Download Lack of Evidence of Measles Virus Shedding in People with

SUPPLEMENT ARTICLE
Lack of Evidence of Measles Virus Shedding
in People with Inapparent Measles Virus Infections
Fabio A. Lievano,1 Mark J. Papania,1 Rita F. Helfand,2 Rafael Harpaz,1 Laura Walls,2 Russell S. Katz,2
Irene Williams,2 Yvonne S. Villamarzo,2 Paul A. Rota,2 and William J. Bellini2
1
Measles Elimination Activity, Child Vaccine Preventable Diseases Branch, Epidemiology and Surveillance Division, National Immunization
Program, and 2Measles Virus Section, Respiratory and Enteric Viruses Branch, Division of Viral and Rickettsial Diseases, National Center
for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
Serological evidence of measles virus infection has been detected among people exposed to measles who do
not exhibit classical clinical symptoms. Throat swabs, lymphocytes, and serum and urine samples were collected
from contacts of individuals with confirmed measles 12–16 days after exposure, during measles outbreaks
occurring in 1998. Follow-up serum samples were drawn 2 weeks later. Samples were tested for measles IgM
antibody by enzyme immunoassays and plaque reduction neutralization testing. Virus isolation and reverse
transcriptase–polymerase chain reaction testing was attempted for all samples. None of the 133 contacts
developed classical measles disease; 11 (8%) had serological evidence of infection. Duration of exposure of
⭓3 h was the only significant risk factor for developing serological response (24% vs. 4% among contacts
exposed for 1–2 h; relative risk, 6.0; 95% confidence interval, 1.9–19.2). None of the 133 contacts had virological
evidence of infection by culture or polymerase chain reaction. We found no evidence that persons with
inapparent measles virus infections shed measles virus.
Measles control strategies are based on the assumption
that measles virus transmission occurs in chains of
transmission of clinically recognizable measles cases.
Surveillance systems rely on the identification of persons with the clinically recognizable symptoms of measles for detecting and responding to outbreaks, vaccinating susceptible contacts, and assessing the efficacy
of vaccines and the impact of vaccination programs.
However, it has been postulated that the occurrence
of measles virus infections in persons without classical
measles symptoms may play an important role in the
transmission of measles. Serological evidence of acute
measles has been documented among people who are
exposed to measles virus but do not develop classical
symptoms [1–10]. This phenomenon has been observed most frequently in highly vaccinated populations
with rates of infection of 4%–42% [2, 4, 5, 8, 9].
Inapparent measles virus infections are clinically un-
Reprints or correspondence: Dr. Fabio A. Lievano, Centers for Disease Control
and Prevention, 1600 Clifton Rd., Mailstop E-05, Atlanta, GA 30333 (flievano@
cdc.gov)
The Journal of Infectious Diseases 2004; 189(Suppl 1):S165–70
This article is in the public domain, and no copyright is claimed.
0022-1899/2004/18909S1-0025
important, because there are no symptoms or only mild
symptoms. However, inapparent infections do provide
a boost in measles antibody titers, which some investigators have suggested provide increased protection
against future exposures to measles [9]. However, this
increased protection is probably not necessary. People
with inapparent infections, by definition, have been exposed to measles virus and did not develop measles
disease.
Several different terms have been used to describe
measles virus infections in persons without classical
symptoms, including subclinical measles [7, 9], asymptomatic measles [2, 4, 5, 6], modified measles [1, 4, 8],
mild measles [1, 5, 6], and secondary immune response
[2, 3, 4, 6]. Herein we use the term “inapparent infection” to indicate serological evidence of measles virus
infection in any person who does not meet the clinical
case definition of measles (the clinical case definition
of measles is a case of generalized maculopapular rash
lasting for ⭓3 days, with fever of ⭓38.3C, and cough,
coryza, or conjunctivitis [11]).
