Hantavirus cardiopulmonary syndrome (HCPS) is caused by infection with New World hantaviruses.
First described in 1993 in the southwestern United States, HCPS has been documented
throughout the Americas (
1
,
2
). For human cases, the mean incubation period of hantavirus infection from exposure
to illness onset is 18.5 (range 7–42) days (
3
). As of December 31, 2013, a total of 848 human HCPS cases had been reported in Chile;
the case-fatality rate has ranged from 32% to 35% per year (
4
).
The sole confirmed etiologic agent of HCPS in Chile is Andes virus (ANDV). Human infection
with this virus occurs from exposure to contaminated excreta and secretions of rodents
of the family Cricetidae. Transmission of ANDV between rodents has been experimentally
documented after exposure of seronegative rodents to inhalation of aerosolized infected
rodent secretions (
5
). ANDV is endemic in Chile and Argentina and is the only hantavirus for which person-to-person
transmission has been documented. Person-to-person transmission of ANDV occurs mainly
in family clusters or, less commonly, after activities in which close contact with
an infected case-patient has occurred, primarily during the disease prodrome (
6
–
8
). A prospective study in Chile found that sexual partners and other close household
contacts of ANDV-infected persons showed a 10-fold higher risk of acquiring the virus
than household contacts who did not share bed or bedroom with the index case-patient
(
3
,
9
).
Nosocomial transmission of ANDV has been a matter of concern for infection control
practice and for health care workers who provide care for these patients, and in particular
for workers who perform invasive procedures. In Argentina, person-to-person transmission
of ANDV was documented in a physician who acquired infection after exposure to an
ANDV-infected patient shortly after onset of the febrile prodrome (
7
,
8
). Although person-to-person transmission in Chile has been epidemiologically documented
(
10
), nosocomial transmission has not been reported. Seroprevalence studies conducted
among health care workers in hospitals in Chile where patients with ANDV infection
have been treated have reported that health care workers exhibited ANDV IgG antibody
at a proportion similar to that of the general population (
11
,
12
). Similarly, a study in the southwestern United States found no evidence of nosocomial
transmission of another hantavirus, Sin Nombre virus (
13
).
We describe an outbreak of 5 cases of ANDV infection that occurred in a small, rural
community in southern Chile in 2011. We present the epidemiologic and the clinical
features of the cases, along with the molecular analysis of the virus strains from
each case. Epidemiologic and virus sequence analyses support person-to-person transmission
of ANDV in 4 of these cases, including nosocomial transmission in 2 cases.
Materials and Methods
Study Population
A case cluster of 5 human case-patients, including 2 persons involved in health care,
occurred in Corral, Los Rios, Chile, during February–April 2011. Clinical history
and information from epidemiologic questionnaires were obtained for each patient;
all 5 had an acute febrile illness and signs and symptoms compatible with hantavirus
infection. Acute infection was confirmed by detection of IgM against viral nucleoprotein
antigen and real-time reverse transcription PCR (RT-PCR) targeting the small RNA segment
of ANDV in blood samples obtained from these patients during the acute illness (
14
,
15
). Samples from 7 additional patients who had had HCPS in the same geographic region
in previous years were used as controls for virus sequence analysis. All participants
signed an informed consent approved by an ethics committee.
Geographic and Demographic Features of Corral
Corral is a coastal town (39°52′0″ S, 73°25′60″ W) located 15 km west of Valdivia,
the capital of the Los Rios region in Chile; the town is in the foothills of a coastal
mountain range in the Valdivian rainforest ecoregion (
16
). The population is ≈5,433 inhabitants. Corral has 1 primary care hospital with 5
beds (hospital I); all patients with complications are transferred to a regional care
center in Valdivia that has intensive care facilities (hospital II). Since 1997, a
total of 13 cases of hantavirus infection have been reported in Corral, including
the 5 cases described in this report (
17
). Prior to this report, the last 2 confirmed cases were in 2008 and 2010.
