Secondary Logo

Journal Logo

Original article

Natural herbal medicine Lianhuaqingwen capsule anti-influenza A (H1N1) trial: a randomized, double blind, positive controlled clinical trial

DUAN, Zhong-ping; JIA, Zhen-hua; ZHANG, Jian; LIU, Shuang; CHEN, Yu; LIANG, Lian-chun; ZHANG, Chang-qing; ZHANG, Zong; SUN, Yan; ZHANG, Shu-qin; WANG, Yong-yan; WU, Yi-ling

Author Information
doi: 10.3760/cma.j.issn.0366-6999.2011.18.024
  • Free

Abstract

In March 2009, the first case infected with 2009 pandemic influenza A (H1N1) virus was identified in Mexico, this novel influenza virus spread-rapidly around the world.1-3 Due to being highly contagious and wide spreading, it is more lethal and has already caused social disruption.4,5 According to the most recent report from the WHO, as of 13 December 2009, more than 208 countries and overseas territories or communities have reported laboratory confirmed cases of pandemic influenza H1N1 2009, including at least 10 582 deaths.6 Facing a novel virus, vaccination is considered to be the most effective approach, nevertheless we usually encounter two difficult problems, undersupply and/or oversupply,7-9 intensifying the pressure on healthcare services.

Oseltamivir phosphate (Oseltamivir) and Zanamivir, inhibitors of neuraminidase, have been recognized as the most effective treatments presently available.10 But there are limitations to such antiviral drugs: a low clinical response in the patients if the initial administration of Oseltamivir is 48 hours after the onset of illness,11 viral resistance in some patients,12-14 the shortage of drug supply and the comparatively high cost in some under-developed countries.7-8 Therefore, there is an urgent need to explore new approaches and drugs for disease control.15

Alternative therapies, including nature herbal medicines (NHMs) have been recommended as a practice measure to treat influenza A (H1N1) in some countries and regions of the world.16 For example, traditional therapies have been used in China for treatment of infectious diseases for thousands of years.17 Because of the fundamental theoretical differences between traditional medicine and modern medicine, and also due to the therapeutic effects usually coming from the synergistic action of certain ingredients of NHMs, it is more difficult to evaluate NHMs efficacy and safety. The role of NHMs in treatment is still controversial. Lianhuaqingwen capsule (LHC), a kind of NHM, approved by the China State Food and Drug Administration (SFDA) in 2004, has been recommended as one of the drugs to treat influenza A (H1N1) by the China health authority in 2009.16 Before this study, there were several experiments that showed the anti-influenza A action of LHC. For example, an in vitro study showed that the treatment index of LHC anti-influenza A with three drug delivery options, including pre-treatment, co-treatment and post-treatment, were higher than Oseltamivir phosphate. Another in vivo study showed that LHC could provide protection to Balb/C mice infected by influenza A virus, and the survival rate and survival time of the LHC group were prior to model group. In September 2009, influenza A broke out in Langfang city of Hebei province. A randomized controlled clinic trial showed that there were no significant differences between LHC treated and Oseltamivir treated patients in the duration of viral shedding. However, LHC significantly reduced the duration of symptoms including fever, cough, and so on.18 To further confirm its therapeutic effects, we conducted a randomized, double blind, and positive controlled clinical trial to evaluate the efficacy and safety of LHC in patients infected with 2009 influenza A.

METHODS

Patients

Patients had to meet all the following criteria for inclusion: with confirmed influenza A virus infection by real time RT-PCR (rRT-PCR), age between 16-65 years old, axillary temperature ≥37.4°C and with at least one of the following symptoms, cough, sore throat, headache, running nose, fatigue, nasal obstruction, myalgia, chills and sweating. Illness onset had to be within 36 hours, women must be urine pregnant test negative and must use effective contraceptive methods during the study and within 30 days after the end of the study, and informed consent was obtained.

Patients were excluded from the study if they met any one of the following conditions: age younger than 16 or older than 65 years old, axillary temperature lower than 37.4°C, those with underling primary disorders, such as cardiovascular disease, cerebral vascular disease, hepatic disease, renal disease, hematological disease or psychological disorders, those with accompanying pharyngitis, acute chronic obstructive laryngitis or acute tracheitis, or brochures and pulmonary disease, lung cancer, bronchiectasis or with concomitant respiratory diseases, allergy to the study medication(s), those already vaccinated or who will receive influenza vaccine, women who are pregnant or may possibly become pregnant or are lactating with a positive urine pregnant test, obesity (a body mass index (BMI) of 40 kg/m2 or more), have participated in other clinical trial within one month before study randomization, and other reasons not suitable for enrollment based on the investigator's discretion.