Inapparent measles virus infections could be epidemiologically important if infected persons are capable
of transmitting measles virus. Many countries, includShedding and Inapparent Infection • JID 2004:189 (Suppl 1) • S165
ing the United States, have the elimination of measles as a
public health goal [12]. Recent mathematical models have suggested that eliminating measles would be impossible if measles
virus can be transmitted in the absence of classical measles
disease [13]. However, there is very little information available
regarding the infectivity of persons with inapparent measles
virus infections. There are no studies of persons with inapparent
measles virus infections that document respiratory shedding of
measles virus, which is a presumed prerequisite for transmitting
the virus to other people. In 1999, Vardas et al. [6] reported
that measles virus was isolated from urine of an asymptomatic
contact of a person with measles. The nucleotide sequence of
the measles virus isolated from the contact was identical to that
of the virus isolated from the individual with measles. Although
genotypically identical viruses were recovered, the authors
could not rule out cross-contamination.
To estimate the potential for measles virus transmission from
persons with inapparent infections, we studied a cohort of people exposed to confirmed measles cases. We collected blood,
urine, and respiratory specimens to identify those with inapparent infections and to determine whether any of the exposed
cohort had detectable measles virus.
METHODS
Study population. Persons exposed to confirmed measles
cases during outbreaks in the fall of 1998 in Anchorage [14]
and Phoenix were contacted to identify those who met the
following study criteria: contact with a person with confirmed
measles for ⭓1 h in the same room during the infectious period
(from 3 days before rash onset through 3 days after rash onset),
availability for specimen collection at the times defined by the
protocol, and no receipt of measles vaccine and no diagnosis
of measles within 2 months before specimen collection. Eligible
contacts were invited to participate in the study. Written consent was obtained from participants, and the protocol was approved by the Institutional Review Board of the Centers for
Disease Control and Prevention (CDC).
We collected demographic information; previous history of
measles, rubella, or chickenpox; and vaccination history. We
also collected details of the exposure and symptoms experienced
after exposure. We obtained samples of blood and urine and
throat swabs from the participants 12–16 days after their exposure to measles. Viral shedding is most intense in a classical
measles case during this period [12]. We estimated that this
period would give us the highest likelihood of detecting measles
virus in this cohort. Two days later, throat swabbing was repeated to increase the probability of detecting pharyngeal shedding of measles virus. About 14 days after the collection of the
initial blood sample, a follow-up blood sample was drawn. All
specimens were shipped immediately in a cold box with ice
S166 • JID 2004:189 (Suppl 1) • Lievano et al.
packs to Atlanta and processed immediately upon arrival at the
Measles Laboratory, CDC.
Laboratory analysis. All serum specimens were tested for
the presence of IgM antibodies as evidence of recent infection
by means of EIA capture test [15–17]. Plaque reduction neutralization (PRN) assays were done on paired serum samples
as described elsewhere [18]. We used the initial PRN antibody
titer as an estimate of baseline immunity.
Lymphocytes were separated from whole blood specimens
by using Lymphocyte Separation Medium (ICN Biochemicals).
Lymphocytes were stored at ⫺70C in cryoprotective medium.
Before being used for RNA extraction or virus isolation, lymphocytes were washed with 1 mL of sterile PBS (pH 7.5) and
resuspended in 200 mL of Dulbecco’s modified Eagle medium
(Gibco BRL Life Technologies). Urine and throat swab eluates
were centrifuged at 770 g for 15 min at 4C, and the pellet was
resuspended in 1 mL of Dulbecco’s modified Eagle medium
and stored at ⫺70C.
Virus isolation was done by inoculating 24-well plates containing B95a cells [19]. Duplicate wells were infected with 100
mL of each specimen and observed microscopically for the presence of viral cytopathic effect. Infected cells were passaged at
least 3 times. If no cytopathic effect was observed after the third
passage, the sample was considered negative.
RNA was extracted from the specimens by use of the guanidinium acid–phenol procedure, and the presence of measles
virus RNA was detected by reverse transcriptase–polymerase
chain reaction (RT-PCR) as described elsewhere [19]. The
primer sequences were the same as described elsewhere [20],
except that the primers were 5-labeled with a fluorescent dye
(FAM). The RT-PCR reactions were performed with the
GeneAmp EZ rTth RNA PCR system (Perkin-Elmer), and PCR
products were analyzed on an ABI PRISM 310 Genetic Analyzer
(Applied Biosystems). The sensitivity of this procedure was
determined to be 104 molecules of RNA.