Outbreak Description
On March 20, 2011, two suspected cases of hantavirus infection were reported. The
patients were a 31-year-old woman (case-patient B) who worked as a nursing assistant
at hospital I and a 53-year-old woman (case-patient C). Both lived near Corral. In
addition to these cases, in late February, a 73-year-old man (case-patient A), the
spouse of case-patient C, had been transferred from hospital I to hospital II for
treatment of a pulmonary disease and evaluated for hantavirus infection; initial serologic
testing results at a national reference laboratory were negative. On March 22, a fourth
patient (case-patient D), a 60-year-old female housekeeper at hospital I, was admitted
to hospital II with respiratory failure; she died a few hours later. A fifth patient
(case-patient E), a 34-year-old man who was the husband of case-patient B, was hospitalized
on April 3 at hospital II. On April 3, an epidemiologic investigation was initiated
by the Health and Epidemiology Service, including investigation of infection control
measures used at hospital I.
Genetic Characterization of the Virus
RNA was obtained from patients’ leukocytes from diagnostic samples and extracted by
using the High Pure Viral RNA Kit (Roche Diagnostic GmbH, Roche Applied Science, Mannheim,
Germany), according to the manufacturer’s instructions. For segment amplification,
heminested RT-PCR was performed as described previously (
16
). Two portions of the medium segment, Gn and Gc, were amplified (Table 1), and the
amplicons underwent agarose gel purification and sequencing in both directions. The
chromatogram of each sequence was analyzed and aligned to generate a consensus sequence
by using BioEdit version 7.1.11 (http://www.mbio.ncsu.edu/bioedit/bioedit.html). Twelve
sequences were aligned by using ClustalW (http://www.clustal.org). Sequences were
phylogenetically analyzed by conducting maximum-likelihood (ML) and Bayesian methodology
on the concatenated Gn and Gc sequences. For ML, PAUP* version 4.0 (
18
) was used for a heuristic search with 100 random additions and branch swapping via
tree-bisection-reconnection (
19
). jModeltest 3.7 was used to choose the best-fitting model of sequence evolution
(
20
). The corrected Akaike information criterion (Akaike 1974) identified the Kimura
81 unequal base frequencies + gamma model (K81uf + Γ) as optimal (−lnL = 1251.2770,
Akaike information criterion = 2515.7539, G = 1.5780), with base frequencies A = 0.2868,
C = 0.3132, G = 0.0670, and T = 0.3329. Reliability of nodes in the ML tree was estimated
by bootstrap analysis (
21
) obtained after 1,000 pseudo-replicates. The tree was rooted on the basis of the
outgroup criterion by using the ANDV sequence available in GenBank (accession no.
NC_003467.2). Sequences also were analyzed in a Bayesian framework to estimate the
posterior probabilities of phylogenetic trees. Ten million phylogenetic trees were
generated; the first 1,000 trees of the sample were removed to avoid including trees
before convergence of the Markov Chain. As 2 independent molecular markers were used,
a general likelihood–based mixture model of sequence evolution was applied as described
(
22
). This model accommodates cases in which different sites in the alignment evolved
in qualitatively distinct ways but does not require prior knowledge of these patterns
or partitioning data. These analyses were conducted by using Bayes Phylogenies software
(
22
). To find the best mixture model of evolution, the number of general time reversible
matrices was estimated by using a reversible-jump Markov chain Monte Carlo method
(
23
).
Table 1
Primers used for M segment amplification and sequencing of Andes hantavirus
Primer identification
Primer sequences, 5′ → 3′
GN1+
TAGTAGTAGACTCCGCAAGAAGAAG
GN534−
TCCTGCTKKTAAACACACTAGCCAT
GC94+
TGCAAATGATTGTGTTAGTAACACCA
GC674−
GTATTAGAGCCCCTAGCACAGGTT
Results
Laboratory and Epidemiologic Investigations
IgM and IgG against ANDV were detected in serum samples, and ANDV RNA was detected
by RT-PCR in blood for all 5 patients in the cluster (Table 2; Figure 1). Case-patient
A, the 73-year-old man, was identified as the index case-patient of the cluster.