The study protocol was approved by the central ethics committee and the local ethics committee. An informed consent was signed by each subject before the screening.

Study design

A multi-center, double blind, parallel and randomized study was conducted in eight hospitals crossing seven provinces in mainland China, with LHC as the investigational medication and Oseltamivir as the positive control. The randomization number for each site and study medication was developed using a computer random number generator with software SAS®9.13. Each block contained four numbers.

Subjects were randomly enrolled either in LHC or Oseltamivir group in a 1:1 ratio; each subject was assigned a randomization number in ascending order and treated with the correspondingly identified study medication until the enrolled subjects in each site reached the scheduled number. Each block comprised four subjects randomly assigned to either LHC or Oseltamivir. Each site was scheduled to enroll no less than eight subjects (two blocks) and no more than 56 subjects (14 blocks); based on the actual recruitment, the sponsor adjusted them timely.

The nasal swab from each suspected case of influenza A infection was first screened by the One Step Influenza Virus A Paper Test (manufactured by Guangzhou WondFo Biotech. Co., Ltd., China). The pharyngeal or nasopharyngeal swab from patients with positive results by paper test were collected and tested by rRT-PCR (Beijing KINGHAWK Pharmaceutical Company, China) to confirm the infection of influenza A (H1N1) virus. If patients were positive for both tests and met the inclusion/exclusion criteria, the subject would be randomly assigned to either the LHC or Oseltamivir group for administration study medications for five days and observation for seven days.

Although the on site paper test only took about 15-20 minutes to get results, laboratory diagnosis by rRT-PCR usually took about 6 hours, or even 12 hours in some cases. To ensure subjects received the study medication within 36 hours of the onset of illness and to improve patient compliance, the study procedure was adjusted: patients positive for the paper test who met the inclusion/exclusion criterion were eligible to enter the study and randomly assigned to either LHC or Oseltamivir. Later on, if rRT-PCR tested negative, the study medication was stopped immediately and the patient was considered as not fulfilling the inclusion/ exclusion criteria and withdrawn from the study.

Rationale for sample size

The study was powered as a non-inferiority study, and the primary endpoint was relief time of influenza-like symptoms. Non-inferiority boundary value was 24 hours. Sample size was based on a common standard deviation of both groups was 60 hours, with an error probability of 0.025 (single-tailed alpha error) and 0.9 for beta, computation formula described following yielded a sample size of 107 subjects per treatment arm. Considering a possible 20% expulsion rate, the designed sample size should be 256 128 subjects per treatment arm.

Study medications

Patients in LHC group received four capsules (0.35 g of ingredients/capsule) of LHC/TID and one capsule of Oseltamivir analogue/BIO orally for five days. Patients in Oseltamivir group received one capsule (75 mg) of Oseltamivir/BIO and four capsules of LHC analogue/TID orally for five days.

All the study medications (LHC, LHC analogue and Oseltamivir analogue) were prepared in identical- appearing capsules (including color, weight and smell) by the sponsor. Oseltamivir was manufactured by Shanghai Roche Pharmaceutical Company. The personnel involved in the manufacture and package were not allowed to have any contact to the study subjects and the relevant study personnel.

Detection of influenza A (H1N1) virus

Collection of nasal swabs and the One Step Influenza Virus A Detection kit

The nasal swab was inserted in the nasal cavity having lots of secretions, gently turned and pushed it to the turbinate with the distance about 2.5 cm from the nostril. After turning three times, take the nasal swab out then follow the instruction of the test. The result was obtained in 15-20 minutes.

Collection of swab of pharyngeal and influenza A (H1N1) virus nucleic acid detection

The swab was taken out from the tissue culture tube, then gently and quickly wiping the double transpalatal arch, pharynx and tonsils. Sterilizing the test tube was performed by heating under an alcohol lamp. Take the swab and insert it into the test tube and plug the hole with the cork. On the tube note the patient information and patient's number and date. Put the swab of the pharyngeal into a sterilized solution and keep at -65°C. Transport the specimen to the virology laboratory of the local CDC or virology laboratory of the local site in container with coal pack within 12 hours of collection and assayed by rRT-PCR.