RESULTS
A total of 44 confirmed cases of measles occurred during the
2 outbreaks. We studied 6 case patients, all of whom had laboratory-confirmed measles that occurred during the study period and agreed to provide contact information. Of the 364
exposed contacts of these 6 case patients, 153 met eligibility
criteria, and of these, 133 (87%) consented to participate.
Among the 133 cohort members, 12 (9%) were not vaccinated, 36 (27%) were previously vaccinated with 1 dose, and
85 (64%) were previously vaccinated with ⭓2 doses. Twelve
participants (9%) reported previous history of measles. One
hundred four persons (78%) were exposed for 1–2 h and 29
(22%) for ⭓3 h. The median age of the 133 participants was
16 years (range, 13–55 years). Eighty-nine participants (67%)
Table 1.
Patient
no.
1
Description of source measles cases in Anchorage and Phoenix in 1998.
Age,
years
Place of
exposure
Doses of
measles-containing
vaccine
Time since
last dose of
MMR, years
Positive for
IgM antibody
by EIA capture
25
Cruise ship
1
120
Yes
No. of
people
exposed
No. of
people
in study
F + R + C1 + C2
21
15
Symptoms
2
17
High school
2
11
Yes
F + R + C1
60
13
3
14
High school
1
14
Yes
F + R + C1 + C2 + C3
60
8
4
16
Home
1
15
Yes
F + R + C1 + C2 + C3
3
1
5
17
High school
0
NA
Yes
F + R + C1 + C2 + C3
110
55
6
45
High school
0
NA
Yes
F + R + C1 + C2 + C3
110
41
NOTE.
C1, cough; C2, coryza; C3, conjunctivitis; F, fever; MMR, measles-mumps-rubella vaccine; NA, not available; R, rash.
were female, 110 (83%) were white, and 112 (84%) were
students.
The 6 patients with measles exposed the study participants
in different settings. Contacts of 4 patients were exposed in the
high school setting. One case patient exposed contacts on a
cruise ship and 1 exposed household contacts (table 1).
None of the 133 study participants developed classical measles symptoms. Among initial and follow-up blood samples, 11
(8%) of 133 tested positive for measles-specific IgM, indicating
recent measles virus infection (95% confidence interval, 0%–
29%).
No measles virus was isolated from any of the initial or
follow-up serum samples or from lymphocytes, urine, or throat
swabs (table 2). The observed virus isolation rate was 0 among
all contacts. The upper 95% confidence intervals for this rate
were calculated as 2.2% among all 133 contacts, 10% among
Table 2. Contacts of measles patients described in table 1 who
had any positive result of testing for measles but who did not
meet the clinical case definition for measles.
Contact
no.
Exposed
Virological
to case Initial Convalescent 4-fold
results of
no.
IgM
IgM
increase throat swab 1
1
2
⫺
+
⫺
⫺
2
3
⫺
+
⫺
⫺
3
5
+
+
⫺
⫺
4
5
+
⫺
⫺
⫺
5
5
+
⫺
⫺
⫺
6
5
+
+
⫺
⫺
7
6
+
+
⫺
⫺
8
5
⫺
+
⫺
⫺
9
1
⫺
+
⫺
⫺
10
1
⫺
⫺
+
⫺
11
1
⫺
⫺
+
⫺
12
1
⫺
⫺
⫺
…a
5
7
2
1
Total
NOTE. No viruses were identified in urine, lymphocytes, or in the second
throat swabs. +, positive; ⫺, negative.
a
Nucleotide sequence (D3) did not match outbreak sequence (D6).
the 29 contacts exposed for ⭓3 h, and 29% among the 11 with
serological evidence of infection.