Table 2
Clinical and epidemiologic features of 5 patients involved in outbreak of ANDV infection,
Chile, 2011*
Feature
Case-patient A†
Case-patient B
Case-patient C
Case-patient D
Case-patient E
Age, y/sex
73/M
31/F
53/F
60/F
34/M
Occupation
Farmer
Nursing assistant at hospital
Teacher
Cleaning personnel at hospital
Car mechanic
Relationship to other case-patients
Husband of case-patient C
Health care provider for case-patient A
Wife of case-patient A
Health care assistant for case-patient A
Husband of case-patient B
Date of symptom onset
Feb 21
Mar 17
Mar 18
Mar 18
Apr 2
Date of hospitalization
Feb 24
Mar 20
Mar 20
Mar 22
Apr 3
Signs and symptoms
Fever
Yes
Yes
Yes
Yes
Yes
Respiratory symptoms‡
Yes
Yes
No
Yes
Yes
Gastrointestinal symptoms§
No
No
Yes
Yes
No
Other symptoms¶
No
Yes
Yes
Yes
Yes
Mechanical ventilation, d
28
8
0
1
6
Hospitalization, d
30
22
12
1
17
Outcome
Died
Survived
Survived
Died
Survived
Days from environmental exposure to onset of symptoms
16
25–26
41
7–45
41–42
Days from exposure to hantavirus case-patient to onset of symptoms
NA
19–21
22–25
18–20
13–27
Laboratory test results on admission
Platelet count, × 103/μL
51
56
108
101
147
Leukocytes, × 103 cells/μL
4,67
5,46
1,21
3,92
11,46
Hematocrit, %
52
39
39
45
44
Lymphocytes, %
12
7
39
19
7
Immunoblasts, %
Yes
Yes
Yes
NR
Yes
IgM/IgG for ANDV
Negative#
Positive
Positive
Positive
Positive
RT-PCR ANDV in blood cells
ND
Positive
Positive
Positive
Positive
*ANDV, Andes virus; NA, not applicable; NR, not reported; RT-PCR, reverse transcription
PCR; ND, not done.
†Index case-patient.
‡Dry cough, dyspnea, cyanosis, crepitus.
§Vomiting,
diarrhea, nausea.
¶Severe headache, meningeal signs, myalgia, arthralgia, conjunctival
infection, chills, photophobia, facial edema.
#On hospital admission. Repeat testing
after 24 days yielded positive results.
Figure 1
Timelines showing progression and key events related to each case-patient (A–E) in
a cluster of 5 Andes hantavirus cases, southern Chile, 2011. Blue boxes along timeline
for index case-patient (A) indicate date of illness onset for subsequent case-patients;
green boxes indicate environmental exposures (exposure for case-patient A was the
same as for case-patient C); red boxes indicate contact with other case-patients.
Case-patient A lived in a small settlement near Corral. His main risk activity was
the cleaning of a home cellar where he was moving tiles on February 5. The cellar
was heavily contaminated with rodent feces. The patient was admitted to hospital I
on February 24 after 3 days of fever, dry cough, weakness, and progressive dyspnea.
During hospitalization, he experienced progressive respiratory compromise, productive
cough, and intense sweating that required frequent changes of gowns, sheets, and blankets.
On February 26, he was transferred to the critical care unit at hospital II for mechanical
ventilation. Serum samples were sent to the National Reference Laboratory 11 days
after onset of his symptoms; results were negative for ANDV IgM. When the epidemiologically
related hantavirus case-patients were admitted to hospital II, ANDV IgM testing was
repeated, 24 days after onset of his symptoms, and results were positive. Case-patient
A died on March 26 after 28 days of mechanical ventilation and use of vasoactive drugs.
Case-patient B, a nursing assistant at hospital I, exhibited a fever on March 17.
She was hospitalized on March 20 and the same day was transferred from hospital I
to the intensive care unit at hospital II. Severe shock and respiratory failure developed,
and high doses of vasopressors and mechanical ventilation were required. A diagnosis
of HCPS caused by ANDV infection was confirmed after 8 days of symptoms, and she was
discharged on April 11. This patient had direct contact with case-patient A at hospital
I from February 24–26, during his febrile prodrome and progression to the cardiopulmonary
phase. She changed the patient’s clothes, sheets, and blankets because he perspired
profusely. In addition, having met the patient previously, she greeted case-patient
A with a kiss on his cheek several times during his hospitalization. She also had
close contact with her husband at their home from the time she cared for the index
case-patient through the first 3 days of her illness. She recalled possible environmental
exposure from camping at 2 local beaches during February 1–4 and February 19–20; she
collected wood and cleaned the area to set up tents.