Data collection and follow-up

Clinical laboratory tests included routine blood tests, urine tests, hepatic function, renal function, serum myocardial enzymes and blood chemistry examination. Physical examination, vital signs, chest X-ray, 12-head electrocardiograph (ECG) and laboratory tests were measured at baseline and the last follow-up or the day when patient's influenza-like symptoms were completely resolved and laboratory test negative by rRT-PCR. Patients' specimens were collected and assayed daily by rRT-PCR. Patients with any presenting symptoms were evaluated by themselves based on the predefined four levels of severity (0 absent, 1 mild, 2 moderate, 3 severe) and axillary temperature was measured every six hours with the maximum interval less than one hour and recorded in the patients' diary once daily.

Investigators collected and assessed the adverse events and assessed any complications or any concomitant medications including acetaminophen and antibiotics on daily basis. At the last follow-up, patients returned all medication containers and the remaining pills were counted. If any symptom was not resolved, recording continued until all the symptoms were resolved and sustained for 24 hours.

Clinical outcomes

The primary end point was the duration of illness which was defined as the time from onset of symptoms to the alleviation of the nine influenza-like symptoms including nasal obstruction, running nose, cough, sore throat, headache, fatigue, myalgia, chills and sweating.19,20

The secondary end points included: viral shedding duration, defined as the time from the illness onset to the first time the viral nucleic acid test was negative; the time to defervescence, defined as the time from the first dose of study medication to the time when the body temperature declined to lower than 37.3°C and was sustained for at least 24 hours; the severity of the disease, assessed by an area under the curve (AUC) analysis of a total of eight influenza-like symptom scores, the AUC was calculated as the product of the daily symptom scores times the duration of illness;20 time to individual symptom alleviation, defined as the time from the onset of the individual symptom to the time the symptom was alleviated. Accuracy of measurement was one hour.

Safety evaluation

The safety population included all the subjects who received at least one dose of study medication and for whom post-baseline safety data were available. Safety evaluations included adverse events and clinical laboratory examination.

Statistical analysis

The clinical trial was analyzed by the principle of intention to treat (ITT). ITT included all the subjects who were randomly assigned to one of the group and received at least one dose of study medication and for whom one follow-up efficacy data point was available. We used the last-observation-carried-forward method in the analysis of all outcomes among patients who made at least one follow-up visit but who did not complete follow-up. The safety population included all the subjects who received at least one dose of study medication and for whom post-baseline safety data were available. The Cochran Mantel-Haenszel (CMH) chi-square test or Fisher's exact test was used to analyze the difference in qualitative parameters between groups. Two-group t test or paired t test was used to compare the quantitative parameters of normal distribution for two-group; and Wilcoxon rank sum test was used to compare the quantitative parameters of non-normal distribution. For statistical comparison of the difference in time to relief of all flu-like symptoms between the two treatment groups, a non-parametric approach was chosen: the Hodges-Lehmann estimator and the one-sided 97.5% CI according to Moses.21 Non-inferiority was concluded if the lower boundary of the 97.5% CI was greater than 24 hours, as the difference of 24 hours was considered as the clinically relevant margin. Significant difference was established when P values <0.05. All analyses were performed using SAS software, version 9.1.3 (SAS Institute, USA).

RESULTS

Patient characteristics

From October 24, 2009 to November 23, 2009, a total of 405 suspected influenza A (H1N1) virus infected subjects were screened by nasal swabs, 362 were found influenza A positive by paper test. Of them 256 subjects that met the inclusion/exclusion criteria and were randomly assigned to treatment. Ten subjects, including five from each group, failed the laboratory test. One subject from the LHC group was administrated other NHMs after entry into the study and the pharyngeal swab from one subject in the Oseltamivir group did not detect virus by rRT-PCR due to inappropriate transportation. The study medication administration was stopped immediately for those 12 patients and they withdrew from the study. Final numbers were 244 subjects, with 122 in each group included in ITT analysis, with one from the LHC group and three subjects from the Oseltamivir group lost to follow-up (Figure 1).

Figure 1.
Figure 1.:
Study work flow chat.

The proportion of patients quarantined in a designated dormitory of a college campus, in the hospitals and at home was 54.5% (133/244), 31.1% (76/244) and 14.3% (35/244), respectively. All the patients were instructed by study investigators to measure their body temperature, to score and record the severity of symptoms, and to administrate study medications by themselves. All the measurements were performed based on the standard operating procedure attached to the patient's diary card and recorded on the patient's card once daily. Of the patients who were not hospitalized, the study investigator visited those patients in the designated dormitory or at home once daily to collect the records of the severity of symptom scores and the patient's specimens.