A throat swab from one contact without serological evidence
of infection yielded positive results by PCR. However, the nucleotide sequence obtained (D3) was not consistent with the
genotype associated with the outbreak (D6) [21], suggesting
that the result was due to a laboratory artifact. Genotype D3
has been detected only 4 times in the United States since the
1989–1991 resurgence and, in each case, was the result of importation of virus. A genotype D3 virus was used to produce
the positive control used in the RT-PCR assays [22].
We examined hours of exposure to a measles case, years since
last dose of measles vaccine, baseline immunity (as estimated
by PRN), presence of any symptom, measles history, and vaccination status as potential risk factors for inapparent infection.
As seen in table 3, only hours of exposure was found to be
statistically significantly associated with inapparent infection.
There were 4 persons (4%) with serological evidence of infection among 104 persons exposed for 1–2 h, compared with 7
(24%) of 29 exposed for ⭓3 hours (relative risk, 6.0; 95%
confidence interval, 1.9–19.2). Among those with initial PRN
titers 11052 (n p 82), 5 developed inapparent infection; of
those with titers !1052 (n p 48), 6 developed inapparent infection (relative risk, 2.0; 95% confidence interval, 0.7–6.4). No
association was found for any of the above-described variables
when an analysis was conducted stratifying the cohort by hours
of exposure.
DISCUSSION
Measles virus was not detected in respiratory, urine, or blood
specimens collected from persons exposed to measles, despite
serological evidence of measles virus infection in 8% of the
exposed cohort. Studies showing serological evidence of measles
virus infections in persons without classical measles symptoms
have raised the concern that transmission of measles virus in
persons without clinically apparent measles may be an impediment to global measles elimination and eradication. The rate
Shedding and Inapparent Infection • JID 2004:189 (Suppl 1) • S167
Table 3.
Risk factors for inapparent measles virus infections.
Percentage with
positive serological
results
Relative
risk
103
4
Ref.
30
23
6.0
!15
95
6
Ref.
⭓15
24
13
2.0
Variable
N
95% confidence
interval
Hours of exposure
!3
⭓3
1.9–19.2
Years since last MCV
0.5–7.4
Postexposure immunity (PRN titer)
⭓1052
82
6
Ref.
!1052
48
13
2.0
0.7–6.4
Symptoms
Yes
81
6
Ref.
No
51
12
1.9
No
105
8
Ref.
Yes
10
10
1.2
0.6–5.8
Measles history
0.2–8.7
MCV received
Yes
121
8
Ref.
No
12
8
1.0
NOTE.
MCV, measles-containing vaccine; PRN, plaque reduction neutralization; Ref., referent.
of these serological responses among exposed persons has been
estimated in a range from 4% to 42% by statistical and laboratory methods (table 4). The use of different diagnostic methods and different degrees of exposure may have affected the
calculated rate of inapparent measles virus infections in highly
vaccinated populations.
This was the first attempt to detect measles virus in a highly
vaccinated group of people who had been exposed to measles.
The collection of multiple specimens for virus detection (throat
swabs, urine, and lymphocytes), repeated on separate days during the period when viral shedding was most likely, was a
unique approach to evaluate the potential for spread of measles
from persons with inapparent measles virus infections [20].
Addition of highly sensitive RT-PCR testing to standard virus
Table 4.
0.1–7.2
isolation techniques maximized the likelihood of detecting
measles virus RNA present in the specimens [22].
The absence of detectable measles virus among persons with
clinically inapparent measles in this study suggests that infectious transmission from such cases is rare, if it occurs at all,
and is unlikely to be an impediment to measles elimination
and eradication efforts.
Our study suggests that the intensive exposure to persons
with symptomatic measles is required for a substantial proportion of previously immune exposed contacts to develop
inapparent infections, which reduces the likelihood that these
infections will be epidemiologically important. If persons with
inapparent infections are capable of transmitting measles virus,
they are likely much less infectious than classical cases, on the
Studies of susceptibility to inapparent measles virus infections.