Case-patient C was the spouse of case-patient A. She shared the same bed and cared
for him during his febrile prodrome but denied that they had sexual activity after
symptom onset. She entered the contaminated cellar with her husband but did not participate
actively in his work in this area. On March 18, twenty-five days after her husband’s
illness onset and 41 days after they entered the cellar, she exhibited mild fever,
severe headache, myalgia, and photophobia . She sought medical attention at hospital
II while her husband was still hospitalized, and acute ANDV infection was confirmed
on March 24. Her chest radiograph results were normal. Her most remarkable symptoms
were headache and irritability, and she had meningeal signs. Testing of cerebrospinal
fluid (CSF) showed 8 white mononuclear cells, normal glucose levels, and a slightly
elevated protein level of 0.5 g/L. CSF testing by RT-PCR for ANDV and ELISA for ANDV-specific
IgG yielded negative results.
Case-patient D, a housekeeper at hospital I, had fever, abdominal pain, and vomiting
develop on March 18. Two days later, she was hospitalized at hospital I, and 4 days
later, she was transferred to hospital II, where severe shock and respiratory failure
developed. She died a few hours after admission to hospital II. Her diagnosis was
confirmed by positive results of serologic testing and RT-PCR for ANDV. She had direct
and indirect contact with case-patient A while he was at hospital I. She entered his
room and helped the nursing assistant (case-patient B) change his clothes and remove
his sheets and bedclothes for washing.
Case-patient E, the husband of case-patient B, had fever, headache, myalgia, and back
pain develop on April 2, and he was admitted to hospital II on April 3. Serologic
testing for ANDV IgM and IgG after 5 days of symptoms yielded negative results, but
results of RT-PCR for ANDV RNA were positive. IgM and IgG seroconversion were confirmed
10 days after symptom onset. The person-to-person exposure period for this patient
was March 6–20; his possible environmental exposure exceeded the known incubation
period for ANDV (
11
). Shock and respiratory failure developed, and he required mechanical ventilation
and vasopressors but survived.
Environmental Investigation
Rodent trapping was performed for 2 and 3 nights, respectively, at the 2 sites where
case-patients reported possible environmental exposure: the cellar of the home of
case-patients A and C and a camping area used by case-patients B and E (Table 3).
Rodent serum samples were tested for ANDV antibodies by strip immunoblot assay (
24
); results were positive for 1 Abrothrix longipilis rodent trapped at the camping
site. However, RT-PCR results for this sample were negative, and testing of rodents
trapped at the home of case-patients A and C yielded negative results.
Table 3
Results of environmental investigation for 4 cases of ANDV infection, Chile, 2011*
Case-patients
Days after case-patient diagnosis
No. trapping nights
No. trapped rodents
No. traps per night
Rodent species trapped
SIA results, n = 24
RT-PCR results
B and E
63
3
46
57, 40, 40
Abrothrix longipilis, A. olivaceus, other Abrothrix sp., Oligoryzomys longicaudauts
1 positive (A. longipilis)
Negative
A and C
90
2
9
68, 68
A. olivaceus, O. longicaudauts, Rattus norvegicus, R. rattus
Negative
ND
*SIA, strip immune assay; RT-PCR, reverse transcription PCR; ND, not done.
Viral Molecular Analysis
A portion of 942 bp of the ANDV small RNA segment was amplified and sequenced from
samples of each of the 5 patients in the case cluster. Sequences aligned by using
ClustalW showed 100% identity (data not shown), an observation consistent with the
high degree of conservation of the small segment among hantaviruses (
7
,
25
).
Virus variability was established by comparing a portion of 914 bp of the highly variable
ANDV medium RNA segment. The sequences obtained for the 2 medium segments encoding
the ANDV glycoproteins Gn and Gc were compared separately (data not shown) and concatenated.
Results were visualized in the identity matrix of concatenated sequences and showed
that the concatenated sequences derived from the 5 cases in the cluster were similar
to each other but differed from viral sequences from 7 patients who acquired ANDV
in the same community in previous years (Table 4). The molecular identity of the concatenated
Gn and Gc sequences between cases ranged from 99% to 100%, whereas the comparison
with control sequences from the same geographic region ranged from 97% to 99%. These
values show higher identity between the sequences derived from the cluster cases compared
with other human cases from the same geographic region from previous years. All sequences
obtained in this study have been deposited in GenBank (accession nos. KC567258–KC567281).