There were no significant differences between the groups at the baseline in demographic variables including duration of illness, body temperature before the initial dose and physical examination, laboratory test (except lymphocyte counts), chest X-ray film and ECG. The baseline data and distribution for subjects were comparable (Tables 1 and 2).

Table 1
Table 1:
Demographic characteristics of the patients at the baseline
Table 2
Table 2:
Distribution of the patients

Time to symptom alleviation

The Hodges-Lehmann estimator for the median difference in time to relief of all flu-like symptoms between treatment groups was 10 hours and the one-sided 97.5% CI according to Moses was (2; ∞) (ITT population). Considering that the non-inferiority margin was set at -24 hours, the lower boundary of the CI was clearly higher. A sensitivity analysis was also performed for 18 subjects in the ITT population with non-remission at the end of the study or who withdrew from the study. In the sensitivity ananlysis,22 168 hours was assigned to missing values in the LHC group and 0 hour to those of the control group. The one-side 97.5% CI for the median difference in time to relief of flu-like symptoms between groups was (-11; ∞) and the lower boundary was still higher than the non-inferiority margin (-24 hours).

Analyzing the total clinical response, as shown in Figure 2, the two Kaplan-Meier curves were completely overlapped within the first 12 hours after enrollment, but were split thereafter and remained parallel until the end of the observation. This indicates better relief in LHC treated patients, although there was no statistically significant difference (median 69 vs. 85, P >0.05).

Figure 2.
Figure 2.:
Alleviation time of all symptoms in influenza A (H1N1)-infected.

When it came to the alleviation of the severity of illness, a significant difference was observed between the groups (P=0.047). The areas under the curve were 281.2 for LHC treated patients and 318.5 for Oseltamivir treated patients. This indicated that in comparison with Oseltamivir, LHC significantly reduced the severity of illness and the duration of illness.

To eliminate any potential effects of analgesics and antipyretics on the duration of illness, patients who administrated analgesics and antipyretics during observation were excluded, 15 from the LHC and 19 from the Oseltamiviri groups. Still no significant difference was observed in the median duration of the illness between LHC and Oseltamiviri groups (68 hours vs. 83 hours, respectively; P >0.05). But Log-rank examination demonstrated that LHC had a significant effect on the reduction of the average duration of illness ((73±37) hours for LCH vs. (86±35) hours for Oseltamivir, P=0.0354).

Duration of viral shedding

The average duration of viral shedding in LHC treated patients and Oseltamivir treated patients was (108±36) hours and (101±34) hours, respectively, and Log-rank test showed no statistically significant difference was observed (P=0.1546). As shown in Figure 3, from 72 hours after illness onset to the end of the observation period, the two Kaplan-Meier curves were completely overlapped with each other. This confirms that from three days of illness onset, no significant difference in viral shedding was observed between the groups.

Figure 3.
Figure 3.:
Duration from onset to the first negative of H1N1 virus by rRT-PCR.

Time to defervescence

The average time to defervescence for LHC treated patients was (17±14) hours (n=122) and (23±17) hours (n=122) for Oseltamivir treated patients, a significantly improved defervescence effect for LHC (P=0.0059).

To eliminate any potential effect of analgesics and antipyretics on the time to defervescence patients administrated analgesics and antipyretics during observation were excluded, 15 from the LHC group and 19 from the Oseltamivir group. LHC had a significant effect on reduction of the average time to defervescence over Oseltamivir (n=107, (15±12) hours for LHC treatment vs. n=105, (19±15) hours for the Oseltamivir group).

Based on body temperature, these patients were subgrouped into a ≤38°C group and a >38°C group. We found no significant difference in the average time to defervescence between LHC treated patients (n=54) and Oseltamivir treated patients (n=55) in the ≤38°C group ((12±13) hours and (15±14) hours, respectively, P=0.1770). Meanwhile, significant difference was observed between the LHC (n=53, (17±10) hours) and Oseltamivir treated patients in the average time to defervescence (n=50, (24±15) hours) in the >38°C subgroup (P=0.0077).

Average duration of each individual influenza-like symptom

The average duration of each individual symptom was shown in Table 3. Significant differences were observed in the average duration of symptoms like cough, sore throat, and fatigue (P <0.05). The above data clearly showed that LHC had significantly greater effects on the reduction of the average duration of the above symptoms.

