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Original Article
Relationship between respiratory viral load and lung lesion severity: a
study in 24 cases of pandemic H1N1 2009 influenza A pneumonia
Pu-Xuan Lu1, Ying-Ying Deng1, Gui-Lin Yang2, Wei-Long Liu2, Ying-Xia Liu3, Hua Huang1, Yi-Xiang J Wang4
1Department of Radiology, The Shenzhen No. 3 People’s Hospital, Guangdong Medical College, Shenzhen 518020, China; 2Medical
Laboratories, The Shenzhen No. 3 People’s Hospital, Guangdong Medical College, Shenzhen 518020, China; 3Department of Infectious
Diseases, The Shenzhen No. 3 People’s Hospital, Guangdong Medical College, Shenzhen 518020, China; 4Department of Imaging and
Interventional Radiology, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, China
Corresponding to: Dr. Puxuan Lu. Department of Radiology, The Shenzhen No. 3
People’s Hospital, Guangdong Medical College, Shenzhen 518020, China. Email:
lupuxuan@126.com; Dr. Yixiang J Wang. Department of Imaging and Interventional
Radiology, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin,
NT, Hong Kong, China. Email: yixiang_wang@cuhk.edu.hk.
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Abstract
Objective: To investigate the relationship between respiratory viral load and lung lesion severity of patients with pandemic H1N1 2009 influenza A pneumonia.
Study design: Cross-sectional observation study.
Methods: 24 consecutive H1N1 influenza patients with viral pneumonia (13 males, 11 females, mean age: 17.5 years)
during their presentation to hospital were retrospectively analysed. Viral load were first measured on average 5.2 days after
the onset of symptoms. The initial CT and viral load measurement was carried on the same day in 13 patients. The rest were
carried out with a mean interval time of 1.5 days. All patients had viral load follow-up till turned negative. Thirteen patients
had radiological follow-up.
Results: There was no significant correlation between the initial lung lesion severity and viral load (P=0.4). Both viral load
and lung lesion severity decreased over time, being highest value at initial presentation. The patients had higher initial viral
load or higher initial lung lesion severity tended to be slower in resolving. The lung lesion decreased at a slower rate than
viral load.
Conclusions: While there was no correlation between the initial viral load and lung lesion severity, these two indices
provide valuable information for epidemiological control.
Key words
Chest X-ray; tomography, computed; pandemic; lung, infection; viral load; influenza A (H1N1)2009; real time RT-PCR
J Thorac Dis 2012;4(4):377-383. DOI: 10.3978/j.issn.2072-1439.2012.08.02 |
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Introduction
In April of 2009, a new strain of human influenza A H1N1 virus
was identified in Mexico and was characterized by a unique
combination of gene segments that had not been previously
identified among human or swine influenza A viruses ( 1-5).
The 2009 H1N1 virus is a triple-reassortant influenza virus
containing genes from human, swine, and avian influenza
viruses ( 6, 7). The majority of H1N1 influenza cases have been
mild influenza-like illnesse ( 8). However, large-scale reports of
hospitalized patients with H1N1 influenza in the United States
demonstrated that this strain of H1N1 virus can cause severe
illness, including sepsis, pneumonia, and acute respiratory
distress syndrome ( 9, 10). Cases of severe illness, such as acute
respiratory distress syndrome and death, have been reported
in previously healthy persons ( 2). This phenomenon may be
attributed to the lack of pre-existing cross-reactive antibody
against pandemic H1N1virus in these subjects. On the contrary,
most if not all of these individuals have pre-existing antibody
against the prevailing seasonal influenza virus. Pneumonia is detected in 40% of influenza A H1N1
cases ( 10). The reported radiologic findings included patchy
consolidation and ground-glass opacities consistent with viral pneumonia ( 11, 12). Galit Aviram et al. reported that extensive
involvement of both lungs is associated with adverse prognosis,
while normal initial radiographs cannot exclude adverse
outcome ( 13). The correlation between the virological profile
and clinical characteristics of pandemic H1N1 virus infection
can provide important knowledge for epidemiological
control and clinical management in terms of antiviral therapy
and infection control measures. It has been reported that
in influenza A H5N1 virus infection, serum virus level had
prognostic significance ( 14). The relationship between
respiratory viral load and lung lesion severity of patients with
influenza A H1N1 pneumonia hasn’t been reported. In this
study, this correlation was investigated in 24 consecutive
patients with H1N1 viral pneumonia. |
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Methods
Subjects
The patient data were collected from one single hospital.
All the patient data were collected from this hospital.
