Detection of Adenovirus DNA in Throat Swabs and Blood by SYBR

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Detection of Adenovirus DNA in Throat Swabs and Blood by SYBR Powered By Docstoc
					Jpn. J. Infect. Dis., 59, 394-396, 2006

  Short Communication
                       Detection of Adenovirus DNA in Throat Swabs and
                     Blood by SYBR Green Real-Time PCR Assay in Patients
                             with Adenovirus-Associated Tonsillitis
                                       Urara Kohdera*, Minoru Kino and Masahiro Ito1
             Nakano Children’s Hospital, Osaka 535-0022, and 1Kobe Institute of Health, Kobe 650-0046, Japan
                                      (Received June 26, 2006. Accepted October 17, 2006)
       SUMMARY: The adenovirus DNA load ranged from 105 to 1010 copy/mL and from 104 to 107 copy/mL in throat
       swabs and blood from patients with adenovirus-associated exudative tonsillitis, respectively. The copy number
       of adenovirus DNA in blood was positively correlated with the duration of the fever.

   Human adenovirus causes a variety of diseases including                 patients and 30 whole blood samples from 23 patients were
upper respiratory tract infection, acute conjunctivitis, cystitis          collected. Throat swabs were collected by swabbing the
and gastroenteritis. Acute febrile exudative tonsillitis is caused         tonsils of all patients. Throat swabs from 10 patients and blood
by various agents, including group A -hemolytic strepto-                   from 6 patients were collected in the acute phase (within 4
cocci and viruses. Group A -streptococcal tonsillitis is most              days of the onset of fever) and recovery phase (between 5
common in children 6 years of age or older; by contrast, viral             and 10 days after the onset of fever), respectively. Throat
tonsillitis is most common in children younger than 3 years                swabs were collected and suspended in 1 ml of normal
of age (1). Adenovirus is the most frequent agent among                    saline, and blood was collected in EDTA-coated tubes. Plasma
viral tonsillitis. Adenovirus-associated exudative tonsillitis             was collected after centrifugation at 5,000 rpm for 5 min. In-
induces high grade and prolonged fever, sore throat and                    formed consent was obtained from the parents or guardians
poor appetite; hospitalization is often required for such                  of all patients.
patients. Strong inflammatory responses such as leukocytosis                  DNA was extracted from 200 l of throat swabs, whole
with neutrophilia and high C-reactive protein (CRP) values                 blood and plasma using a High Pure Viral Nucleic Acid Kit
are usually observed. These laboratory findings are different              (Roche Applied Science, Mannheim, Germany), according
from those of common viral infections, making it difficult to              to the manufacturer’s instructions. DNA was eluted in 50 l
distinguish adenovirus respiratory infection from bacterial                of distilled water and stored at –20°C until use. Adenovirus
infection. However, the mechanisms of these findings induced               DNA was tested by quantitative SYBR Green real-time PCR
by adenovirus infection have not been well elucidated. Real-               assay (5). Briefly, 1 l of template DNA was added to a
time polymerase chain reaction (PCR) methods (2) are thought               final volume of 25 l containing 1 SYBR Green PCR
to be useful in the diagnosis and monitoring of Epstein-Barr               Master Mix (Applied Biosystems Japan, Tokyo, Japan) and
virus (EBV) (3) and cytomegalovirus (CMV) infections (4).                  160 nM of the primers Hex3 and Hex4. The sequence of
We have reported SYBR Green real-time PCR assay for the                    primer pair was as follows, Hexon 3: 5´-GACATGACTTTC
quantitative detection of adenovirus DNA (5). Measurement                  GAGGTCGATCCCATGGA-3´, Hexon 4: 5´-CCGGCTGAG
of adenovirus load in clinical samples from adenovirus                     AAGGGTGTGCGCAGGTA-3´ (6). PCR amplification was
infections may provide important information for analyzing                 performed using an ABI PRISM 7900 HT. Amplification was
the pathogenesis of various adenovirus infections. In this                 performed with 40 cycles of denaturation at 94°C for 1 min,
study, we evaluated the adenovirus DNA load in patients with               annealing at 57°C for 1 min and elongation at 72°C for 1.5
adenovirus-associated exudative tonsillitis and analyzed the               min. During thermal cycling, emission from each sample was
correlation between the viral load and clinical findings.                  recorded and SDS software processed the raw fluorescence
   Thirty patients at Nakano Children’s Hospital, Osaka,                   data to produce threshold cycle (Ct) values for each sample.
Japan, between June 2003 and October 2004 were clinically                  The SDS software then computed a standard curve from the
diagnosed as having acute adenovirus-associated tonsillitis                Ct values of the diluted standards and extrapolated absolute
based on their high fever (>38.0°C) and exudative tonsillitis,             quantities for the unknown samples based on their Ct values.
and were admitted for fluid transfusion because of poor fluid              The minimum detectable adenovirus DNA level in this assay
intake. These patients did not have chronic tonsillitis as an              is 104 copy/mL using adenovirus type 2 quantification stan-
underlying illness. Etiologic diagnosis was confirmed using an             dard plasmid DNA as previously reported (5).
immunochromatographic rapid diagnostic kit (Adenoclone;                       The mean duration of fever in patients was 5.5 days. The
TFB, Tokyo, Japan) with throat swabs. Patients included                    mean values of CRP and leukocyte (WBC) counts at the acute
20 boys and 10 girls aged from 8 months to 8 years (mean                   phase were 4.8 mg/dl and 14,200/ l, respectively. Changes
age, 4 years and 11 months). Forty-one throat swabs from 30                in the copy number of adenovirus in throat swabs and blood
                                                                           are shown in Fig. 1(A). Adenovirus DNA was detected in
  *Corresponding author: Mailing address: Nakano Children’s                throat swabs from 30 patients (100%) in the acute phase. The
   Hospital, 4-13-17 Shinmori, Asahi-ku, Osaka 535-0022, Japan.            copy number of adenovirus DNA ranged from 105 to 1010/
   Tel: +81-6-6952-4771, Fax: +81-6-6954-8621, E-mail: kohdera             mL. Adenovirus DNA was detected in 16 (88.9%) out of 18                                                    samples in the recovery phase. Changes in the copy number