Table 3
Table 3:
The median disappear time (hour) and their 95% CIs of symptoms

Safety analysis

There were no significant differences in the baseline data of laboratory test between the groups. There were a total of 11 non-serious adverse events with four from the LHC group and seven from the Oseltamivir group, which were assessed as possibly unrelated to medication. A patient who was isolated at home developed pneumonia at study day seven, and antibiotics and supporting therapy were applied immediately at home, and the event was resolved on day 18. The causal relationship with Oseltamivir was assessed to be unrelated and the event assessed as a serious adverse event by the study investigator. The reporting of serious adverse events was in accordance to the relevant regulation of China health authority. There was no significant difference in the incidence rate between the LHC and Oseltamivir groups. No study medication was discontinued due to adverse events in the study. There were no adverse events judged to be related to LHC, while there were four adverse events, nausea and vomiting, judged to be related to Oseltamivir.

DISCUSSION

Although vaccination and antiviral medicines, Oseltamivir and Zanamivir, are the most effective measures, certain limitation still exists for the control of the pandemic outbreak of novel influenza.23-26 Researches intent on finding more natural drugs against influenza A have demonstrated in vitro or in mouse models the anti-influenza virus activity of different plant extracts. The known antiviral actions include causing virus inactivation, interference with viral replication, or blocking the spread of infection.27-30 LHC is a combination of 45 ingredients extracted from 13 kinds natural herbs including honeysuckle flower, isatis root, weeping forsythia capsule, rhubarb, radix glycyrrhizae. Although LHC has been used for treating seasonal influenza in China since 2004, as an alternative therapy against influenza A (H1N1), its clinical effectiveness and safety evaluated by randomized clinical trial was largely untested. We conducted the random, double blind and Oseltamivir positive control study to evaluate its efficacy and safety in patients infected with influenza A (H1N1).

The clinical features of influenza A (H1N1) are similar to seasonal influenza: fever, cough, sore throat as the most common symptoms, then headache, running nose, fatigue, nasal obstruction, myagia and chills, nausea, vomiting and diarrhea seldom occur.8,31 A recent observational study in Chinese patients showed that the most common symptoms were fever, cough, and sore throat. The other symptoms, like nausea, vomiting and diarrhea, were comparatively uncommon.32 Accordingly these clinical manifestations were regarded as the main symptoms in the management guideline of influenza A (H1N1) issued by the China Health Authority.19 Influenza is a self-limiting disease, therefore, symptoms alleviation and quality of life improvement are the goal of therapy. In the present study, the most common symptoms in the study population, except for fever (100%), were cough (86.1%), sore throat (80.7%), headache (62.7%), running nose (57.8%), fatigue (57.0%), nasal obstruction (52.5%), myalgia (47.5%), chills (45.1%), and sweating (24.2%). Therefore, the relief of these symptoms was used to evaluate the clinical response.

In the present study, 85.7% of patients were college students with median age of 21 years old (16-65 years), which reflects the demographic features of the second outbreak of influenza A (H1N1) in China, and was similar to previous reports from the United States.33 In comparison with seasonal influenza, influenza A (H1N1) was more likely to disproportionately infect individuals in a younger age group.

This study demonstrated that no statistically significant difference was observed in the median duration of illness. Furthermore, no difference was observed between the two groups, even after the patients who were administrated analgesics and antipyretics medications excluded. It seems worthwhile to notice that, in terms of AUC analysis, LHC had a more significant effect on relieving the severity of the disease. Meanwhile, we also noticed that LHC had a significant effect on the relieving of individual influenza-like symptoms like fever, cough, sore throat, and fatigue. The average time to defervescence for LHC treated patients and Oseltamivir treated patients was 17 hours and 23 hours, respectively. The six hour difference in patients receiving LHC strongly suggested that LHC had a better antifebrile effect than Oseltamivir. Further stratified analysis failed to show any difference of the time to defervescence in patients with body temperature ≤38°C. However, we observed a significant difference in LHC treated patients with body temperature >38°C in comparison to Oseltamivir treated patients, which again indicated a better antifebrile effect of LHC. In terms of the patients not administered analgesics and antipyretics medications, the average duration of illness and the time to alleviation of fever were comparatively shorter in LHC treated patients than in Oseltamivir treated patients. This indicated the effect of LHC was not affected by this factor.