Consecutive 24 patients diagnosed with pandemic H1N1
2009 influenza A from May 2009 to December 2009 and had
H1N1 virus induced lung lesions during their presentation
to the hospital were retrospectively analysed. The individuals
of this study consisted of 13 males and 11 females with age
ranging from 3 to 49 years (mean, 17.5 years). During this
period, there were additional 20 H1N1 influenza patients
without radiological lung lesions during their initial
presentation. These 20 cases were not included in this
analysis due to the absence of radiological findings of the
lung. The institutional review board of the Shenzhen No. 3
People’s Hospital approved this study. Informed consent was
waived owing to its observational nature. All the patients
presented with an influenza like illness, including fever,
headache, dry cough or productive cough, shortness of
breath, rhinitis. The patients fulfilled the clinical criteria
for confirmed pandemic H1N1 2009 influenza A infection,
as established by the U.S. Centers for Disease Control and
Prevention. These criteria included influenza-like symptoms
and a real-time reverse transcriptase polymerase chain
reaction (RT-PCR) assay with positive results for H1N1
virus.
Among these 24 patients, one case had chronic hepatitis
and one case had liver cirrhosis; one case had history of gastric
ulcer; one case had history of kidney calculus; one case had
history of partial thyroidectomy due to hyperthyroidism; one
case had bronchial asthma during early childhood. The rest
patients had no other significant medical history, and none of the
other patients had any symptoms, signs, or laboratory findings
indicating the presence of other additional acute illness.
The initial viral load measurement and first time CT were
both performed prior to anti-viral treatment. Respiratory viral
load was first measured on average 5.2 days after the onset of
symptoms (range, 2-9 days). The first CT and first viral load
measurement was carried on the same day in 13 patients. For the
remaining patients, the first CT and first viral load measurement
were carried out with an interval time of one day to 3 days (mean,
1.5 days). Once the diagnosis was confirmed, all the patients
were administered orally with Oseltamivir (Tamiflu®, Roche) or
Zanamavir (Relenza®, GlaxoSmithKline) and general supportive
treatments.
All the 24 patients had viral load follow-up. 13 patients had
additional radiological follow-up following the initial examination,
with chest X-ray or CT. Radiological follow-up varied from once
to four times (average 2 times) and the average follow-up interval
was 7.2 days. This 13 patients’ respiratory viral load measurement
follow-up varied from twice to 18 times (average 4.4 times), and
the average follow-up interval was two days.
One case developed bacterial pneumonia during the course of
hospitalization and antibiotic was administered. After treatment,
all the patients’ clinical symptoms disappeared and virus load
turned negative, and were then transferred to community health
centers for further follow-up. No recurrence of symptoms or
other illness was observed in the community health centers for
these subjects.
Quantitative real- time RT-PCR for detection of influenza A
H1N1 virus
Respiratory specimens were collected from nasopharyngeal
swab or bronchoalveolar aspirate and viral load was examined
by real-time RT-PCR method. Primers and probes specific for
2009 human H1N1 influenza A virus (H1 and H3 subtypes)
were developed and quantitative real-time RT-PCR tests were
performed for detection of the virus according to published
guidelines by World Health Organization (WHO). These
reagents allowed for a rapid detection of H1N1 infuenza A
virus. Technical details on this assay have been published on
the WHO Global Influenza Program Website (www.who.int/
csr/disease/influenza/en). In order to establish copy number
as a unit of viral load, the primers were designed to establish
PCR assay to amplify a part of the M gene of 2009 H1N1
influenza A virus according to the published sequences online
by WHO, then the amplified PCR products were cloned into
vector PMD18-T and subsequently produced a new plasmid
containing M gene. Concentration of the resultant new plasmid
was serially diluted from 109 to 103 copies/mL and quantitative
real-time PCR assay was performed to detect copy number of
viral load in nasopharyngeal-swab specimens of the patients
using the resultant new plasmid containing M gene as a standard
curve. The unit of mol can be transformed into copy number according to the following equation: (6.02×1023 copies/mol) ×
(concentration of plasmid g/mL)/(MW g/mol) = copies/mL.
According to institutional reference, viral load value of less than
1.7 log10 copies/mL was regarded as negative value. Negative
viral load was confirmed with results from two consecutive
measurements.
Imaging techniques and lung lesion score
Chest CT was performed for all patients diagnosed with H1N1
influenza, including 24 patients with positive findings, and 20
patients with negative results who were not included in this
analysis. CT scans were carried out on a dual slice scanner
(Philips MX 4000, Philips, the Netherlands). The protocol
used was as follows: end-inspiratory acquisition, 120 kV, 150-
200 mAs, slice thickness 10mm, interslice gap 0.2 mm. The
images were viewed on a PACS system with both lungs (window
width, 1,500 HU; level, -500 HU) and mediastinal (window
width, 350 HU; level, 40 HU) settings. All the studies were
unenhanced. Chest X-ray or CT was used for follow-up. All
posteroanterior upright radiographs were acquired with digital
radiography (Hologic EPEX/O, Hologic, America), while the
anteroposterior portable chest radiographs were acquired with
a computed radiography machine (CR 850; Kodak Direct View
systems, America).