Table 4
Identity matrix of concatenated Gn and Gc sequences of ANDV isolates from the 5 case-patients
in this study compared with sequences from ANDV samples from previous case-patients
in the same geographic region of Chile*
Sequence
Pan2010
Pai2011
Mar2010
Fut2010
C2012(1)
C2012(2)
Pan2012
C
B
E
D
A
Pan2010
–
0.972
0.991
0.985
0.964
0.971
0.989
0.961
0.961
0.955
0.961
0.961
Pai2011
–
0.974
0.973
0.983
0.994
0.976
0.984
0.984
0.978
0.984
0.984
Mar2010
–
0.987
0.970
0.975
0.995
0.961
0.961
0.955
0.961
0.961
Fut2010
–
0.964
0.974
0.990
0.960
0.960
0.953
0.960
0.960
C2012(1)
–
0.985
0.970
0.970
0.970
0.963
0.970
0.970
C2012(2)
–
0.978
0.981
0.981
0.974
0.981
0.981
Pan2012
–
0.963
0.963
0.957
0.963
0.963
C
–
1.000
0.993
1.000
1.000
B
–
0.993
1.000
1.000
E
–
0.993
0.993
D
–
1.000
A
–
*Geographic location and year are indicated for control cases (numbers in parentheses
indicate multiple cases in the same year; case-patient identification letters (A–E)
are given for cases from this study. – indicates alignment of the same sequence.
The phylogenetic analyses through ML and Bayesian methods revealed similar topologic
results; thus, a single tree is shown (Figure 2). Results show 2 major groups with
strong support provided by the bootstrap and posterior probability values. The group
of samples that included the Corral cases is clearly separated from other major clustering
that includes ANDV sequences from other localities in Chile.
Figure 2
Phylogenetic analyses of the medium RNA segment (Gc and Gn) of concatenated sequences
of Andes hantavirus (ANDV). Isolates from the case-patients (A–E) from the 2011 outbreak
in Chile were compared with control samples from the same geographic region (indicated
by year isolated; number in parentheses indicates multiple isolates from the same
year) and an ANDV sequence from GenBank (bottom isolate on tree; accession no. NC_003467.2).
Scale bar indicates substitutions per site.
Discussion
ANDV is the only hantavirus for which person-to-person transmission has been reported
(
7
). Our study of a case cluster in Chile provides epidemiologic and molecular evidence
that strongly supports the conclusion that 4 of 5 cases resulted from person-to-person
transmission of ANDV, including 2 cases of nosocomial transmission.
Most of the reports of person-to-person transmission of ANDV share common traits that
constitute potential risk factors for virus spread (
7
–
9
). These features were also observed in this cluster. First, the period of the disease
during which the acute case-patient and the household contact or health care personnel
have close contact is primarily the febrile prodrome phase, when symptoms are nonspecific
for hantavirus. Second, the number of days from exposure to an index case-patient
and the onset of symptoms among additional cases ranges from 12 to 27 days (
7
,
26
), consistent with the intervals observed in our report. In the 2 cases for which
environmental exposure was reported, the estimated incubation period after that exposure
exceeded the longest reported incubation range of 42 days for ANDV (
3
,
11
). In contrast, in these 2 cases the estimated incubation periods from exposure to
a case-patient to onset of symptoms was 13–27 days. Finally, the viral genetic characterization
established that viruses from the case cluster shared a high nucleotide sequence identity
in Gn and Gc fragments, the most variable viral genomic regions (
6
).
During the prodrome, when symptoms are nonspecific, consideration of ANDV infection
and early diagnosis might be triggered by a history of environmental exposure (
1
,
2
) or close exposure to another confirmed case-patient within the known incubation
period (
3
,
6
). In this cluster, all the cases appeared in a geographic region that is considered
an endemic risk area for hantavirus (
26
,
27
). However, no other cases had occurred in this town since 2010, and our epidemiologic
and virus sequence analysis showed that the main risk factor for all the 4 additional
cases was the patient’s close contact with a symptomatic, HCPS case-patient (
6
,
28
).
One case of nosocomial transmission of the virus has been previously reported in Argentina
(
7
), and evidence of this transmission has been sought in Chile (
12
). We document 2 cases of nosocomial transmission of ANDV, from the index case-patient
to a nursing assistant and to a housekeeper, even though their contact with the patient
was limited to kissing the patient on the cheek and to handling bedding and gowns
(no invasive procedures). Two seroprevalence studies performed soon after recognition
of hantavirus in Chile did not reveal a higher proportion of antibodies against ANDV
among hospital personnel when compared with the general population (
11
,
12
).