Author, year
[reference]
Chen 1990 [8]
Ozanne 1992 [4]
Huiss 1997 [2]
No. of
persons
studied
Estimated and
observed susceptibility
to inapparent
infections, no. (%)
Age range,
years
18
7 (39)
18–22
496
20 (4)
7–44
44
4 (9)
26–50
Helfand 1998 [5]
44
10 (23)
18–68
Whittle 1999 [9]
102
43 (42)
7 months–7 years
S168 • JID 2004:189 (Suppl 1) • Lievano et al.
Assay used
Plaque reduction neutralization
Country
United States
Complement fixation
Canada
EIA, neutralization, hemagglutination inhibition
Luxemburg
EIA capture
United States
Hemagglutination inhibition
Senegal
basis of the absence of detectable measles virus in respiratory
specimens in our study. Therefore, the likelihood that exposure
to an inapparently infected person would lead to another inapparent infection is probably 0.
Measles surveillance data from the United States are consistent with an extremely limited role of inapparent infections in
measles virus transmission. There are only a few cases of confirmed measles each year in which epidemiological linkage to
a classical measles case is not detected [23–25].
Our study had limited power to detect viral shedding, because we were able to identify only 11 people with serological
evidence of infection for study of possible viral shedding among
the pool of subjects certainly exposed for ⭓1 h to measles virus.
However, we consider that this study closely approximates the
most common exposure settings likely to occur, given the current low measles incidence in United States. We used our best
judgment about timing of collecting the specimens to determine measles virus infection. Because the IgM response is quick
and transient in the secondary immune response, we may have
missed some instances of serological evidence of infection. Likewise, in some persons in the cohort, the IgG titer may have
already begun to rise before our initial serum sample, limiting
our ability to detect a 4-fold increase in titer. Thus, there may
have been many more inapparent infections in this cohort that
we did not detect. However, we did not detect evidence of
measles virus in any member of the exposed cohort, including
those whose inapparent infections we might have missed.
We also used our best judgment about the timing of specimen
collection to identify evidence of viral shedding. It is possible
that viral shedding occurs earlier in persons with inapparent
infections than in persons with classical measles. However, if
that is the case, the duration of shedding would likely be very
transient, because an early and more vigorous immune response
would be expected to shut off viral proliferation earlier, leaving
less time for virus to replicate. Therefore, we consider it very
unlikely that altering the timing of the collection of specimens
for virus isolation would yield different results. We cannot
quantify the relation between detection of measles virus and
infectiousness. However, because we could not detect the viral
type related to the outbreak in any of the specimens, using the
very sensitive RT-PCR and direct isolation, it is unlikely that
the case person would be infectious.
Our findings are not applicable to populations with high
prevalence of HIV. Persons with AIDS may develop severe measles without showing classical symptoms such as rash [26, 27]
and may be infectious. Studies of the rates of undiagnosed
“rashless” measles in patients with AIDS and of shedding of
measles virus and infectiousness in such cases should be undertaken to better characterize their frequency in these populations. However, it appears that these patients do not play
an important role in the epidemiology and control of measles,
because countries in which HIV is hyperendemic in Southern
Africa have recently documented excellent measles control during at least 5 years [28].
Although inapparent measles virus infections did occur
among some persons exposed to symptomatic measles in our
study, intensive exposure was required in this highly vaccinated
population, and we were unable to detect shedding of measles
virus among exposed persons who did not develop classical
measles. These data suggest that inapparent measles virus infections are not likely to be highly contagious and are probably
not important in measles virus transmission in immune populations. Inapparent infections should not be considered an
impediment to measles control and elimination, at least in areas
with low prevalence of HIV infection.
Acknowledgments
We thank study participants, parents, and teachers for their
collaboration; staff of the dental clinic in Anchorage for their
cooperation; Susan Baum and Tracey Lynn for their critical
assistance in gathering subjects for the study; the Alaska Department of Health and Social Services, the Arizona Department of Health and Social Services; the Arctic Investigations
Program, Anchorage; and Jay Butler, Helen Peters, and Marilyn
Getty for their collaboration.
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