.txt
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