In our study, ANDV infection was not diagnosed in the index case-patient until he
had been ill for 31 days, which resulted in a wider time frame of exposure for health
care personnel. The patient had a history of diabetes mellitus but no history of any
other immunodeficiency that might explain his initial negative serologic test. However,
the initial testing was not repeated, so we cannot rule out the possibility of a false-negative
result.
For case-patient C, the clinical manifestation of illness was unusual because she
lacked respiratory symptoms and showed meningeal irritability as the main sign of
the infection. Viral RNA and specific antibodies were not detected, but a slight elevation
in the CSF white blood count and protein level were seen. It is possible that viral
RNA was present before CSF testing or that it was below the level of detection by
RT-PCR, but the timing of her symptoms is probably inconsistent with a postinfectious
process.
It is not clear why person-to-person transmission has been documented for ANDV but
not for other hantaviruses. Risk factors associated with close contact, including
sexual contact, deep kissing, or sleeping in the same bed or room, have been identified
in a prospective study of household contacts of index case-patients with HCPS (
9
). As such, respiratory secretions, saliva, or both may be involved in transmission.
Puumala virus RNA has been detected by RT-PCR but not by cell culture in saliva from
patients who had hemorrhagic fever with renal syndrome (
29
). The antiviral activity of different human saliva concentrations has been experimentally
tested against Hantaan virus, Puumala virus, and ANDV; ANDV was least sensitive to
the antiviral effect of saliva (
30
). RT-PCR testing has found ANDV RNA in previous and ongoing studies in blood and
in body fluids, including gingival crevicular fluid, saliva, endotracheal fluid, and
urine (
31
). ANDV was isolated from blood obtained from a child in Chile before the onset of
symptoms or development of ANDV antibodies (
32
), and studies are ongoing to determine which, if any, of the body fluids positive
by RT-PCR also contain infectious virus.
To characterize and compare the outbreak viral sequences, we used as reference material
a selection of sequences from strains obtained 2 or 3 years earlier in the same ecogeographic
region near Corral. All 5 medium fragments obtained from case-patient isolates in
this cluster were highly similar to each other but were more distantly related to
the reference sequences. The strong relatedness of the viruses in the Corral cluster
is supported by high bootstrap and posterior probability values in the phylogenetic
analyses. Furthermore, the small segment showed 100% identity between the 5 sequences
in this cluster. The dates of exposure to high-risk environments or to persons with
ANDV infection, known incubation periods, and 100% sequence identify all support a
conclusion of person-to-person transmission (
7
). Our data showed 99%–100% identity for a fragment of 913 bp of the medium segment,
supporting identity using different sequences. However, we did not include noncoding
region fragments, which might provide additional confirmation of identity.
Our study documents a small but definite risk of nosocomial acquisition of ANDV infection
for personnel who care for patients, including handling of bedding and gowns. After
this investigation, the Ministry of Health of Chile has recommended, in addition to
strict adherence to universal precautions, the use of droplet precautions when ANDV
infection is suspected. Use of N95 respirator masks, designed to prevent the inhalation
of airborne particles, is recommended for those procedures associated with aerosolization
of viral contaminated secretions (e.g., respiratory, saliva) when procedures such
as suction or intubation are performed. However, this recommendation should be extended
to all personnel who have any kind of direct contact with patients or body fluids,
including bedding and gowns.
Finally, all close household contacts and health care personnel exposed to a confirmed
ANDV case-patient should be closely monitored for signs and symptoms of infection,
such as fever, myalgia, headache, and abdominal pain, during the entire documented
incubation period of 42 days, even though in person-to-person transmission of ANDV,
the onset of symptoms has usually occurred 12–27 days after close contact with a sick
patient (
6
,
9
). ANDV RT-PCR should be performed in addition to testing for specific IgM in any
exposed contact in whom fever develops within the incubation period, particularly
if testing is done within a few days of the onset of fever and before onset of the
cardiopulmonary phase. Results of ANDV RT-PCR on blood cells may be positive as early
as 5–15 days before onset of symptoms or detection of ANDV antibody (
9
). As we have documented, RT-PCR can detect ANDV RNA in the rare, symptomatic patient
in whom seroconversion is delayed.