The previous studies found that, by tissues culture, the highest percentage of active influenza A (H1N1) virus existed within three days of the presenting of influenza-like symptoms. The active virus in tissue culture was even found in 24% of patients until the day seven.34 A study conducted in China from April 2009 to July 2009 found that the median illness duration (from the onset of illness to the first time when the result of rRT-PCR was negative) was six days (range, 1-17 days). At the same time it also indicated that using of Oseltamivir within 48 hours of the onset of illness will reduce the duration of illness and viral shedding.31 In the present study, the median viral shedding duration in LHC treated patients and Oseltamivir treated patients was 108 hours, 101 hours, respectively. Accordingly, administration of either LHC or Oseltamivir within 36 hours of the onset of illness produced a 25.2% or a 29.50% reduction in viral shedding duration compared with the data in Cao's32 reports. The accumulative number of patients with viral nucleic acid clearance (test negative by rRT-PCR) among the LHC treated patients and Oseltamivir treated patients was 17.2% (21/122) and 23.0% (28/122) on the third day of symptoms, which grew to 81.2% (99/122), and 85.3% (104/122) on day 6. In this study the intervention with either LHC or Oseltamivir shortened the duration of the patients' viral shedding compared with the previous reports. This indicated that both study drugs have similar therapeutic effectiveness in inhibition of viral replication, with no statistically significant difference observed.

In the present study, the duration of viral shedding from 79 patients, including 46 LHC treated patients and 33 Oseltamivir treated patients, was over five days. The multivariable logistic-regression analysis under 0.05 examinations found the significance of association of risk fact of sex and the prolonged duration of viral shedding at the edge (P=0.053). No significant difference in the grouping, body temperature, age, BMI, or the duration of illness was observed. In consideration of the characteristics of demography, like age and the time point of administration, the present study results are consistent with the previous reports.31

Oral LHC and Oseltamivir were well tolerated. No difference was observed in laboratory safety evaluations in the two groups. No drug-related serious adverse events occurred in either group, neither of the study drugs were discontinued due to adverse events. There was no-adverse reaction in LHC treated patients, while there were four adverse reactions in Oseltamivir treated patients, which were nausea and vomiting. Overall, LHC and Oseltamivir were well tolerated in the study.

Differently from other clinical trials of NHMs, the current study was designed as a randomized, multicenter double blind clinical trial conduced in China to evaluate the efficacy and safety of NHMs in patients infected with influenza A (H1N1). However, several potential limitations of the present study should be considered. First, since influenza A (H1N1) has the potential to develop secondary severe complications, which are life threatening and even lethal, a placebo group was not considered. Second, the study protocol excluded the high risk population and those patients with severe complications. Third, the observation period was comparatively short, the symptoms of six patients from the LHC group and 12 patients from the Oseltamivir group were not resolved completely on day seven and their eventual outcomes were not included. Fourth, due to the limitation of employing RT-PCR, the viral shedding duration and the viral clearance rate were determined qualitatively, and the dynamic change during viral clearance could not be addressed in this study.

In summary, this randomized and double blind clinical trial demonstrated for the first time LHC's therapeutic effect against influenza A (H1N1), the result showed that LHC had a significant effect on the alleviation of fever, cough, sore throat and fatigue. Regarding to the reduction of illness duration and viral shedding duration, LHC can achieve at least the same therapeutic effectiveness like Oseltamivir. Both drugs were well tolerated.

Acknowledgements:

We thank Dr. ZHENG Qing-shan, LIU Hong-xia and HUANG Zong-han (Shanghai University of Traditional Chinese Medicine Clinical Research Center for Drugs) for kindly guiding us through the protocol, data management, and statistical work. We are also indicated to the patients for their participation and investigators at the hospitals and local CDC for data collection and laboratory testing.

We also thank the Clinical Investigation Group members for their help in collecting samples: LI Xiu-hui, YAN Hui-ping, WU Hao (Beijing You'an Hospital, Capital Medical University); SUN Yan, HAO Yi-bin, MA Chao, WANG Yong-ming (Henan Provincial Infectious Disease Hospital); CHEN Shi-jun, AN Yong (Jinan Infectious Disease Hospital); WANG Li, DENG Ping, CUI Wen-yu (Changchun Infectious Disease Hospital); ZHOU Ji-kun (Shijiazhuang City Center Disease Prevention and Control); DAI Er-hei, JIAO Xiu-kun, ZHOU Ting (Shijiazhuang Infectious Disease Hospital); LIU Fu-qiang (Hunan Provincial Center Disease Control and Prevention); WEN Xian-min, LI Ji-ke, ZHOU Xiao-fei (Chengdu Infectious Disease Hospital); ZHANG Ming-xiang, YAN Ying-chun (Shenyang Infectious Disease Hospital); BAI Wei, LI Bao-fa, LIU Bao-hua, FU Min, YUAN Cui-yun, LI Shu-sen, CAI Shang-yuan (Kaifeng Infectious Disease Hospital).