All chest CT and chest X-ray were assessed blinded to clinical
information and in consensus by two experienced radiologists
regarding lesion location, lesion extent, and lesion morphology.
The two reviewers were blinded to the clinical data and the
patients’ outcomes. In order to assess the lung lesion extent, the
lungs were classified into three zones (upper, middle, and lower);
each zone was evaluated separately. Each of the three zones
corresponded to approximately one-third of the images from the
lung apex to 1 cm below the domes of the diaphragm ( 15, 16).
Each lung zone was assigned a score that was based on the
follows: (I) score 0, 0 involvement; (II) score 1, less than 25%
involvement; (III) score 2, 25% to less than 50% involvement;
(IV) score 3, 50% to less than 75% involvement; and (V) score
4, 75% or greater involvement. Summation of scores provided an
evaluation of overall lung involvement (maximal score for both
lungs was 24). |
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Results
According to our inclusion criteria, all the patients had CT
evidence of lung abnormalities consistent with influenza H1N1
pneumonia. The lung lesions were of typical virus infection,
including ground-glass opacity (hazy areas of increased
attenuation without obscuration of the underlying vessels),
consolidation (homogeneous opacification of the parenchyma
with obscuration of the underlying vessels), reticular opacities,
linear opacities, interlobular septal thickening, and mixed pattern
( Figures 1, 2). The mean viral load from the first time measurement was
4.56 log10 copies/mL (SD =1.62 log10 copies/mL, range,
1.70-8.35 log10 copies/mL). With the five patients whose
virus load was measured within 72 hours after the onset of
symptom, the viral load was 6.5 log10 copies/mL (SD =1.78,
range, 3.96-8.35 log10 copies/mL). The mean lung lesion
scores from the first time CT of the 24 patients was 6.6 (SD
=6.63, range, 1-24). The correlation between the lung lesion and
the virus load viral load was r=–0.14 (n=24 patients, Figure 3). With only the cases’ where first time CT and first time viral
load measurement were performed within 48 hours were
included for analysis, an r=–0.15 was obtained (n=20 patients).
In both cases, the correlation between the lung lesion score and
respiratory viral load was not statistically significant (P=0.4). The viral load time course and lung lesion score time course
of the 13 patients with radiological exam and viral load followup
are shown in Figures 4, 5. Figure 4 showed all the cases’
respiratory viral load decreased over time, with the highest
value at the time of initial presentation. The patients had higher
initial viral load tended to have longer virus shedding period.
Figure 5 showed lung lesion scores were also highest in the initial
presentation and decreased over time, except in one case where
there is a secondary chest infection. The patients had higher
initial lung lesion score were also slower in lesion resolving.
Compared with the respiratory viral load decrease rate (mean
slope =–0.31), the lung lesion scores decreased at a slower rate
(mean slope =–0.27). |
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Discussion
Specific clinical features have been reported for infections
associated with the influenza A H1N1 2009 virus. Most
hospitalized patients are under the age of 18 and very few are
over 65 years ( 10). Our series had a mean age of 17.5 years. This
is similar to the series reported by To et al. where the mean age
was 21 years ( 17). However, the demographics of our series
were different from the series reported by Aviram et al., their
series had a mean age of 40.4 years. All our patients recovered
after treatment. This could be due to most of our patients were in
healthy condition prior to this disease. It has been reported that
between 40% and 70% of patients with severe symptoms have an
underlying medical history ( 3, 10). Dynamic viral load reflects the interaction between viral
replication and clearance by body defense mechanisms. To et al.
reported that in patients with pandemic H1N1 virus infection,
peak viral load occurred on the day of onset of symptoms, and
declined gradually afterwards ( 17). This finding was similar
to the previous studies in seasonal influenza virus infection, in
which viral load was generally low or undetectable by day 5 of
illness, but could persist for up to 21 days in children ( 18). This
is due to patient’s immune system responded to infection ( 19, 20).
Anti-viral treatment may also contribute to the decrease of viral
load ( 21). The mean respiratory viral load during presentation in
our patients was lower than the data reported by To et al. ( 17).