of adenovirus DNA in throat swabs in the acute and recovery                   P < 0.005) (Fig. 2).
phases are shown in Fig. 1(B). The copy number of adeno-                         We have reported that SYBR Green real-time PCR assay
virus in throat swabs decreased in 9 patients in the recovery                 is a useful quantitative tool for analysis of adenovirus DNA
phase compared with the acute phase, but adenovirus                           and more sensitive than the IC kit (5). Strong inflammatory
DNA was still detected in 9 patients in the recovery phase.                   responses such as leukocytosis and a high CRP level are
Adenovirus DNA was detected in blood from 23 patients                         commonly observed in patients with adenovirus-associated
(77%) in either the acute or recovery phases. The copy num-                   exudative tonsillitis. The serum interleukin-6 (IL-6) concen-
ber of adenovirus DNA in blood ranged from 104 to 107/mL.                     tration is higher in patients with adenovirus infection
The changes in the copy number of adenovirus DNA in blood                     compared with influenza virus and respiratory syncytial
in the acute phase and recovery phase are shown in Fig. 1                     virus (RSV) infections. A good correlation between the CRP
(B). No adenovirus DNA was detected in plasma. The WBC                        level and serum IL-6 concentration in adenovirus respiratory
count and CRP level did not correlate with the copy number                    infection has been reported (7). These data suggest that
of adenovirus DNA in either throat swabs or blood (data                       adenovirus stimulates IL-6 production by vascular endo-
not shown). The copy number of adenovirus DNA in blood                        thelial cells, fibroblasts or activated T lymphocytes. In this
was positively correlated with the duration of fever (r = 0.55;               study, the CRP value and WBC number did not correlate with
                                                                              the copy number of adenovirus DNA, suggesting that the
                                                                              clinical severity is not dependent on the adenovirus load in
                                                                              the throat. However, the positive correlation between the
                                                                              duration of the fever and the adenovirus load in the blood
                                                                              suggests that high levels of adenovirus DNA in blood may
                                                                              play an important role in the persistence of fever.
                                                                                 There are a few reports that detect adenovirus DNA in blood
                                                                              from immunocompetent individuals. Aberle et al. reported
                                                                              that adenovirus DNA was detected in 41% of children with
                                                                              an acute respiratory adenovirus infection, and the majority
                                                                              (93%) of serum samples were collected within the first week
                                                                              after the onset of symptoms (8). Shike et al. reported that
                                                                              adenovirus DNA was detected in whole blood from only one
                                                                              out of 6 patients with culture-proven adenovirus infection
                                                                              (9). In our study, 42% of throat swabs and 46% of blood out
                                                                              of adenovirus DNA positive samples were from the recovery
                                                                              phase. Two cases showed an increase of the copy number of
                                                                              adenovirus DNA in the recovery phase. In one patient, the
                                                                              copy number of adenovirus DNA increased to 107 copy/mL
                                                                              and the fever continued for 6 days, then rose again on day 9.
                                                                              This finding may be related to the increase of adenovirus
                                                                              DNA in the blood. These findings indicate that adenovirus
                                                                              replicates in the lymph tissue of the throat and pharynx, and
                                                                              then spreads into the bloodstream. Detection of adenovirus
                                                                              DNA from blood is not unusual phenomenon. For instance,
                                                                              varicella-zoster virus was isolated during the late incubation
                                                                              period and the acute phase (10). Varicella-zoster virus DNA
                                                                              declined gradually with time and was undetectable after 15
                                                                              days from the onset (11). In this study, we evaluated neither
                                                                              serum antibody nor virus isolation. It is not clear whether
  Fig. 1. (A) Detection of adenovirus DNA by SYBR Green PCR in throat         detection of adenovirus DNA in blood indicates the presence
    swabs and blood. Data indicates the copy number of adenovirus DNA         of infectious adenovirus.
    per mL in throat swabs and blood. (B) Changes in adenovirus DNA              It has been reported that adenovirus is an important cause
    in throat swabs and blood in the acute and recovery phases.
                                                                              of morbidity and mortality among allogeneic bone marrow
                                                                              transplantation (BMT) recipients (12-15). Quantification of
                                                                              viremia with the use of real-time PCR is used in routine tests
                                                                              for adenovirus (16,17). It has been suggested that children
                                                                              who are viremic are at the greatest mortality risk from
                                                                              disseminated adenovirus infection (18-20). In immuno-
                                                                              compromised individuals with disseminated adenovirus
                                                                              infection, viral load reflects disease activity and can be used
                                                                              to monitor the response to antiviral treatment. Walls et al.
                                                                              reported that there was no correlation between the quantity
                                                                              of virus in the blood and the severity of symptoms in BMT
                                                                              recipients (21). These data imply that the pathogenesis
                                                                              of adenovirus replication may be different in immuno-
                                                                              competent and immunocompromised individuals. Further
  Fig. 2. Relationship between the copy number of adenovirus DNA in           study is needed to clarify the mechanisms by which adeno-
    throat swabs, blood and duration of fever.                                virus induces fever and inflammatory responses.

                                                                                transplantation in children. Bone Marrow Transplant.,
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