REFERENCES

1. DG statement following the meeting of the Emergency Committee. Geneva: World Health Organization, 2009. (Accessed June 11, 2009 at http://www.who.int/csr/disease/swineflu/4th_meeting_ihr/en/index.htm.)
2. Centers for Disease Control and Prevention (CDC). Swine influenza A (H1N1) infection in two children, Southern California, March-April 2009. MMWR Morb Mortal Wkly Rep 2009; 58: 400-402.
3. Swine influenza update 5. Geneva: World Heath Organization, 2009. (Accessed December 18, 2009 at http://www.who.int/csr/don/2009_04_29/en/index.html.)
4. WHO technical consultation on the severity of disease caused by the new influenza A (H1N1) virus infections. Original short summary posted May 6 2009. Revised full report posted May 9 2009. Geneva: World Heath Organization, 2009. (Accessed December 18, 2009 at http://www.who.int/csr/resources/publications/swineflu/technical_consultation_2009_05_06/en/index.html.)
5. Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team, Dawood FS, Jain S, Finelli L, Shaw MW, Lindstrom S, et al. Emergence of a novel swine-origin influenza A (H1N1) virus in humans. N Engl J Med 2009; 360: 2605-2615.
6. Pandemic (H1N1) 2009 update 79. Geneva: World Heath Organization, 2009. (Accessed December 18, 2009 at http://www.who.int/csr/don/2009_12_18a/en/index.html.)
7. Mossad SB. The resurgence of swine-origin influenza A (H1N1). Cleve Clin J Med 2009; 76: 337-343.
8. Petrosillo N, Di Bella S, Drapeau CM, Grilli E. The novel influenza A (H1N1) virus pandemic: an update. Ann Thorac Med 2009; 4: 163-172.
9. France sells off surplus swine flu vaccine. BBC. One-minute world news. January 3, 2010 (Accessed January 5, 2010 at http://news.bbc.co.uk/2/hi/health/8438663.stm)
10. Centers for Disease Control and Prevention (CDC). Update: drug susceptibility of swine-origin influenza A (H1N1) viruses, April 2009. MMWR Morb Mortal Wkly Rep 2009; 58: 433-435.
11. Updated interim recommendations for the use of antiviral medications in the treatment and prevention of influenza for the 2009-2010 season. Atlanta: Centers for Disease Control and Prevention, 2009. (Accessed December 18, 2009 at http://www.cdc.gov/h1n1flu/recommendations.htm)
12. Lackenby A, Hungnes O, Dudman SG, Meijer A, Paget WJ, Hay AJ, et al. Emergence of resistance to Oseltamivir among influenza A (H1N1) viruses in Europe. Euro Surveill 2008; 13. Pii: 8026.
13. Centers for Disease Control and Prevention (CDC). Update influenza activity — United States, September 28, 2008-January 31, 2009. MMWR Morb Mortal Wkly Rep 2009; 58: 115-119.
14. Centers for Disease Control and Prevention (CDC). Oseltamivir-resistant novel influenza A (H1N1) virus infection in two immunosuppressed patients, Seattle, Washington, 2009. MMWR Morb Morta Wkly Rep 2009; 58: 893-896.
15. Wang YT, Chan CH, Su ZY, Chen CL. Homology modeling, docking, and molecular dynamics reveals HR1039 as a potent inhibitor of 2009 A (H1N1) influenza neuraminidase. Biophys Chem 2010; 147: 74-80.
16. The guideline for the surveillance, reporting, diagnosis, and treatment of pandemic (H1N1) 2009. Beijing: Ministry of Health of the People's Republic of China, 2009. (Accessed December 18, 2009 at http://www.moh.gov.cn/publicfiles/business/htmlfiles/mohyzs/s3585/200905/40478.htm.)
17. Hsu CH, Hwang KC, Chao CL, Chang SGN, Ho MS, Lin JG, et al. An evaluation of the additive effect of natural herbal medicine on SARS or SARS-like infectious diseases in 2003: a randomized, double-blind, and controlled pilot study. Evid Based Complement Alternat Med 2008; 5: 355-362.
18. Liu GX, Zhang YX, Yang JQ, Gao ZQ, Meng YC. The randomized controlled study of Lianhuaqingwen capsule in treating A/H1N1 influenza. Chin J Diffic Ccompl Dis (Chin) 2010; 1: 14-16.