This is likely due to our patients presented to the hospital on
average later after the symptoms than To et al.’s group. With
the patients presented to hospital within 3 days after onset of
symptoms ( Figure 3), respiratory viral loading of 6.5±1.78 log10
copies/mL was similar to To et al.’s data ( 17) ( Figure 4). With
the 22 pandemic H1N1 cases reported by To et al. no virus being
detectable in respiratory specimens by RT-PCR 8 days after the
onset of symptoms, except in one patient. Our patients had a slower
clearance of respiratory viral loading than To et al.’ s group ( 17). Monitoring viral load throughout the disease course has been
used as an objective means of checking the clinical progress
or response to antiviral therapy. de Jong et al. reported fatal
outcome of human influenza A H5N1 is associated with high
viral load and hypercytokinemia ( 14). It has been reported for
seasonal influenza patients hospitalized with severe diseases have
more active and prolonged viral replication ( 22). In severe acute
respiratory syndrome (SARS) cases, it has been reported that
viral load in nasopharyngeal aspirates from day 10 to day 15 after
onset of symptoms was associated with oxygen desaturation,
mechanical ventilation, diarrhea, hepatic dysfunction, and
death ( 23). In SARS coronavirus and influenza A H5N1 virus
infections, there was the inverse correlation between the absolute
lymphocyte count and concomitant viral load level in nontreated
and treated patients irrespective of the days post symptom onset
at the time when the specimens were sampled ( 21). However,
recently Duchamp et al. reported influenza A H1N1 2009 viral
load did not correlate with clinical history or specific clinical
symptoms ( 24). The general radiologic findings of H1N1 pneumonia, similar
to all viral pneumonias, are represented by poorly defined
nodules, patchy areas of peribronchial ground-glass opacity,
and air-space consolidation ( 11, 12, 25). In a series of 272 U.S.
patients, 40% of the hospitalized patients who underwent
chest radiography at admission had radiographic findings
consistent with pneumonia ( 10). In the largest series of patients published, which included 1088 cases of hospitalization or
death in California, 833 (66%) patients who underwent chest
radiography during their hospitalization had opacities suggestive
of pneumonia or acute respiratory distress syndrome ( 9). Our
results agreed with these reports. During the period of May-
December 2009, 24 (54.5%) of the 44 patients diagnosed
with H1N1 influenza in our hospital had associated lung
abnormalities. There are several limitations with this study. The patient
number of this study is small. However, results from this study
were consistent among patient subjects, the authors tend
to believe even there would be a modest increase in patient
number, the conclusions would be the same. The patients were
presented to hospital on average 5.2 days after the onset of
symptoms. This was later than some other series reported ( 13).
However, our additional analysis showed with the five patients
whose virus load and CT were obtained within 72 hours after
the onset of symptom, there was no trend to show a correlation
between initial viral load and initial lung lesion score. As there
was no X-ray exam prior to the onset of symptoms, it could
not be certain that in current series there were no lung lesions
existed before the H1N1 viral pneumonia; other secondary
respiration infections during the treatment course also cannot
be excluded. However, our series were mostly young subjects,
and all the cases’ disease course was consistent with viral
pneumonia except one case with secondary bacterial infection,
the chance of these possibilities is small. Even such cases
did exist in small number, they would not change the overall
conclusion of this study. The value of acquiring CT instead of
chest X-ray for all H1N1 influenza patients is questionable.
Though ground-glass opacity is a well-defined descriptor on a
CT scan, it is a subtle finding with limited demonstrability at
chest radiography. With H1N1 pneumonia, similar to various
other lung diseases, subtle lung opacities may be detected at
chest CT when chest radiographs have normal results ( 11).
The individuals of our series were admitted to our institution
when the epidemic, time course and outcome of this disease
were uncertain, and some extreme containment and mitigation
measures had been taken. To the best of our knowledge, this is the first time
such a study has been carried out. This current study
provides valuable information on H1N1 viral pneumonia
epidemiology. Our data shows both higher initial respiratory
viral load and higher initial lung lesion score are associated
slower resolution of those abnormalities. Respiratory viral
load decreases faster than chest lesion score. However, there is
no correlation between initial respiratory viral load and initial
lung lesion score. This result seems to support Duchamp et
al.’s recent report that, for influenza A H1N1 2009, viral load
does not correlate with clinical history or specific clinical
symptoms ( 24). |
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Acknowledgements
Disclosure: The authors declare no conflict of interest.
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Cite this article as: Lu P, Deng YY, Yang G, Liu W, Liu
Y, Huang H, Wang YX. Relationship between respiratory
viral load and lung lesion severity: a study in 24 cases
of pandemic H1N1 2009 influenza A pneumonia.
J Thorac Dis 2012;4(4):377-383. doi: 10.3978/
j.issn.2072-1439.2012.08.02
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