19. The Guideline for the surveillance, reporting, diagnosis, and treatment of pandemic (H1N1) 2009 (the third edition). Beijing: Ministry of Health of the People's Republic of China. (Accessed October 13, 2009 at http://61.49.18.65/publicfiles/business/htmlfiles/mohyzs/s3586/200910/43111.htm)
20. Treanor JJ, Hayden FG, Vrooman PS, Barbarash R, Bettis R, Riff D, et al. Efficacy and safety of the oral neuraminidase inhibitor Oseltamivir in treating acute influenza: a randomized controlled trial. JAMA 2000; 283: 1016-1024.
21. Hollander M, Wolfe DA. Nonparametric statistical methods. New York: John Wiley & Sons; 1973: 75-82.
22. Points to consider on missing data. EMEA: Committee for Proprietary Medicinal Products, 2001. (Accessed September 20, 2009 at http://www.emea.eu.int/pdfs/human/ewp/177699EN.pdf.)
23. Hayden F. Developing new antiviral agents for influenza treatment: what does the future hold? Clin Infec Dis 2009; 48: 3-13.
24. Jefferson T, Di Pietrantonj C, Debalini MG, Rivetti A, Demicheli V. Inactivated influenza vaccines: methods, policies, and politics. J Clin Epidemiol 2009; 62: 677-686.
25. Lackenby A, Thompson CI, Democratis J. The potential impact of neuraminidase inhibitor resistant influenza. Curr Opin Infect Dis 2008; 21: 626-638.
26. Cheng PK, Leung TW, Ho EC, Leung PC, Ng AY, Lai MY, et al. Oseltamivir- and amantadine-resistant influenza viruses A (H1N1). Emerg Infect Dis 2009; 15: 966-968.
27. Palamara AT, Nencioni L, Aquilano K, De Chiara G, Hernandez L, Cozzolino F, et al. Inhibition of influenza virus replication by resveratrol. J Infect Dis 2005; 191: 1719-1729.
28. Wang X, Jia W, Zhao A, Wang X. Anti-influenza agents from plants and traditional Chinese medicine. Phytother Res 2006; 20: 335-341.
29. Ehrhardt C, Hrincius ER, Korte V, Mazur I, Droebner K, Poetter A, et al. A polyphenol rich plant extract, CYSTUS052, exerts anti influenza virus activity in cell culture without toxic side effects or the tendency to induce viral resistance. Antiviral Res 2007; 76: 38-47.
30. Droebner K, Ehrhardt C, Poetter A, Ludwig S, Planz O. CYSTUS052, a polyphenol-rich plant extract, exerts anti-influenza virus activity in mice. Antiviral Res 2007; 76: 1-10.
31. Human infection with new influenza A (H1N1) virus: clinical observations from a school-associated outbreak in Kobe, Japan, May 2009. Wkly Epidemiol Rec 2009; 84: 237-244.
32. Cao B, Li XW, Mao Y, Wang J, Lu HZ, Chen YS, et al. Clinical features of the initial cases of 2009 pandemic influenza A (H1N1) virus infection in China. N Engl J Med 2009; 361: 2507-2517.
33. Centers for Disease Control and Prevention (CDC). Swine-origin influenza A (H1N1) virus infections in a school — New York City, April 2009. MMWR Morb Mortal Wkly Rep 2009; 58: 470-472.
34. Witkop CT, Duffy MR, Macias EA, Gibbons TF, Escobar JD, Burwell KN, et al. Novel influenza A (H1N1) outbreak at the US air force academy: epidemiology and viral shedding duration. Am J Prev Med 2009; 38: 121-126.

Correction

In the original article entitled Matrix metalloproteinase-9 was involved in the immuno-modulatory defect of mesenchymal stem cell from chronic myeloid leukemia patients published in August 20 issue, 2011 (Chin Med J 2011;124(16):2423-2430), there are two errors in the author information: (1) the last author “LI You-bin” should be “LI Wen-bin”; (2) the corresponding author is changed to be LI Wen-bin, Institute of Medical Oncology, Beijing Shijitan Hospital, Captial Medical University, Beijing 100038, China (Tel: 86-10-63926351. Email: [email protected])

Keywords:

influenza A (H1N1); Lianhuaqingwen capsule; clinical randomized controlled trial

© 2011 Chinese Medical Association